WO2002101382A1 - Dispositif d'analyse d'echantillon chimique ou biochimique, ensemble d'analyse comparative, et procede d'analyse associe - Google Patents
Dispositif d'analyse d'echantillon chimique ou biochimique, ensemble d'analyse comparative, et procede d'analyse associe Download PDFInfo
- Publication number
- WO2002101382A1 WO2002101382A1 PCT/FR2002/001978 FR0201978W WO02101382A1 WO 2002101382 A1 WO2002101382 A1 WO 2002101382A1 FR 0201978 W FR0201978 W FR 0201978W WO 02101382 A1 WO02101382 A1 WO 02101382A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- micro
- fractionation
- columns
- capture
- column
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/466—Flow patterns using more than one column with separation columns in parallel
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6095—Micromachined or nanomachined, e.g. micro- or nanosize
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6034—Construction of the column joining multiple columns
- G01N30/6043—Construction of the column joining multiple columns in parallel
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/76—Acoustical detectors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
- Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
- Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction
Definitions
- Chemical or biochemical sample analysis device comparative analysis set, and associated analysis method.
- the present invention relates to a device for chemical or biochemical analysis of chemical or biological samples, in particular for a comparative analysis of at least two samples.
- the invention also relates to a comparative analysis assembly, as well as to an analysis method.
- the subject of the present invention is a device for chemical or biochemical analysis of samples making it possible to overcome these drawbacks.
- the subject of the invention is a device for chemical or biochemical analysis of samples allowing rapid separation of the constituents of a sample and a rapid analysis of the separate constituents, as well as the comparison of analyzes of different samples.
- the subject of the invention is also a device for chemical or biochemical analysis of samples allowing improved separation of the constituents of a sample, and in particular separations of the constituents of a sample according to different selectivity criteria.
- Such a device for chemical or biochemical analysis of biological or chemical samples comprises a plurality of micro-columns for fractionation of constituents of a sample, each micro-column for fractionation comprising at least a portion of a micro-channel provided with intermediate separation means, the micro-channel portion comprising an orifice for introducing a mobile phase enriched in sample and an evacuation orifice located at a terminal end.
- the device comprises fluid means for capturing fractionation products at the level of a terminal element of each fractionation micro-column situated upstream from its discharge orifice, micro-channels of ' capture intended to recover the captured fractionation products, and sets of selective micro-levers associated with the fractionation micro-columns and located downstream of the capture micro-channels, a micro-lever comprising detection means connected to means of 'analysis.
- the fractionation micro-column designates a portion of micro-channel provided with separation means.
- the micro-channel portion forming the fractionation micro-column can be preceded by downstream or upstream micro-channel portions devoid of separation means.
- the end of the micro-channel portion configured as a fractionation micro-column is designated by the orifice for introducing and removing the fractionation micro-column.
- Separation means can be a phase called stationary phase, of the type used in chromatography, or electrochromatography, or an electrophoresis gel, or electrical means.
- a mobile phase can advantageously be an eluent which, depending on its composition, has a greater or lesser affinity with constituents of the sample, of the molecule or protein type, and therefore more or less able to entrain the constituents, a stationary phase having tendency to slow down the migration of constituents, more or less according to their characteristics.
- a mobile phase circulates in a micro-channel carrying a sample.
- the separation means provided in the micro-channel portion forming a micro- fractionation column causes fractionation or separation of the components of the sample.
- the separation is carried out by differential migrations of each constituent of the sample in the micro-column, as a function of a selectivity of the micro-column and of the mobile phase.
- the mobile phase drives the components of the sample to the micro-channel discharge orifice.
- the fluidic capture means make it possible to take separate constituents from the sample located at the time of sampling in a determined terminal portion of short length of the fractionation micro-column, said terminal portion being located upstream of the orifice. micro-channel discharge.
- the captured constituents are entrained in micro-capture channels towards selective micro-levers, with a view to their detection and analysis.
- the combination of fluidic capture means and micro-levers allows a rapid analysis of the captured components to determine the composition of the sample.
- the means of analysis linked to the micro-levers allow rapid results to be obtained.
- each fractionation micro-column or a group of fractionation microcolumns of the same length may be made for each fractionation micro-column or a group of fractionation microcolumns of the same length to differ in length from the other fractionation microcolumns or groups of fractionation micro-column , the terminal elements being located on each fractionation micro-column at a given distance from the terminal end of the fractionation micro-column.
- the mobile phase enriched in sample flows from the micro-channel introduction opening towards the evacuation opening.
- the terminal elements, where the catches are made are located at different distances from the entry of the micro-columns. The migration rates of the constituents are different.
- micro-columns or micro-column groups of different constituents we will find in the terminal elements.
- the terminal elements of the longest micro-columns we will find the most rapidly migrating molecules.
- the terminal elements of the shortest micro-columns we will find the slowly migrating molecules, the rapidly migrating molecules having then been entrained towards the evacuation orifice of the micro-column thanks to the circulation of the enriched mobile phase.
- each fractionation micro-column differs from an immediately longer fractionation micro-column by an element of given length.
- a gradient of micro-columns is thus obtained allowing differential separation of the samples.
- the device comprises secondary fractionation micro-columns situated downstream of the fluidic capture means and upstream of the set of micro-levers associated with a fractionation microcolumn, and intended for the secondary fractionation of fractionation products captured.
- the constituents captured in a terminal element, or portion of the length of a micro-column are again separated before being analyzed using micro-levers, for more precise detection.
- the device comprises several batches of fractionation micro-columns, each batch of fractionation micro-columns having a selectivity determined by the means for separating its fractionation micro-columns comprising a stationary phase coated or not and / or electrical separation means.
- a separation of a sample in batches of microcolumns of different selectivities makes it possible to highlight, in each batch, different constituents of the sample. The comprehensiveness of the sample analysis is improved.
- the device comprises a support carrying several batches of fractionation micro-columns, capture means and sets of associated micro-levers, and a supply channel for all of the batches of micro-columns fractionation.
- the selective micro-levers comprise means of detection as a function of their surface state or of the surface state of a coating, of their chemical nature or of the chemical nature of a coating. Separate constituents, such as proteins, may or may not react with one or more specific micro-levers, which will indicate their presence in the sample.
- the micro-levers comprise means for detection by adsorption of molecules.
- micro-levers are intended for the detection of molecules attached to it. Antibodies grafted onto microlevers will make it possible to detect the presence of a specific molecule, such as a specific protein, the nature of which is already known. Micro-levers intended to detect molecules which are adsorbed there allow detection of molecules and in particular of proteins whose nature is not known a priori.
- the micro-columns have a diameter of between 1 micron ( ⁇ m) and 100 microns ( ⁇ ).
- the device comprises a fractionation support carrying the fractionation micro-columns and a detection support carrying the micro-levers, the supports being substantially planar, and the supports being substantially parallel or perpendicular to each other.
- the upstream of the fractionation micro-columns at least one preliminary fractionation stage comprising at least one prior fractionation micro-column, fluid capture means at the level a terminal element of the preliminary fractionation micro-column, and a recovery channel for the preliminary extracts provided for bringing the preliminary extracts to the fractionation micro-columns.
- the preliminary extraction stage allows an analysis of a portion of the sample comprising a reduced number of constituents to be detected.
- the selectivity of preliminary fractionation micro-columns is adapted as a function of the selectivity of fractionation micro-columns to promote the prior extraction of extracts containing constituents which will be well separated in the associated fractionation micro-columns, given their own selectivity.
- a plurality of preliminary extraction stages of different selectivities may be provided, each associated with batches of fractionation micro-columns.
- regular successive catches can be provided in a preliminary extraction stage.
- a pre-fractionation stage comprises a plurality of pre-fractionation micro-columns, each intersected by a capture micro-channel, the capture micro-channels being connected to a recovery channel.
- a prior fractionation stage comprises a plurality of micro-columns of prior fractionation, and a capture microchannel successively intersecting the micro-columns of prior fractionation, and opening into a recovery channel.
- a terminal portion of the fractionation micro-column may include terminal separation means different from the intermediate separation means .
- the constituents arriving substantially at the same instant downstream of a fractionation micro-column have the same characteristics in relation to the selectivity of the intermediate separation means. A change in selectivity of a terminal portion makes it possible to separate these constituents.
- fluidic capture means associated with a fractionation micro-column comprise a capture micro-channel comprising an upstream portion opening at a downstream end of a capture segment of the fractionation micro-column and a downstream portion opening at an upstream end of the capture segment.
- the offset of the upstream and downstream portions of a capture micro-channel allows the capture of constituents of a sample lying on a length segment of a fractionation micro-column.
- Such capture ico-channels with offset portions can be provided for a main fractionation micro-column or a prior fractionation micro-column.
- a capture on a preliminary fractionation micro-column a greater variety of constituents is captured.
- a capture on a preliminary extraction micro-column During a capture on a fractionation micro-column, the counter-current circulation in the capture segment of a capture eluent, different from a mobile phase circulating in the micro-column, can allow a secondary fractionation of the fractionation product present in the capture segment at the time of capture.
- the analysis device comprises a micro-washing channel for selective micro-levers opening into capture micro-channels directly upstream of selective micro-levers.
- a micro-washing pipe allows to bring a washing buffer or an eluent directly on the micro-levers.
- the micro-levers retain certain molecules according to their surface properties.
- the washing eluent is chosen for its affinity with the molecules retained on certain micro-levers, in order to entrain the molecules previously retained by these microlevers.
- a washing buffer removes any molecule attached to the microlevers.
- the invention also relates to a set of chemical or biochemical comparative analysis of at least two biological or chemical samples comprising at least two devices comprising a plurality of micro-columns for fractionation of constituents of a sample, each micro-column for fractionation comprising a micro-channel provided with an orifice for introducing a mobile phase enriched in the sample, an evacuation orifice situated at a terminal end and intermediate separation means.
- a device further comprises fluid means for capturing fractionation products at a terminal element of each fractionation micro-column situated upstream from its discharge orifice, capture micro-channels intended for recovering the products of fractionation captured, and sets of selective micro-levers associated with the fractionation microcolumns and located downstream of the capture micro-channels, a micro- lever comprising detection means connected to analysis means.
- a set comprising separation and analysis devices allows rapid comparison of the samples to determine the differences in composition, for example as a function of differences in the physiological or pathological state of cells making up the samples.
- the invention also relates to a method of chemical or biochemical analysis of biological or chemical samples, in which differential fractionations of a mobile phase enriched in sample are carried out, different fractionation products obtained are captured simultaneously, and each of the fractionation products using a set of selective micro-levers. Differential fractionation allows rapid separation of the components of the sample, and simultaneous capture of the fractionation products.
- a captured fractionation product is fractionated before analyzing it.
- the fractionation product includes certain fractional components of the sample.
- constituents of a fractionation product are detected using micro-levers, according to characteristics of polarity, solvophobicity or porosity of the material which constitutes them or of a coating of the microlevers, or according to characteristics of polarity, solvophobicity, ion exchange or affinity with functional groups grafted onto the micro-levers.
- the sample can be fractionated by chromatography, by micro-electrophoresis, or by interaction with nano-electrodes.
- the deviation or the frequency of vibration of the micro-levers is analyzed.
- a molecule such as a protein or a peptide can be fixed on a micro-lever, according to a selectivity due for example to a surface state or a coating.
- micro-lever can also be excited in vibration at a certain frequency, for example its resonant frequency.
- a protein binds to the micro-lever, a change in vibration frequency is measured.
- the fractionation elements are analyzed by mass spectrometry, before or after the analysis using micro-levers.
- a first sample is analyzed
- a second sample is analyzed, and the analysis results of the two samples are compared.
- the samples are analyzed using suitable micro-levers, which will be able to detect differences in the composition of the samples, taking into account the difference in state of the latter, and the difference in composition that l 'we presuppose.
- a preliminary extraction is carried out on a sample before a differential fractionation of the sample.
- Figure 1 is a partial schematic view of an analysis device comprising micro-columns according to one aspect of the invention
- Figure 2 is a schematic view of a first variant of the analysis device according to Figure 1;
- Figure 3 is a schematic view of a second variant of the device according to Figure 1, where fluid capture means are shown, according to one aspect of the invention
- Figures 4 and 5 are partial schematic views of an analysis device showing a particular arrangement of supports
- Figures 6 and 7 are partial schematic views of an analysis device showing another arrangement of supports
- Figure 8 is a schematic overview of an analysis device according to one aspect of the invention.
- Figure 9 is a partial schematic view of a stage of prior extraction of a support;
- FIGS 10 and 11 are partial schematic views of alternative pre-extraction stages according to Figure 9;
- FIG. 12 is a view of a circuit for washing selective micro-levers
- FIG. 13 is a partial view of an analysis support with mobile phase supply and separate sample supply
- Figure 14 is a variant of a device according to Figure 3.
- a support 1 comprises a plurality of micro-columns 2 arranged in parallel and having a length gradient.
- the micro-columns 2 are shown in thick lines.
- Each micro-column 2 comprises a micro-channel portion 3 provided with an introduction orifice 3a and with an evacuation orifice 3b. Each portion of micro- channel 3 is provided with intermediate separation means.
- a feed channel 4 connects all of the introduction orifices 3a of the fractionation micro-columns 2.
- the feed channel 4 is connected to the introduction orifices 3a of the fractionation micro-columns 2 by intermediate micro-channels 5 shown in fine lines to differentiate them from the fractionation micro-columns 2.
- the intermediate micro-channels 5 are in fact portions of micro-channels devoid of separation means and located upstream of the portions of micro-channels 3 forming the micro - fractionation columns 2.
- An evacuation channel 6 connects all of the evacuation orifices 3b of the microcolumns 2.
- the portions of micro-channels 3 forming the fractionation micro-columns 2 have different lengths, each micro-channel 3 differing from the following micro-channel 3 by an element of determined length delta 1.
- the micro-channels 3 therefore have a length gradient.
- the shortest fractionation micro-column 2 has a length L1.
- the longest fractionation micro-column 2 has a length L2.
- the length L2 of the shortest fractionation micro-columns 2 varies by way of example in no way limiting in a range 1 to 20 centimeters.
- the length L1 of the longest fractionation micro-columns 2 varies by way of example in no way limiting in a range 5 to 40 centimeters.
- Said fractionation micro-columns 2 have a diameter of around 1 to 100 microns, and in particular 10 to 100 microns.
- the difference in length between a fractionation micro-column 2 and an immediately longer fractionation micro-column 2 is understood, by way of example in no way limiting, between 1 to 100 microns.
- An integrated support 1 is provided with groups of fractionation micro-columns 2.
- the fractionation micro-columns 2 of the same group have the same length.
- the fractionation micro-columns 2 of a group differ from the fractionation micro-columns 2 of another group by their length. More precisely, the fractionation micro-columns 2 of a group differ from the fractionation micro-columns 2 of the following group by a very small element of length. In other words, there is a length gradient between the groups of fractionation micro-columns 2.
- a supply channel 4 directly connects the introduction orifices 3a of the fractionation micro-columns 2, being devoid of intermediate micro-channels.
- micro-channels 3 are entirely configured as fractionating micro-columns 2, being provided over their entire length with separation means.
- a support 1 comprises micro-columns for fractionation
- the introduction orifices 3a are connected directly to the supply channel 4.
- the fractionation micro-columns 2 have a length gradient.
- the fluidic capture means 7 comprise capture micro-channels 8 intersecting the fractionation micro-channels 3 at a terminal element or terminal portion 9 of each fractionation micro-channel 3, at a determined distance from the end end of the fractionation micro-channel 3, that is to say at a determined distance from its discharge orifice 3b.
- Each fractionation micro-channel 3 is associated with a capture micro-channel 8.
- the input ends of the capture micro-channels 8, located upstream of the intersection with the micro-channels 3, are connected to a channel secondary supply 15, intended for supply with secondary eluent.
- the support 1 also comprises secondary fractionation micro-columns 10 located in downstream portions of the capture micro-channels 8, being located upstream of detection zones 11.
- a secondary fractionation micro-column 10 is associated with a microphone -capture channel 8 and a detection zone 11.
- a secondary fractionation micro-column 10 comprises fractionation means similar to those of the fractionation micro-column 2 with which it is associated.
- a detection zone 11 comprises a circulation channel 12 passing through one or more selective micro-levers 13.
- a mobile phase preferably in the form of an eluent, enriched in the sample, is brought in by the feed channel 4 and enters the fractionation micro-columns 2 through the introduction orifices. 3a.
- the mobile phase enriched in sample circulates from the introduction orifice 3a to the evacuation orifice 3b while being separated in the micro-channel 3.
- the terminal elements 9, where the catches are made, are located at distances different from the introduction orifices 3a of the fractionation micro-columns 2.
- the migration rates of the constituents of a sample are different. Therefore, at a given time after the start of migration, we will find in the various terminal elements 9 of the microcolumns fractionation 2 sets of different constituents.
- the rapidly migrating constituents will be found.
- the terminal elements of the shortest micro-columns one will find constituents migrating more slowly.
- the molecules protruding from the terminal elements 9 are evacuated through the evacuation orifices 3b and the evacuation channel 6. It will be noted that the migration speeds of the constituents and therefore the separations depend on the selectivity of the means of separation of a micro-column and on the nature of the mobile phase or eluent entraining the constituents.
- the separation means tend to retain the constituents, more or less according to their characteristics, while the eluent tends to entrain the constituents, also according to their characteristics.
- a micro or nano-flow of secondary eluent is circulated simultaneously in all of the capture microchannels 8.
- a nano-flow of secondary eluent flowing in a capture micro-channel 8 crosses the terminal element of length Delta L contained in the associated micro-channel 3.
- the nano-flow of secondary eluent is recovered by a downstream portion of the capture micro-channel 8 after it has passed through the terminal element.
- the components of the sample present in the terminal element at the time of capture are entrained in the capture micro-channel 8.
- a secondary eluent is preferably chosen which is capable of entraining the constituents retained in the fractionation micro-column. 2.
- fractionation product includes a plurality of components of the sample.
- the fractionation products are in particular separate molecules, non-separated molecular complexes and non-disaggregated molecular aggregates.
- the fractionation products are brought by the capture micro-channels 8 to the secondary fractionation micro-columns 10. By passing through these secondary fractionation microcolumns 10, the fractionation products undergo a new separation. In the secondary fractionation micro-columns, the fractionation products can undergo secondary, terminal, parallel, simultaneous micro- or nano-extractions or separations, and / or secondary, enzymatic, terminal, parallel micro- or nano-digestions, concurrent.
- the products of secondary micro or nano-elution, of secondary micro or nanodigestion and of secondary micro or nano-extraction, hereinafter called secondary fractionation products circulate in the capture micro-channels 8 downstream of the micro-columns secondary fractionation 10, towards the detection zones 11.
- the components present in the secondary fractionation products are detected on the selective micro-levers 13.
- the retention of the constituents present in the secondary fractionation products on the micro-levers 13 is measured by measuring the deviation of the micro-levers 13 or by measuring the variation in the frequency of vibration of the micro-levers 13.
- the means for separating the secondary fractionation micro-columns 10 are similar to those for the fractionation micro-columns 2. However, provision may be made for the means for separating a secondary fractionation micro-column 10 to be different from those of the associated fractionation micro-column 2.
- the selectivity of the separation means may be different, to promote separation of the constituents present in the fractionation products. These constituents, which were captured at the same time in a fractionation product after a first separation, have similar migration characteristics in relation to the selectivity of the fractionation micro-column 2 from which they are derived. A second separation with different selectivity allows effective additional separation.
- the secondary eluent is chosen to favor this secondary separation.
- fractionation micro-columns 2 comprising identical separation means. It is also possible to provide different batches of fractionation micro-columns 2, each batch comprising fractionation micro-columns provided with particular fractionation means, for example having a different selectivity. Thus, according to the selectivity of a batch, a particular constituent is better separated in this batch and can be more easily detected downstream of this batch.
- the fractionation micro-columns 2 can be fed from a collective enrichment column, that is to say a feed channel for enriched mobile phase, or from a plurality of enrichment columns, a specific batch being associated with a specific enrichment column, for example circulating a specific eluent, corresponding to the particular means of separation of the fractionation micro-columns 2 of the lot. Indeed, depending on the selectivity of the fractionation micro-columns and the nature of the eluent, the migration speeds of the molecules may be different.
- fractionation micro-columns of each batch can have length gradients between micro-columns or groups of microcolumns.
- a fractionation of a sample followed by a capture of fractionation products, and the detection of their constituents makes it possible to recover an "imprint" of the sample. You can make a series of imprints.
- To allow successive captures and detections using the same micro-levers detection it is possible to provide for successive washing steps of the micro-levers 13, in particular by passing a particular eluent capable of entraining molecules retained in on the micro-levers.
- the molecules entrained by a mobile phase in the fractionation micro-columns 2 are retained, according to the selectivity of the means of separation of the fractionation microcolumns.
- the constituents of the sample are separated as a function of their speed of migration, a function of their characteristic, of the selectivity of the separation means, and of their affinity with a mobile phase of the eluent type.
- a variation in the composition of an eluent allows for different entrainment of the constituents and improved separation.
- the variation in composition can be carried out in successive steps, or continuously. In this case, we speak of an eluent gradient.
- the capture of the fractionation products is carried out in step-by-step mode, each capture step being based on a precise physical or chemical or hydrodynamic condition prevailing at the intersection of a fractionation micro-column 2 with said micro-channel of capture 10.
- the series of successive fingerprints of a first sample is compared to the series of successive fingerprints of a second sample using analysis means of the computer type, the series of detection fingerprints then being archived in a computer database.
- (n) is a number between 1 and 5
- (m) is a number between 1 and 5
- (x) is a number between 5 and 50
- (z) is a number between 1 and 5
- (t) is a number between 10 and 10 000, in particular between 10 and 1000.
- a set of supports is used for the analysis of a sample, another set of supports being used for the analysis of another sample.
- the fingerprint made up of (n.m ⁇ .z.x) detections of the first sample is compared to the footprint made up of (n.m.t.z.x) detections of the second sample.
- the primary and secondary elutions are made by elution gradient.
- several fingerprints on micro-levers are made, at fixed or varied time intervals. Between each impression, micro-lever washes can take place.
- the method of analysis of the sample gives rise, for each set of supports, to a passage of (p.t.z.x) different fractionation products over detection zones.
- the separation in the fractionation micro-columns 2 or the secondary fractionation micro-columns 10 can be carried out by way of nonlimiting example by electrophoresis, chromatography or electrochromatography.
- organic solvents can be used such as a mixture of dichloromethane-hexafluoro-2-propanol containing traces of pyridine with a linear gradient formic acid-2-propanol and formic acid-water on a stationary phase.
- non-polar such as Vydac C4 (Cf. Bollhagen R, Schmiedberger M, Grell E. High performance liquid chromatography purification of extremely hydrophobic peptides: transmembrane segments Journal of Chromatography A, 1995, 711, 181-186).
- nonaqueous solvents it is also possible to use nonaqueous solvents, as mentioned in combined use with methods of separation by non-capillary electrophoresis.
- aqueous Cf. Cottet H, Struijk MP, Nan Dongen JLJ, Claessens HA, Cramers CA.
- Electrophoresis 20 (1999) 111-117; Jussila, M., Sinervo, K., Porras, SP and Riekkola, M.-L. Modified liquid junction interface for nonaqueous capillary electrophoresis-mass spectrometry. Electrophoresis 21 (2000) 3311-3317; Koch, JT, Beam, B., Phillips, KS and Wheeler, JF Hydrophobic interaction electrokinetic chromatography for the separation of polycyclic àromatic hydrocarbons using non-aqueous matrices. J. Chromatogr. A 914 (2001) 223-231; Li, S.
- C10-APSO4 respectively 3 (nonyl-dimethyl-ammonio) propyl sulfate and 3 (decyl - dimethyl-ammonio) propyl sulfate, for the extraction-preconcentration of hydrophobic molecular species (Cf. Saitoh T, Hinze WL. Concentration of hydrophobic organic compounds and extraction of proteins using alkylammoniosulfate zwitterionic surfactant mediated phase separations. Anal. Chem 1991, 63 (21): 2520-5.)
- Basic analytes can also be separated in combination with nonaqueous capillary electrophoresis (Cf. Karbaum, A. and Jira, Th. Nonaqueous capillary electrophoresis: Application possibilities and suitability of various solvents for the separation of basic analytes. Electrophoresis, 1999 , 20, 3396-3401).
- size exclusion chromatography can also be used with stationary apolar phases and elution with a ternary mixture such as (chloroform-methanol-trifluoroacetic acid) (Cf. Bunger H, Kaufner L, Pison U. Quantitative analysis of hydrophobic pulmonary surfactant proteins by high performance liquid chromatography with light-scattering detection. J. Chromatogr A, 2000, 18, 870 (1-2), 363-9.)
- micellar retro-extraction in which the proteins encapsulated inside micelles are recovered after destruction of the micelles by a surfactant having a counter-electrostatic action (Cf. Jarudilokkul S,
- Extraction and chromatography are based in particular on the concept of polarity, which comes from an asymmetric distribution of electronic clouds within molecules.
- Polarity scales are designed in several independent ways which refer to the various consequences of polarity phenomena:
- this method gives, by measurement of adsorption on alumina, the following polarity orders: water> methanol> ethanol> 2-propanol> dimethyl sulfoxide> acetonitrile> methyl ethyl ketone.
- solubility parameters Hildebrand and Scott
- solubility parameters Hildebrand and Scott
- this method gives the following polarity orders: water> methanol> ethanol> dimethyl sulfoxide> acetonitrile> 2-propanol> methyl ethyl ketone.
- the partition chromatography is based on the differential solubilities of the solutes between two liquid phases, more precisely between a mobile liquid phase and another liquid phase, called stationary, matching the meshes of a porous solid phase of fine particle size.
- the solid phase may be polar, such as for example consisting of silica gel grains grafted with aminopropyl or paranitrobenzyl, or alkylnitrile, or glyceropropyl groups.
- the weakly polar mobile phase as can be a mixture (95% hexane, 5% dichloromethane) will receive a "polar modifier" to give a mixture with increased polarity, such as (80% hexane, 20% dichloromethane ), until moving polar solutes interacting with the stationary polar liquid phase. This is normal phase sharing chromatography.
- the solid phase can also be apolar, such as matrices such as a styrene-divinybenzene copolymer matrix, or else a pyrocarbon matrix, or silica gels grafted with apolar functional groups, such as, for example, alkyl or phenyl groups.
- the mobile, polar phase as a mixture can be (40% methanol or acetonitrile, 60% water), will receive a "polar modifier" to give a mixture with less polarity (60% methanol or acetonitrile, 40 % water), until displacing apolar solutes interacting with the stationary apolar liquid phase. This is reverse phase partition chromatography.
- the other chromatographic separation techniques are based on a differential retention of solutes contained in a mobile, liquid or gaseous phase, which passes through a solid stationary phase.
- the retention mode is size, adsorption or affinity.
- Size exclusion chromatography is based on a stationary phase consisting of porous beads forming a gel. The distribution of the pore diameters inside the porous beads corresponds to a fairly wide range. Depending on their steric hindrance, the molecules may or may not pass inside a greater or lesser number of pores of the porous beads. Those which pass most easily inside the pores of the porous beads are the most delayed. In practice, the phenomenon is biased by ionic or hydrophobic interactions between the solutes and the stationary phase.
- Size exclusion chromatography in denaturing or non-denaturing conditions can also help, while minimizing the use of detergents, in the characterization of molecular aggregates in which membrane proteins are involved (see Lôster K, Baum O, Hofman W, Reutter W. Characterization of molecular aggregates of alpha 1 betal integrin and other rat liver membrane pro teins by combination of size exclusion chromatography and chemical cross-linking. Journal of Chromatography A, 1995, 711, 187-199.) Techniques Particularly preferred chromatography are those based on a differential adsorption of solutes contained in a mobile phase, liquid or gas, which passes through a solid stationary phase.
- the selectivity in adsorption chromatography is based on a complete process for each of the solutes: entrainment by the mobile phase and interaction of specific energy with the stationary phase.
- the polarity of the solute is intermediate between that of the mobile phase and that of the stationary phase. If the polarity of the solute is too far from that of the mobile phase, there is not sufficient solubility of the solute in the mobile phase to prevent irreversible retention on the stationary phase. If the polarity of the solute is too far from that of the stationary phase, there is no interaction with the stationary phase.
- a solvent is all the more eluting as its polarity approaches that of the solute, and finally that of the stationary phase, since the latter is supposed to be close to that of the solute.
- the selectivity is all the better when a small variation in the polarity of the solvent results in a selective change in the adsorption equilibrium of solutes of similar polarities.
- matrices comprising a stationary phase, the grafts of which consist of polymers of molecules having an apolar side and a polar side, such as a macroporous copolymer produced from an equilibrium between the two monomers, divinylbenzene, apolar, and polar N-vinylpyrrolidone.
- Normal phase adsorption chromatography is based on the differential adsorption of solutes on a solid and polar stationary phase, such as in particular based on alumina but especially on silicates, or hydrophilic polymers, such as agarose or dextran gels , the mobile phase being, it, apolar.
- a solid and polar stationary phase such as in particular based on alumina but especially on silicates, or hydrophilic polymers, such as agarose or dextran gels , the mobile phase being, it, apolar.
- micro-beads are obtained by a hot emulsion-gelling process first using a water-immiscible solvent, then a stabilizer, said process ending with the removal of the solvent by suction filtration.
- Agarose gels can be crosslinked with crosslinking agents such as epichlorohydrin, 2,3 dibromopropanol or divinylsulfone.
- crosslinking agents such as epichlorohydrin, 2,3 dibromopropanol or divinylsulfone.
- 2B, 4B, 6B are examples of the names 2B, 4B, 6B respectively.
- Sepharose CL is crosslinked with 2,3-dibromopropanol under strong alkaline conditions, operation followed by alkaline hydrolysis of its sulfate groups under very reducing conditions, so as to make it non-ionic or very weakly ionic.
- Sephadex is a dextran gel crosslinked with epichlorohydrin which is stable under alkaline, saline and weakly acidic conditions, but which is hydrolyzed under marked acid or oxidizing conditions.
- Sephadex (LH-20) and (LH-60) gels can be grafted with hydroxypropyl groups which bind by ether linkages to the glucose units of the dextran chains, with the effect of modulating their polarity.
- Silica is insoluble in water for a pH varying from 2 to 8. Its polarity is provided by the presence on its surface of silanol groups (SiOH), 4.6 in number per nm 2 , of which the OH group is polar and proton donor in hydrogen bonds.
- a silanol group can remain free (free silanol), or else initiate a hydrogen bond with a neighboring silanol group (bound silanol), or else initiate a hydrogen bond with a water molecule.
- the OH group of a free silanol can also be a proton donor to a water molecule (free silanol hydrated by a layer of monomolecular water), or to another polar molecule.
- bound silanols can attract water molecules: we then silanols hydrated by a layer of pluri-molecular water, these are highly hydrated silicon gels. Sihce gels are very porous. Depending on whether their specific surface is more or less large (it varies from 200 to 600 m 2 per gram), they comprise pores of a more or less large diameter and consequently a more or less large masking of the free silanols.
- the free silanol groups are “strong” adsorption sites, completely available for hydrogen bonding. Hydrated free silanol groups and linked silanol groups are still adsorption sites.
- silanol groups hydrated by a layer of pluri-molecular water are rather sites of partition chromatography.
- highly hydrated silica gels whose specific surface exceeds 550 m 2 per gram and whose water content exceeds 5%, it is considered that partition chromatography becomes preponderant compared to adsorption chromatography.
- the silica gels sold have a varied particle size and mention the number of silanols remaining free per unit area (for example Lichosorb Si 100 has 2.95 free silanol groups per nm 2 for a specific surface of 309 m 2 per gram, while that Lichosorb Si 80 contains 2.20 for a specific surface of 482 m 2 per gram).
- adsorption chromatography In adsorption chromatography, an attempt is made to keep the adsorption capacity of the absorbent constant, whatever the mobile phase. To do this, we adjusts the water content of a solvent to a level called "isoactivating water content" so that the adsorption energy of the adsorbent is equivalent to what it can have with a reference solvent having a content of given water (for example, the reference solvent could be ethyl acetate at 0.06% water when we want a reference for the absorbency of a silica with a specific surface of 550 m_ per gram .)
- the polarity of the silicas can be modified by grafting.
- the polar grafts can be of the aminopropyl, paranitrobenzyl, alkylnitrile (nitro), glyceropropyl (diol) type.
- the grafts can be carried out by silanization, that is to say by reactivity of mono, di- or tri-functional alkoxysilanes or chlorosilanes. For this reaction to take place, the silane molecules must penetrate into the pores of the silica, which supposes a pore diameter greater than 10 nm.
- the non-polymerized grafted commercial phases have a grafting rate of 3.5 to 3.7 micromoles per nm 2 .
- Reverse phase adsorption chromatography is based on the differential adsorption of solutes on a stationary solid and nonpolar phase, such as in particular silicas grafted with nonpolar groups, the mobile phase being more or less polar, according for example to various proportions of more or less polar solvents, for example water and methanol or alternatively water and acetonitrile.
- a stationary solid and nonpolar phase such as in particular silicas grafted with nonpolar groups
- the mobile phase being more or less polar, according for example to various proportions of more or less polar solvents, for example water and methanol or alternatively water and acetonitrile.
- apolar functional groups grafted onto the stationary phases such as sihce or Sepharose can be alkyl groups Cl 8 or C8 or C4, or phenyl groups .
- the grafting procedure for grafted silicas with nonpolar groups is obtained by silanization, as is the grafting procedure for grafted silicas with polar groups. It should be noted, as in the case of virgin (polar) silicas or polar grafted silicas, the presence of residual silanols originating from the hydrolysis of reactive groups of trifunctional silanes which have not reacted during the synthesis.
- the organic solvent molecules of a mixture are preferentially fix on the surface of the apolar grafts
- the solute molecules interact with the stationary liquid phase.
- the interaction mechanism is either a solute sharing mechanism between the mobile phase and the adsorbed liquid phase, or based on a hydrophobic interaction between the solute molecules and the stationary apolar phase.
- the solute molecules can displace molecules from the stationary polar liquid phase.
- the accessible residual silanol groups can be eliminated (this process is called "end-capping") by a treatment with trimethylchlorosilane (TMCS).
- TMCS trimethylchlorosilane
- Apolar matrices other than silica grafted with C18 or C8 or C4 or phenyl alkyl groups can be used.
- Phenyl and Octyl-Sepharose can be used in chromatography of hydrophobic interactions, and are obtained when the crosslinking of Sepharose CL is coupled with a derivation with phenyl or octyl groups.
- Styrene-divinylbenzene or pyrocarbon copolymer matrices can be used, which have the advantage over grafted silicas of being stable in a much wider pH range (1 to 13) against (2 to 7.5), because silica is attackable by OH ions. This silica defect can be resolved by applying a silicone coating to the surface of the pores found in stationary commercial phases such as Capcell Pak.
- a weak point of the copolymer matrices can be improved by using macroporous copolymer matrices.
- macroporous copolymer matrices comprise both a very highly crosslinked part, impermeable to solvents, and a weakly crosslinked part favorable to exchanges between the stationary phase and the mobile phase, and macro-pores, without the presence of polymers.
- Other stationary phases such as porous zirconium oxide or porous graphite naturally offer the qualities of stability (pH from 1 to 14) and mechanical resistance.
- copolymeric matrices Another characteristic of the copolymeric matrices mentioned is the presence of aromatic groups, capable of interacting in the formation of donor-acceptor complexes of electrons with the solutes.
- Other copolymer matrices can be used, such as for example those based on vinyl alcohol or polymethylmethacrylates.
- the stationary phase which comprises a matrix to which are grafted fixed, ionized functional groups capable of fixing counterions.
- the microparticles of the ionized stationary phase accept ions of opposite charge within them, and exclude ions of the same charge.
- the matrices of the stationary phase can be grafted silicas or copolymeric matrices.
- the matrices By their role of exclusion, as well as by their own composition, such as for example the presence of aromatic nuclei in the polystyrene divinyl benzene copolymer matrices giving rise to interactions by pi electrons, the matrices contribute to the process which is added to those of their ionized functional groups.
- a competition for binding to the stationary phase is established between the solute ions of the mobile phase and counterions releasable by said stationary phase and therefore exchangeable.
- the mobile phase is a buffer solution whose pH makes it possible to control the electrostatic attractions of the solutes, insofar as a certain value of the pH will correspond to a certain charge of the solutes.
- the amino acids of proteins can be present in the solution in the form of zwitterionic molecules or in the form of anions or in the form of cations.
- the grafted matrices are porous (they are micro-particles of porous layer or organic copolymers of microporous or macro-porous structure of poly (styrene / divinylbenzene) or polyacrylate type), which gives rise at the same time to a non-ionic separation mechanism, for example mechanisms for sharing molecules with a given polarity. For example, nonionic solutes do not undergo electrostatic repulsion to penetrate inside the pores of the matrix.
- a matrix can be a cation exchanger or anion exchanger, weak or strong.
- Strong cation exchangers (SCX for Strong Cation Exchangers), of the strong acid type, can be sulfonic, that is to say grafted with sulfonate SO3- functional groups.
- the weak cation exchangers, of the weak acid type can be carboxylic, that is to say grafted with functional groups CO2- carboxylates.
- the strong anion exchangers (SAX for Strong Anion Exchangers), of the strong base type, can be quaternary ammonium, that is to say grafted with NR3 + functional groups, such as for example trimethylammonium.
- the weak anion exchangers of the weak base type can be non-quaternary ammonium, that is to say grafted with protonated forms of primary, secondary or tertiary amines (NHR2 + functional groups, such as for example diethylaminoethylammonium).
- NHR2 + functional groups such as for example diethylaminoethylammonium.
- the abbreviations of everyday language relate, for cation exchangers, the CM, weakly acid, for Carboxymethyl, as well as SP and S, strongly acid, for respectively Sulphopropyl and methylSulfonate.
- the eluting force partly depends on the nature of the developer ion conveyed by the mobile phase.
- a second mode of ion pair chromatography or ion interaction chromatography advantage is taken of the presence in the mobile phase of large ions (they are called counterions) comprising an apolar part and a charge opposite to that of the solute. Electro-neutrality is ensured by the presence of co-ions of the same sign as the ions of the solute.
- each counterion can form a pair with a solute molecule by hydrophobic interaction.
- a nonpolar stationary phase such as silica grafted with alkyl groups
- the counterion can be adsorbed on the nonpolar grafts of the stationary phase, while having to leave free 60 to 70% of them.
- the ions of the solute are sufficiently hydrophobic, it is then also possible to assist in the sharing of pairs of ions (solute / counterion) which are fixed on the alkyl groups which remain free from the stationary phase and the solubilization of these same ions (solute and storytelling) in the mobile phase.
- solutes The retention of solutes depends on their degree of ionization, the content of organic solvent, and the concentration of counterions in the mobile phase.
- solutes When the solutes are capable of forming complexes with a cation (Cu_ +, Zn_ +, Cd2 +, Ni_ +) or a donor or acceptor complex, it is possible to carry out chromatography by ligand exchange respectively, or chromatography with transfer of loads.
- the metal cation for example copper
- the stationary phase for example a virgin silica
- a covalent bond of copper with silica is obtained in the presence of ammonia, giving rise to silicas covered with cupri-diamine silicates.
- These cupri-diamine silicates attached to the stationary phase are capable of exchanging ammonia with a doubling-giving solute, which becomes the new ligand by forming a hedge with the copper of the stationary phase.
- cupri-diamine silicates can solvate water molecules, which makes them very hydrophilic.
- solute The retention of a solute will depend on its donor character (complexing character) and on its hydrophilic character, as well as on the ammonia content of the mobile phase, generally a ternary water-acetonitrile-ammonia mixture whose water content does not exceed step 50% in order to preserve the stability of the stationary phase.
- the mobile phase contains a complex of a transition metal with a ligand comprising a hydrophobic chain
- the stationary phase for example a silica grafted with nonpolar groups, such as groups C18 alkyl
- the transition metal is in excess with respect to the hydrophobic ligand, so as to be free to also be able to preserve weak hedge sites with solvent molecules.
- solutes are capable of forming complexes with the transition metal, they are divided between bonds with the metal in the mobile phase and bonds with the metal engaged in complexes with the hydrophobic ligand, itself adsorbed on the stationary phase hydrophobic.
- the solvent molecules can also solvate the grafts of the stationary phase. Consequently, there can be, to receive electrons from the stationary phase, a two-way competition (solute, polar modifier) with a bias which is that the solute can also interact with the grafts solvated by the polar modifier. Finally, there is competition between the solutes and the polar modifier to give (or accept) electrons to (or from) free, unsolvated grafts from the stationary phase.
- the retention of solutes is all the stronger as the number of free grafts of the stationary phase is high, and the number of aromatic nuclei per graft and the spatial density of these nuclei are high. All things being equal, the competition for binding to the stationary phase will be decided on the content of the polar modifier of the mobile phase.
- the hydrophilicity of a protein or peptide depends on its amino acid composition.
- the proportion of hydrohile or polar amino acids is high, the hydrophobic or apolar amino acids (isoleucine, valine, leucine, phenylalanine) are pushed back inside the molecule.
- the proportion of non-polar or hydrophobic amino acids is high, there is a more direct interaction between certain hydrophobic amino acids and the aqueous medium.
- a first means of increasing selectivity is to use changes in the composition of binary or ternary mixtures of solvents of different polarities to obtain a variation in polarity of the mobile phase, in order to completely modify the spatial conformation of the peptide or protein.
- a second means of increasing selectivity is to use solvation variators of functional groups, such as salts.
- functional groups such as salts.
- hydrophobic or apolar functional groups are surrounded by self-organizing water molecules.
- a strong ionic force unmasks the hydrophobic or apolar functional groups, by disorganizing the water molecules which surround them.
- Chromatography of hydrophobic interactions consists of starting with a strong ionic strength and then decreasing it until the hydrophobic or apolar functional groups of the peptides or proteins find their mask in an aqueous medium of weak ionic strength.
- a stationary apolar phase such as grafted with alkyl groups Cl 8, C8 or C4, or phenyl
- the proteins which have the most hydrophobic functional groups are those which are the most delayed.
- Phenyl and Octyl-Sepharose are often used for this type of chromatography.
- Several models have attempted to describe the laws of separation in chromatography, in particular by attempting to configure the theoretical plateau height in the micro-column.
- Nan Deemter equation is of the form:
- A is a term which accounts for axial diffusion
- B is a term which accounts for incomplete mass transfers between mobile phase and stationary phase
- C is a term which accounts for, on the one hand, paths of unequal length to cross the column on the other hand difficulty for the mobile phase and the solutes to access the meshes formed by the stationary phase: it is optimal that the mobile phase and the solutes reach said meshes by convection rather than by diffusion
- d is the flow rate of the mobile phase through the column.
- Altria KD Overview of capillary electrophoresis and electrochromatography, Journal of Chromatography A, 1999, 856, 443-463; Quirino JP, Terabe S. Electrokinetic chromatography, Journal of Chromatography A, 1999, 465-482; Smith NW, Carter-Finch AS, Electrochromatography, Journal of Chromatography A, 2000, 892, 219-255; Bartle KD, Carney RA, Cavazza A , Cikalo MG, Myers P, Robson MM, Roulin SCP, Sealey K. Capillary electrochromatography on silica columns: factors inflencing performance. Journal of Chromatography A, 2000, 892, 279-299; Pyell U.
- Micellar electrochromatography can be performed on miniaturized supports as described in the following document: (Cf. Culbertson CT, Jacobson SC, Ramsey JR. Micro-chip device for high efficiency separations. Anal. Chem, 2000, 72, 5814 -5819).
- Elution gradients can be used, and in particular micro-gradients of elution (cf. AH, Kahle N, ⁇ ovotny MN. A micro-gradient elution system for capillary electrochromatography. J. Microcolumn separations, 2000, 12 (1 ), 1-5.).
- the separation and in particular the separation of the peptides or the proteins can be carried out using separation methods by micro-chromatography, micro-electrochromatography or micro-electrophoresis.
- a separation of peptides and proteins can be used by reverse phase chromatography on 1.5 micron non-porous non-porous silicas (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Retention and selectivity of proteins and peptides in gradient elution of non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 71-80; Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Mobile phase and surface mediated effects on recovery of native proteins in gradient elution on non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 80-89.)
- Peptides and proteins can be separated by anion exchange chromatography on a 3-micron non-porous polymeric phase of 3 microns of poly (styrene-divinylbenzene) (Cf. Régnier FE, Rounds MA. Synthesis of a non-porous, polystyrene-based anion-exchange packing material and its application to fast high-performance liquid chromatography of proteins. Journal of Chromatography A. 1988. 443. 73-83).
- Peptides and proteins can be separated by hydrophobic interaction chromatography on 1.5 micron non-porous non-porous silicas (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Performance of non -porous monodisperse 1.5 micron bonded silicas in the separation of proteins by hydrophobic interaction chromatography Journal of Chromatography A. 1987, 397, 91-97).
- Proteins can be separated in solutions of pH 5, 7 and 9 by affinity chromatography for cupric ions immobilized on stationary Sepharose CL-4B phases thanks to an epoxy coupling procedure and using the tridentate chelating ligand N- (2- pyridylmethyl) aminoacetate (Cf. Chaouk H, Hearn MTW. New ligand, N- (2-pyridylmethyl) aminoacetate, for use in the immobiled metal iion affinity chromatography separation of pro teins. Journal of Chromatography A, 1999, 852, 105-115 ).
- Synthetic peptides having a greater or lesser number of histidine residues can be retained in affinity chromatography for metal ions immobilized on stationary phases Sepharose CL-4B and using the iminodiacetic acid ion as tridentate chelating ligand or the acid ion.
- nitrilotriacétqiue as tetradenté chelating ligand Cf Kronina NN, Wirth HJ, Hearn MTW. Characterization by immobiled metal ion affinity chromatography procedures of the binding behavior of several synthetic peptides designed to have high affinity for Cu (U) ions. Journal of Chromatography A, 1999, 852, 261-272.
- micellar chromatography One can use a peptide chromatography by micellar chromatography as that was presented in the following article: (Cf. Kord AS, Khaledi MG. Selectivity of organic solvents in micellar liquid chromatography of amino-acids and peptides. Journal of Chromatography A. 1993, 631, 1255-132).
- the separation of biological samples can be carried out by micro-electrophoresis with a prior enrichment phase (Cf. Lichtenberg J, Nerpoorte E, de Rooij Sample. Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis. Electrophoresis 2001, 22, 258-271; Wu XZ, Hosaka A, Hobo T. An on-line electrophoretic concentration method for capillary electrophoresis of proteins. Anal. Chem, 1998, 70, 2081-2084; Tragas C, Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface.
- Isoelectric focusing that is to say a pH gradient over the length of the separation capillary
- This gradient can be obtained by a set of small ampholyte molecules charged according to their pi, such as those synthesized based on acrylic acid and polyamines or obtained by coupling with epichlorohydrin.
- the pH gradient can generally vary from 3 to 10.
- the gradient can also be obtained by grafting, in a polyacrylamide gel, modified acrylamide monomers carrying ionizable chemical groups of acidic or basic PK.
- the pH gradient can vary from 1 to 12.5 (Cf Kawano Y, Ito Y, Yamakawa Y, Yamashino T, Horii T, Hasegawa T, Ohta M.
- a free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000; 14 (14): 1269-74; Wen J, Lin Y, Xiang F, Maison DW, Udseth HR, Smith RD. Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry. Electrophoresis. 2000 Jan; 21 (l): 191; Rossier JS, Schwarz A, Reymond F, Ferrigno R, Blanchi F, Girault HH. Microchannel networks for electrophoretic separations. Electrophoresis.
- the fractionation micro-columns comprise means for separation by chromatography.
- the particular selectivity of a batch is determined by the nature of the stationary phase of all the chromatographic fractionation micro-columns 2 forming the batch.
- the selectivity of the fractionation micro-columns 2 of a batch is determined by the intrinsic polarity and the solvophobicity of a stationary phase and by the polarity, the amphipaticity and the solvophobicity of the functional groups which therein. are grafted.
- the selectivity of the fractionation micro-columns 2 is determined secondarily by other criteria such as the micro- porosity, macro-porosity, the ability to exchange ions or to the interaction of pairs of ions or to exchange ligands or to charge transfer or to the affinity reactions of said stationary phase or the granting of a pH gradient over the length over the entire length of said fractionation micro-columns 2, said pH gradient extending over a range all the wider as said fractionation micro-columns 2 are long.
- Said selectivity is determined thirdly by an electric field applied to stationary phases of said fractionation micro-columns 2 (Cf. Method of electric field flow fractionation which the polarity of the electric field is periodically reversed. US Patent N ° 6113819).
- each batch of fractionation micro-columns 2 receives a mobile phase, of the eluent type, which is specific to it and in relation to the nature of the stationary phase of its fractionation micro-columns 2 .
- the separation in fractionation micro-columns 2 is carried out by a first method of chromatography, and that the separation in the corresponding secondary fractionation micro-columns 10 is carried out by a second chromatography method different from the first.
- a second chromatography method is therefore preferably chosen according to a different selectivity allowing effective separation of the molecules of the fractionation product obtained with the first method.
- the first chromatography method is an ion exchange method, the second method being a hydrophobic interaction chromatography method.
- the detection is carried out on the detection zones 10 by detection by micro-levers, followed or optionally preceded by additional detection by one of the methods known to those skilled in the art for the detection of eluates from chromatography, directly or after hyphenation, for example the mass spectrometry mentioned previously.
- the fractionation micro-columns comprise means of separation by micro-electrophoresis, of the zone micro-electrophoresis type, or of the micro-isochatophoresis type, or of the micellar micro-electrophoresis type, or of the micro-electrophoresis type with isoelectric focusing obtained by the granting of a pH gradient over the length over the entire length of said micro-channels, said pH gradient extending over a range that is wider the longer said micro-channels.
- the detection is carried out using micro-levers and possibly by spectrometry.
- the separation in the fractionation micro-columns is carried out by a method called Field Flow Fractionation (cf. Suslov SA, Roberts AJ., Modeling os sample dynamics in rectangular asymetrical flow field flow fractionation channels. Anal Chem 2000, 72 (18), 4331-45).
- micro-channels of micro-columns are provided along their length with nano-electrodes (cf. US 6,123,819).
- the molecules entrained by an eluent are all the more braked as their charges interact with an electromagnetic field created by the nano-electrodes. Rectilinear and parallel fractionation micro-columns have been described.
- fractionation micro-columns 2 can for example be rectilinear, curved or sinuous. They are manufactured in part or in whole thanks to the techniques used in micro-manufacturing on silicon or glass or ceramic or plastic. In one embodiment, the fractionation micro-columns are monoliths.
- micro-column beds that is to say grooves formed on a support and intended to be covered and closed using another support having a flat surface where corresponding grooves can be directly etched when said supports are based on silicon or glass or ceramic.
- the fractionation micro-columns 2 and said secondary microcolumns 10 can be manufactured in part or in whole thanks to the techniques used in micro-manufacturing such as photoengraving, micromolding, micro-stamping, photolymerization, thermopolymerization.
- micro-manufacturing such as photoengraving, micromolding, micro-stamping, photolymerization, thermopolymerization.
- the microparticulate network can be obtained by micro-molding or micro-stamping or photopolymerization or thermopolymerization in situ stamping when said supports are plastic-based, or else can be made up of micro or nano-rods inserted in the ht of said micro-columns (Cf. Gusev I, Huang X, Horvath C. Capillary columns with in situ formed porous monolithic packing for micro- high performance liquid chromatography and capillary electrochromatography, Journal of Chromatography A, 1999, 855, 273-290; Yu C, Svec F., Fréchet J.
- Micro-particles can also be immobilized in a continuous bed (Adam T., Unger KK, Dittmann MM, Rozing GP. Towards the column bed stabilization of columns in capillary electroendosmotic Chromatography. Immobilization of microparticulate silica columns to a continuous bed. J.
- micro-particulate network of micro-columns which constitutes the stationary phase of a thin film of hydrophobic or hydrophilic nature and can be subjected to coupling chemistries known to those skilled in the art to graft characterized molecules. mainly by their polarity and their amphipathicity. Chemical etching increases the retention properties, as indicated in the following document: (Cf. Pesek, Protein and peptides separations on high surface area capillaries, Electrophoresis, 1999, 20, 2343-2348).
- micro-channels can be provided with micro-rods of polymer monoliths suitable for the separation of proteins both by electrochromatography and by micro-HPLC (Cf. Hjerten, Electroosmosis and Pressure-driven chromatography in chips using continuous beds. Anal. Chem, 2000 , 72, 81-87).
- Stationary chromatography phases can be obtained by molding using silicon molds.
- the very high resolution of micro-plastic molding techniques is directly linked to that of silicon molds. It is therefore at the level required to consider the manufacture of stationary chromatography phases directly molded in the plastic using silicon molds dimensioned to manufacture stationary phases comprising a very fine microparticulate plastic network, such as for example by being made of cubes. 5 micron polymeric edges separated by spaces of 500 nanometers.
- the conduits and components that guide or receive the fluids are miniaturized, on the other hand that the components which manage the flow of fluids and reagents (micro-valves, micropumps, micro-sensors, micro-heaters, etc.) are also miniaturized, and finally that connections can be made set up inside and outside the device.
- components which manage the flow of fluids and reagents micro-valves, micropumps, micro-sensors, micro-heaters, etc.
- connections can be made set up inside and outside the device.
- the desired micro-system can hold in a more or less flattened volume represented by the superposition of subcomponents themselves clearly flattened.
- wet chemical etching techniques of photolithography dry etching with various photonic or particulate radiation, micro-shaping with micro-tools or lasers, cutting, ablation, fusion assembly or anodic assembly , gluing, welding, molding, hot stamping (hot-embossing in English), punching, drilling, electroplating, chemical vapor deposition, production by progression by successive sheets (lamination in English ).
- wet etching is applied in a known manner to silicon and its derivatives in the microelectronics industry. It can be isotropic. It can also be anisotropic when one seeks to take advantage of the orientation of the crystals and the properties of the gravants to control its direction. (S ato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. Characterization of anisotropic etching properties of single crystal silicon: surface roughening as asolution of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206).
- the wet etching techniques both isotropic and anisotropic, have many variants.
- Knowledge in materials physics, orbital chemistry, radiation physics, doping of materials allow to take advantage of the atomic structure of different materials used, help to design methods for controlling the direction, depth and stopping of engravings on different layers.
- the techniques cited have many variations.
- Knowledge of surface treatment makes it possible to improve the qualities required of materials during manufacture or the qualities required of the finished product.
- thermophysics and differential thermochemistry between two materials makes it possible to envisage new techniques of fusion, molding, stamping, punching, in particular plastics.
- a technique of micro-manufacturing of polymers by stereolithography can be used, in particular for rapid 3D prototyping.
- micro-manufacturing techniques are applicable not only to the manufacture of the finished products, but also to those of the tools used to carry out these micro-manufacturing, as well as to the micro-molds and to the hot stamping micro-matrices used. to micro-replicate en masse a micro-object.
- the other criteria which will help to select a manufacturing method and material are the intrinsic qualities of the materials making up the finished object, and the prospects for controlling manufacturing costs.
- Some techniques assume a manufacturing method that is less suitable for mass production: dry etching by photonic or particulate radiation (Bean. Anisotropic etching of silicon. 1978. vol ED-25 (10), pp 1185-1193. IEEE Transactions of Electron devices.), laser ablation, etching with micro-tip.
- Microfluidic devices for ⁇ -TAS applications fabricated by polymer hot embossing. Proceedings of SPLE. Microfluidic Devices and Systems. 21-22 Sept 1998, Santa Clara, ppl77-182 _ Grzybowski BA, Haag R, Bowden N, Whitesides GM. Generation of micrometer-sized patterns for microanalytical applications using a laser direct-write method and microcontact printing. Anal. Chem, 1998, 70, 4645-4652 _ Martynova L,
- micro-manufacture replication masters for example micro-molds for injection molding or for reactive molding, or hot stamping micro-dies
- two qualities are combined. : a high aspect ratio and a surface compatible with the requirements of the replication process. Indeed, certain steps in replication are crucial, in particular the separation of the replication matrix from the newly replicated object.
- the complexity of the method chosen for manufacturing a replication matrix is taken into account.
- new techniques of dry etching and especially of wet etching with increased performances can prove to be more flexible with aspect ratios which approach more and more the LIGA technique.
- anisotropic wet etching has progressed a lot (Hôlke A., Henderson HT. Ultra-deep anisotropic etching of (110) silicon; J. Micromech. Microeng. 1999, 9, 51-57).
- Other results also show progress in the performance of wet chemical isotropic etching procedures of silicon - a possibility for the production of deep structured microcomponents. Schwesinger N, Albrecht A .. SPLE vol 3223, p 72- 79 ).
- Certain unitary techniques can be adapted to mass production when the manufacturing instruments themselves used to implement them are miniaturized and can be used in a massively parallel manner. This is a close prospect for laser ablation (thanks to the manufacture of micro-lasers) and microtip etching, more distant for certain dry etching techniques.
- Manufacturing in large quantities is possible with certain techniques such as: wet etching on silicon and derivatives, and on glasses, UN photolithography on photoresists, production by progression by successive layers of polymers with the use of sacrificial layers according to Webster and Mastrangelo cited below in reference, molding of poly (dimethylsiloxane) (PDMS), molding of plastics by injection with micro-mold, molding of ceramics and metals, hot stamping of polymers with micro-matrix of stamping.
- PDMS poly (dimethylsiloxane)
- wet etching can be applied to all types of silicon and quartz derivatives, as well as to different types of glass (for example pyrex, borophospho-silicate glasses, etc.).
- micro-fluidics an important criterion is compatibility with the use of micro-electrophoresis, 2D micro-electrophoresis and micro-electro-chromatography to separate the molecules.
- Compatibility with electro-osmosis to move fluids is also and above all important, this technique having the advantage of avoiding components such as micro-valves and micro-pumps.
- electro-osmosis as well as micro-electro-chromatography combined with electro-osmosis require the application of significant potential differences. Consequently, they are not very compatible with the use of silicon. However, they are compatible with glasses and plastics.
- thermo-capillary force Bosset MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT .
- Microfabricated structures for integrated DNA analysis. PNAS 1996, vol. 93, pp5556-5561 or the forces coupled to alternating surfaces or hydrophobic-surface lines or hydrophilic lines (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Selective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers.
- Device for fluid supply of a micro-metering device US Patent N ° 5805189 _ Beckton Dickinson. DNA microwell device and method. US Patent No. 5795748).
- Transparency a quality sought after in biological analysis, is a quality shared between glasses (Kricka L, Wilding P, et al. Micromachined Glass-Glass Microchips for In Nitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) and some plastics.
- the glasses offer, among other things for biochemical analysis, compatibility with fluorescence detection and a good heat exchange coefficient. They are however etched only in an isotropic mode, which for example today limits the shape of the micro-channels on glass to a circular shape.
- Plastics even if they have less compatibility with fluorescence detection and a lower heat exchange coefficient than glasses, have many other qualities, including the low cost price.
- the very low cost of manufacturing micro-manufactured plastic objects comes from the low price of the raw material, the simplicity of the production processes which can be envisaged, and the ability to replicate by molding or by hot stamping, even for plastics photoresists to photolithography.
- metal can be deposited once the product is finished.
- the support can also be marked with a conductive ink.
- - photoresists which can be machined among other things by photolithography, including for example PMMA for X-ray lithography, SU-8 (negative photoresist) and Novolac de Hoescht and AZ 9260 (positive photoresists ) for UN photolithography (Lorenz H, Despont M, Fahrni ⁇ , LaBianca ⁇ , Renaud P, SU-8: a low-cost negative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121-124. _.
- - silicone elastomers including poly (dimethylsiloxane) (PDMS), usable among others by simple molding, (Mac Donald JC, Duffy DC, Anderson JR, Chiu DT, Hongkai Wu, Schueller O, Whitesides GM, Fabrication of microfluidic Systems in poly (di ethylsiloxane), Electrophoresis 2000, 21, 27-40.
- PDMS poly (dimethylsiloxane)
- PA polycarbonates
- PC polyoxymethylenes
- POM polyoxymethylenes
- COC cyclopentadienenorbomen copolymer
- PMMA polymethylmethacrylates
- PE-ld low density polyethylene
- PE-hd high density polyethylene
- PP polypropylene
- PS polystyrenes
- COC polyetheretherketone
- Still other plastics can be micro-manufactured: polybutyleneterphthalate (PBT), polyphenylene ether (PPE), polysulfone (PSU), liquid crystal polymer (LCD), polyetherimide (PEI).
- PBT polybutyleneterphthalate
- PPE polyphenylene ether
- PSU polysulfone
- LCD liquid crystal polymer
- PEI polyetherimide
- Biodegradable polyactide can also be microfabricated.
- PMMA and PC are widely used in injection molding and hot stamping. COC is commonly cited in hot stamping.
- photohthography of photoresists including for example X-ray lithography for PMMA, UN photohthography for the Epson SU-8 photopolymer.
- the "fill process” This is done by filling with a sacrificial layer, such as, for example, the Ciba-Geigy Araldite GT6063 between the second and the third photoresist layer. At the end of the process, the sacrificial layer is dissolved. - the "mask process”. A layer of metal is interposed on the second layer of photoresist that is not developed. This second layer of metal masks the microchannel. A third layer of photoresist is deposited and then illuminated. Then the photoresist is developed inside and outside of said micro-channel.
- a sacrificial layer such as, for example, the Ciba-Geigy Araldite GT6063 between the second and the third photoresist layer.
- the sacrificial layer is dissolved.
- the "mask process” A layer of metal is interposed on the second layer of photoresist that is not developed. This second layer of metal masks the microchannel.
- a third layer of photoresist
- the "lamination process” a process without dissolution, where a layer of dry film of SU-8 is unwound on the construction made from the first layer of photoresist to seal it.
- Plastic surface treatments depend on the application and the material used. For example, a hydrophobic surface must often be made hydrophilic.
- Several methods exist for assembling and sealing plastic micro-products with a cover We can cite among others:
- Microcolumn Separations 2000, 12 (7), 407-11; Alarie JP, Jacobson SC, Ramsey JM. Electrophoretic injection bias in a microchip valving scheme. Electrophoresis. 2001. Jan; 22 (2 ): 312-7; Rocklin RD, Ramsey RS, Ramsey JM. A microfabricated fluidic device for performing two-dimensional liquid-phase separations.
- Electrophoresis 2001 Mar; 22 (5): 864-73; Colyer C. Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection. Cell Biochem Biophys. 2000; 33 (3): 323-37; Bonneil E, Waldron KC. On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis. J Capillary Electrophor. 1999 May-Aug; 6 (3-4): 61-73; Horvath J, Dolnik N. Polymer wall coatings for capillary electrophoresis.Electrophoresis.
- Beds of fractionation micro-columns 2 can be etched on a support based on silicon or glass or ceramic. Beds of fractionation micro-columns 2 can be micro-molded or micro-stamped using silicon matrices when the support is plastic-based. Fractionation micro-column beds 2 can be coated with a thin film of hydrophobic or hydrophilic nature.
- the micro-particulate network can be obtained by micro-molding or micro-stamping or photopolymerization or thermopolymerization in situ, or can be made up of micro or nano-rods fitting into said ht of said micro -colonnes.
- a micro-particulate network of fractionation micro-columns 2 which constitutes the stationary phases can for example be obtained by photoengraving when the support is based on silicon or glass or ceramic.
- a micro-particulate network of fractionation micro-columns 2 which constitutes the stationary phases can for example be obtained by micro-molding, micor-stamping, photopolymerization or thermopolymerization in situ, or may consist of micro or nano-rods fitting into the bed of said micro-columns.
- a microparticulate network can be coated with a thin film of hydrophobic or hydrophilic nature.
- a microparticulate network can be subjected to coupling chemistries known to those skilled in the art to graft molecules characterized by their polarity and their amphipathicity.
- the stationary phases can be coated with peptides by grafting, using direct coupling chemistries or with spacer arms known to those skilled in the art, such as via cyanogen bromide, or carbodiimide or carbonyldiimidazole, or oxirane or azlactone.
- An increasingly used method is the immobilization on tentacular gels of peptides via fixation by activation of epoxy gel and azlactone derivative (Pribl M. Beêt der Epoxyend phenomenon in mod en chromatographischen Sorbentien un Gelen. Anal. Chem. 1980. 303. 113 -116.).
- the stationary phases can be coated with peptides by peptide synthesis in solid phase, the solid phase serving for the synthesis being also said stationary phase (Kumar KS, Rajasekharan Pillai NN, Das MR. Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS TTEGDA support fot the investigation of HCN infection in human blood J. Peptide Res., 2000, 56, 88-96)
- Stationary phases can be co-grafted into microcolumns for fractionation 2 of lipid monolayers of cell membranes such as, for example, phosphatidylcholines.
- lipid monolayers of cell membranes such as, for example, phosphatidylcholines.
- micro-levers In general, the detection of specific interactions is possible thanks to the measurement of the variation of mechanical properties of microstructures. In most cases, these microstructures are in the form of micro-levers.
- Detection using micro-levers can be performed in static mode or in dynamic mode.
- static mode the formation of a layer on the surface of the lever during a specific interaction generates a mechanical stress effect which results in a curvature of the lever.
- the sensitivity is dependent on the stiffness of the micro-lever. It is of the order of 0.1 N / m in general or even lower.
- the addition of a mass following a specific interaction on a resonant micro-lever causes a reduction in its resonant frequency.
- the stiffnesses are greater between 1 and 100 N / m and the quality factors are between 10 and 500 in air, between 1 and 10 in liquids.
- the sensitivity of such a detection is greatly increased if the measurements are carried out under vacuum (the quality factor can reach values greater than 104).
- a first approach is based on the principle of laser optical deflection which is used as a detection system in commercial atomic force microscopes. It is an external detection. This technique is very sensitive and allows access to variations in deflection less than the angstrom or variations in resonant frequency of a few hertz. It is used in most cases (cf. patents WO 00/14539 or US 5,445,008 or J. Fritz et al., Science 288, 316, 2000).
- the second approach consists in integrating the function of detection. They are generally of the piezoresistive type (cf.
- the micro-levers can be coated with a particular molecule which will give it adsorption or affinity properties.
- the positioning of the active part (specifically treated for molecular recognition particuhere) on the surface of the micro-lever, it can extend over the entire surface of the micro-lever in the case of a static measurement ( constraint effect) .
- constraint effect being maximum when the micro-lever is embedded, an active surface reduced to the micro-lever mounting area may be sufficient.
- the active part In the case of a dynamic measurement, if it is considered that the added mass does not modify the stiffness properties of the micro-lever, the active part must be positioned at the end of the micro-lever. However, an active part covering the entire surface of the micro-lever is conceivable.
- micro-levers in the case where they are used with external optical detection.
- Surface and volume micro-machining associated with deposits of thin layers make it possible to develop levers in silicon, in silicon oxide, in silicon nitride.
- These micro-levers can also be metallized (gold, platinum ).
- the dimensions of these micro-levers are typically a few hundred microns for the length, a few tens of microns for the width and a few tenths of a micron (for a static detection) or a few microns (for a dynamic detection) for the thickness.
- the mechanical properties of the materials used as well as the dimensions of the micro-levers will modify their stiffness and their natural resonance frequencies.
- the number of connections is equal to 2 per micro-lever, the first for the upper electrode and the second for the lower electrode.
- the lower electrode is generally grounded and all of the lower electrodes are connected together to form a common ground. There are therefore (n + 1) electrical connections for n piezoelectric lever arms, which significantly reduces the number of electrical connections.
- the other advantage of a piezoelectric detection is that it ensures not only the detection function but also the actuation function (resonance in the case of a dynamic measurement) thanks to the piezoelectric effect direct and reverse.
- the second drawback concerns the stability of the ferroelectric and piezoelectric properties which are subject to thermal drifts, hysteresis effects and especially aging and fatigue which very strongly limits their lifespan for dynamic uses.
- the number of connections is at least 2 for each micro-lever, i.e. 2n electrical connections for n micro-levers . This number can be equal to 4 if the piezoresistors are connected in Wheatstone bridge which gives 4n electrical connections.
- the integration of a Wheatstone bridge makes it possible to reduce the compactness of the complete system compared to an assembly with an external Wheatstone bridge. In addition, it eliminates the effects of thermal drifts.
- the disadvantage of a piezoresistive detection in the case of a dynamic measurement is that it supposes that there is an external mechanical excitation or that the quality factors of the microlevers are sufficiently important (> 100) to that the resonant frequency is detectable in white noise (thermomechanical excitation due to Brownian motion).
- the selectivity of the micro-levers 13 depends on the intrinsic polarity, the solvophobicity and the porosity of the material which constitutes the micro-lever or a thin film coated on the micro-lever , and according to the polarity and the solvophobicity of the functional groups grafted onto the micro-lever.
- the selectivity of the micro-levers 13 also depends on criteria such as the ion exchange and the affinity of the functional groups, the successive conditions of secondary micro-elution in the fractionation micro-columns 2 as well as the successive conditions of micro- extraction and micro-digestion carried out upstream of said micro-levers 13.
- the capture of a molecule, and in particular of a protein, by a microlever 13 can be carried out by affinity. This is the case, for example, when the microlever has been coated with an antibody. In this case, the micro-lever captures a precise, known protein, and indicates its presence.
- the capture of a molecule, and in particular of a protein, by a microlever 13 can be carried out by adsorption.
- the same micro-lever will be able to detect a category of proteins having similar adsorption properties. Proteins not known or researched a priori can be detected.
- fingerprint comparisons By carrying out fingerprint comparisons on different samples, it will be possible to identify differences in fingerprints, in particular differences in detection on adsorption micro-levers 13. Subsequently, by reproducing the capture with the same steps of selection, we can isolate the different proteins thus obtained to analyze them more specifically.
- a stepwise or gradient elution allows the passage of different secondary eluents entraining different molecules according to their affinity with these molecules and the affinity of micro-levers with these same molecules. Successive fingerprints are recorded, at each elution step for a step-by-step elution, or at different times for an elution with an elution gradient. As already indicated, successive washes on the micro-levers 13 can be carried out, the retention of secondary micro-eluates or secondary micro-extracts or secondary digestion products of the fractionation products on the micro-levers 13 being measured by the deviation or by the vibration frequency of said micro-levers 13, several successive fingerprints being recorded.
- the series of successive fingerprints for detecting the first sample on micro-levers 13 is for example then compared to the series of successive fingerprints for detection on micro-levers 13 of a second sample.
- the passage of washing eluent into the detection zones can be carried out using micro-capture channels 8, into which a washing eluent is sent.
- an additional washing circuit is provided, making it possible to bring a washing eluent directly upstream of a detection zone.
- the analysis device comprises secondary fractionation micro-columns.
- a fractionation micro-column 2 is intersected at the level of a terminal element by a capture micro-channel 8, connected upstream of the intersection with a secondary eluent supply conduit 15, and comprising downstream of the intersection a secondary fractionation micro-column 10.
- a detection zone 11 comprising micro-levers 13 is located on the capture micro-channel 8 downstream of the secondary fractionation micro-column 10.
- a washing micro-duct 70 comprises an inlet orifice 71 for supplying washing eluent, and an outlet orifice 72 opening into the micro- capture channel 8, downstream of the secondary fractionation micro-column 10, and upstream of the detection zone 11.
- washing eluent If a washing eluent is brought in by the capture micro-channel 8 passing through the secondary fractionation micro-column 10, the washing eluent will entrain molecules retained in the secondary fractionation micro-column 10.
- the micro-duct of washing 70 makes it possible to bring the washing eluent directly upstream of the detection zone for washing the micro-levers 13, without washing the secondary fractionation micro-column 10.
- additional detection can be provided downstream of the detection zones li using micro-levers 13, for example by mass spectrometry.
- mass spectrometry methods are discussed below.
- a second variant after a comparison of successive fingerprints of two samples, provision is made for detection of secondary micro-eluates or secondary micro-extracts or secondary digestion products of the fractionation products to be carried out by mass spectrometry, but essentially on the detection zones 11 where the series of fingerprints by micro-levers 13 of the first sample differs from the series of fingerprints by micro-levers 13 of the second sample.
- Mass spectrometry detection can be carried out in a known manner, as described in the following documents: (Cf. Dongré AR, Eng JK, Yates JR LU. Emerging tandem-mass spectrometry techniques for the rapid identification of proteins.
- Detection by mass spectrometry can also be coupled in a known manner to a separation of peptides and proteins in capillaries or on miniaturized supports with micro-channels or micro-columns by micro-chromatography, micro-electrochromatography or micro-electrophoresis.
- Documents describe more particularly the detection of peptides, proteins and carbohydrates by mass spectrometry.
- polypeptides are analyzed by mass spectrometry before or after enzymatic digestion (Cf. Roepstroff P. Mass spectrometry in protein studies from genome to function. Current Opinion in Biotechnology, 1997, 8, 6-13), by mass spectrometry techniques using ionization in nebulization or desorption.
- Post-translational modifications of proteins can be studied by subjecting the analytes to phosphatases or glycosylases (Cf. Qin J, Chait BT. Identifications and characterization of posttranslational modifications of proteins by MALDI Ion Trap mass spectrometry. Anal Chem, 1997, 69 , 4002-4009.) When the analysis by mass spectrometry takes place after digestion with a given endopeptidase, one can compare the spectra of the masses observed with the banks of the spectra of theoretical masses of the digestion residues with said endopeptidase.
- the samples can be deposited on poly (vinylidene difluoride) or polyurethane membranes (Cf. Me Comb ME, Oleschuk RD, Manley DM, Donald L, Chow A, O'neil JD, Ens W , Stabding KG, Perreault H.
- polyurethane membranes Cf. Me Comb ME, Oleschuk RD, Manley DM, Donald L, Chow A, O'neil JD, Ens W , Stabding KG, Perreault H.
- non-porous polyurethane membrane as a sample support for matrix-assisted laser désorption ionisation time-of-flight mass spectrometry of peptides and proteins. Rapid Commun Mass Spectrom, 1997, 11 (15), 1716 -22).
- we can plan to analyze glycoproteins by mass spectrometry Cf.
- a first support 20 in the form of a flat plate, comprises a supply channel 4 intended for supply in the mobile phase and comprising an enrichment zone 21 of the mobile phase in the sample.
- the first support 20 comprises a fractionation micro-column 2 comprising a micro-channel portion 3 provided with an introduction orifice 3a in fluid communication with a supply channel 4 downstream of an enrichment zone 18 d 'a mobile phase in sample, and an evacuation orifice 3b.
- the first support 20 includes a capture micro-channel 8 intersecting the fractionation micro-column 2 at a terminal element 9, and a secondary eluent supply channel 15 in fluid communication with an inlet of the micro-channel of capture 8 upstream of the intersection with the fractionation micro-column 2.
- a second support 22, in the form of a flat plate, comprises a micro-conduit 23 opening at one end into a secondary fractionation micro-column in fluid communication on the opposite side with a detection zone 11 provided with micro-levers 13.
- a third support 24, also in the form of a flat plate, comprises a discharge channel 6.
- the second and third supports 22, 24 are arranged on either side of the first support 20, parallel to the first support, being attached to the latter, so that the discharge channel 6 of the third support 24 is in fluid communication with the discharge orifice 3b of the fractionation micro-column 2, and the micro-conduit 23 of the second support is in fluid communication with the capture micro-channel 8, downstream of the intersection with the micro-column fractionation 2.
- a first support 20 comprises a plurality of fractionation microcolumns 2, and a plurality of associated capture micro-channels 8.
- the second support 22 comprises a plurality of micro-conduits in fluid communication with the capture micro-channels 8, and a plurality of detection zones 11.
- the third support 24 comprises a discharge channel 6 in fluid communication with all of the discharge orifices 3b of the micro-channels 3.
- the first, second and third supports 20, 22, 24 are arranged in parallel.
- the second and third supports 22, 24 are arranged perpendicular to the first support 20.
- the first support 20 comprises a single fractionation micro-column 2.
- the first support 20 comprises a plurality of fractionation micro-columns 2, the second and third supports 22, 24 being adapted accordingly.
- a support comprises four lots of fractionation micro-columns 2 arranged substantially in a star.
- the fractionation micro-columns 2 of the first batch fall within a range of small lengths.
- the fractionation microcolumns 2 of the second batch fall within a range of longer lengths, those of the third batch fall within a range of even longer lengths, those of the fourth batch fall within a range of even longer lengths.
- the support comprises a central enrichment micro-column 25 of square shape, in fluid communication from which each of the batch fractionation micro-columns comes, at the center of which opens a channel for introducing the sample located in a vertical plane, and not shown in FIG. 8.
- it is possible to provide for preliminary extractions of the constituents of the sample before introducing the sample into the fractionating microcolumns.
- a preliminary extraction stage 30 comprises a support 31 shown partially and provided with a plurality of prior fractionation mico-columns 32 rectilinear parallel and of the same length, with a supply channel 33 in fluid communication with the fractionation micro-columns 32, and a discharge channel 34 in fluid communication with the fractionation micro-columns 32 on the side opposite to the feed channel 33.
- the fractionation micro-columns 32 are formed by portions of micro-channels provided with intermediate separation means.
- the support 31 comprises fluid capture means 35 in the form of capture micro-channels 36, supplied by eluting on one side from a supply line 37, and in fluid communication on the opposite side with a supply line recovery 38 common.
- Each capture micro-channel transversely passes through a fractionation micro-column 32 at the level of a terminal element situated near an evacuation orifice of the fractionation micro-column 32 on the side of the evacuation channel 34.
- the sample transported by a mobile phase is brought through the feed channel 33, circulates in the fractionation micro-columns 32, where it undergoes separation according to a selectivity of the separation means of the fractionation micro-columns 32
- the association of a plurality of fractionation micro-columns 32 allows a significant separation in fractionation micro-columns 32 of small diameter, without limiting a flow rate of a mobile phase enriched in sample.
- the capture means 35 allow successive captures of the constituents of the sample present at a given capture time at the terminal element of intersection of the capture micro-channels 36 with the fractionation micro-columns 32.
- a portion of the constituents of the sample is recovered in the recovery conduit 38, which is conveyed to fractionation micro-columns and detection zones as described above.
- the constituents separated during the prior extraction in a batch of preliminary fractionation micro-columns 32 having a particular selectivity can be better separated thereafter by adapting the selectivity of one or more batches of analysis fractionation micro-columns to which the captured portion is routed.
- the micro-columns are of equal length in order to recover at the end of each micro-column substantially the same constituents.
- the products captured on different portions are collectively evacuated in a separation conduit.
- the fractionation micro-columns can be of different length to allow differential fractionation, and each capture micro-channel communicates with an associated detection zone.
- an analysis device it is possible to provide a plurality of batches of preliminary fractionation microcolumns each having a different separation selectivity, and each communicating with a plurality of batches of analysis fractionation micro-columns, each batch of micro- analysis fractionation columns having a particular selectivity, preferably adapted as a function of the selectivity of the batch of prior extraction.
- a support 31 comprises a single capture micro-channel 36 successively passing through the prior fractionation micro-columns 32 of the same batch, and finally opening into the recovery duct 38.
- each capture micro-channel 36 comprises an upstream portion 36a situated between the supply duct 37 and the intersection with the preliminary fractionation micro-column 32, and a downstream portion 36b situated between the preliminary fractionation micro-column 32 and the recovery duct 38.
- the upstream portion 36a and the downstream portion 36b open onto the preliminary fractionation micro-column 32 at offset points, at the ends of a capture segment 40.
- the upstream portion 36a opens at the downstream end of the capture segment 40, the downstream portion 36b opening at the upstream end of the capture segment 40.
- the separate components of the sample located at the capture segment 40 will be captured.
- a larger number of constituents is captured during the same capture.
- a support 1 comprises fractionation micro-columns 2, associated capture micro-channels 8 provided with upstream portions 8a and downstream portion 8b offset, opening into each fractionation micro-column 2 respectively downstream and upstream of a capture segment 40.
- the downstream portion 8b of a capture micro-channel 8 leads downstream to a detection zone 11 provided with micro-levers 13.
- An outlet channel 45 connects all of the capture micro-channels 8 downstream of the zones of detection 11, for evacuation of the mobile phases and of the constituents not retained by the selective micro-levers.
- the support 1 comprises a washing micro-duct 46 in fluid communication with each of the capture micro-channels 8, directly upstream of the detection zones 11.
- a fractionation micro-column to comprise a terminal portion. provided with a selectivity different from the selectivity of the upstream portion of the micro- column. Given the selectivity of the upstream portion, it is known that constituents having certain characteristics will migrate more quickly and reach the terminal portion first. The selectivity of the terminal portion is then adapted for additional separation of the constituents arriving at substantially the same time at the end of the fractionation micro-column.
- This difference in selectivity of a portion of a fractionation micro-column can be applied to a fractionation micro-column, a secondary fractionation microcolumn, a prior fractionation micro-column.
- terminal portion is meant a portion located just upstream of an outlet or of capture means.
- an analysis support 1 comprises means for supplying the sample and the mobile phase separately.
- a support 1 partially shown comprises a sample supply channel 4 comprising an introduction orifice 4a and a discharge orifice 4b.
- the support 1 also includes a mobile phase supply channel
- mobile phase supply micro-conduits 42 comprising an inlet orifice 43 opening into the mobile phase supply channel 41, and an outlet orifice 44 opening into the sample supply channel 4.
- Each discharge orifice of a mobile phase supply micro-duct 42 opens into the sample supply channel 4 opposite an introduction orifice 3a of a fractionation micro-column 2.
- Each mobile phase supply micro-duct 42 extended by a fractionation micro-column 2 forms a micro-channel intersecting the sample-raising channel 4.
- a phase supply micro-duct mobile 42 can be envisaged as a portion of a micro-channel devoid of separation means, and located upstream of a portion of micro-channel provided with means of separation and thus forming a fractionation micro-column 2, the sample supply micro-channel 4 intersecting all of the micro-channels at the level of the introduction orifice 3a of the fractionation micro-columns 2.
- a sample circulates in the sample supply channel 4, from the introduction orifice 4a to the discharge orifice 4b.
- a circulation of mobile phase is caused in the mobile phase supply micro-conduits 42
- the mobile phase crosses the sample supply channel 4 transversely, enriching itself in the sample, then is recovered downstream by the fractionation micro-columns 2.
- the mobile phase supply micro-conduits 42 allow at a given instant an injection simultaneously into all of the fractionation micro-columns 2 of the same quantity of mobile phase enriched in sample.
- a difference in mobile phase flow rate enriched from one fractionation micro-column 3 to another could lead to detection variations upstream of the fractionation micro-columns 3, in particular if provision is made for a simultaneous capture of fractionation products at the level of terminal elements of the fractionation micro-columns.
- an analysis device comprising a preliminary extraction stage, it is of course possible to provide, upstream of the preliminary extraction stage, a sample capture zone as described above.
- a detection of proteins or peptides has been described indifferently.
- a biological cell contains a large number of proteins, which can generate an even greater number of peptides after digestion.
- one or more sorting micro-columns can be provided upstream of micro-columns, and in particular a micro-column for sorting by size exclusion chromatography, already mentioned previously.
- Example 1 examples of possible analyzes using an analysis device according to one aspect of the invention are provided.
- Two biological samples can be analyzed, each using 8 supports of the type shown in FIG. 8. On the supports, the samples are separated by electrochromatography supplemented by additional pressure.
- each of the supports contains 4 lots of 1000 fractionation microcolumns having a length gradient, with a minimum length difference of 20 microns between two micro-columns, so that between the earliest and the last of the 1000 micro-columns columns, there is a length difference of 20 mm.
- the 1000 microcolumns in the first batch have lengths between 12 and 14 cm.
- the 1000 microcolumns in the second batch have lengths between 14 and 16 cm.
- the 1000 microcolumns in the third batch have lengths between 16 and 18 cm.
- the 1000 microcolumns in the fourth batch have lengths between 18 and 20 cm.
- FC fractionation microcolumns having a length gradient, with a minimum length difference of 20 microns between two micro-columns, so that between the earliest and the last of the 1000 micro-columns columns, there is a length difference of 20 mm.
- the 1000 microcolumns in the first batch have lengths between 12 and 14 cm.
- the media used include micro-capture channels.
- the fractionation products adsorbed at time t on a fractionation micro-column at the point of intersection with said corresponding capture micro-channel are captured simultaneously, and undergo secondary, orthogonal, parallel micro or nano-elutions, terminal, simultaneous.
- the capture micro-channels lead to detection zones with microlevers with optical detection.
- the stationary phases of the fractionation micro-columns of the first support (FC, 4, 1000, 20, 12-20) are grafted with C30 alkyl chain molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) -C30.
- the stationary phases of the fractionation micro-columns of the second support (FC, 4, 1000, 20, 12-20) are grafted with butyl molecules. (FC, 4, 1000, 20, 12-20) -butyl.
- the stationary phases of the fractionation micro-columns of the third support (FC, 4, 1000, 20, 12-20) are grafted with cyclo-hexyl molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) -cyclohexyl.
- the stationary phases of the fractionation micro-columns of the fourth support (FC, 4, 1000, 20, 12-20) are grafted with phenyl molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) - phenyl.
- the stationary phases of the fractionation micro-columns of the fifth support (FC, 4, 1000, 20, 12-20) are grafted with ethyl molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) -ethyl.
- the stationary phases of the fractionation micro-columns of the sixth support (FC, 4, 1000, 20, 12-20) are grafted with amino-propyl molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) -amino-propyl.
- the stationary phases of the fractionation micro-columns of the seventh support (FC, 4, 1000, 20, 12-20) are grafted with dihydroxypropyl molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) -dihydroxypropyl
- the stationary phases of the fractionation micro-columns of the eighth support (FC, 4, 1000, 20, 12-20) are grafted with cyanopropyl molecules.
- Such a support can be called (FC, 4, 1000, 20, 12-20) - cyanopropyl.
- Each detection zone associated with a capture micro-channel comprises eight micro-levers each provided with a specific coating.
- the first may have a coating based on C30 alkyl chains, the second a coating based on octadecyl chains, the third a coating based on octyl chains, the fourth based on butyl chains, the fourth based on chains phenyl, the fifth based on cyclo-hexyl chains, the fifth based on ethyl chains, the sixth based on amino-propyl chains, the seventh based on dihyroxypropyl chains, the eighth based on cyanopropyl chains.
- the basic solvents for the primary elutions of the fractionation chromatographies carried out on format supports can be ternary mixtures of water, trifluoroacetic acid (TFA), acetonitrile.
- TFA trifluoroacetic acid
- Six primary elution steps can be provided, the eluent used in each step having successively the following composition variants: (water, 10% acetonitrile, 0.1% TFA). (water, 15% acetonitrile, 0.1% TFA), (water, 20% acetonitrile, 0.1% TFA), (water, 25% acetonitrile, 0.1% TFA), (water, 30% acetonitrile, TFA 0.1%), (water, 35% acetonitrile, 0.1% TFA).
- the successive series of imprints mentioned above are produced for each sample.
- the series of successive fingerprints of the first sample is then compared to the series of successive fingerprints of the second sample, the series detection fingerprints then being archived in a computer database.
- the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
- the method is applicable to any search for differential expression of proteins for a given tissue, in particular for the comparison of a healthy individual and an individual suffering from a pathology. It is also applicable to the comparison of protein expression in two different physiological situations. It is also applicable to the comparison of protein expression in two strains of microorganisms (viruses, bacteria, yeasts), or applicable to the detection of differential expression of proteins on microorganisms (viruses, bacteria, yeasts) subjected to specific stimuli.
- the device can be used to compare fingerprints of basic proteins between two samples. Each sample is analyzed on a support of the type shown in FIG. 8, and of format (FC, 4, 1000, 20, 12-20), in accordance with the name used in Example 1.
- fractionation micro-columns are strongly zwitterionic in nature and contain copolymers based on sulfoalkylbetaine (N, N-dimethyl-N-methacryloyloxyethyl-N- (3-sulfopropyl) ammonium betaine (Cf. Niklund C, Sjorgen A, Irgum K , ⁇ es I. Anal. Chem. 2001. Feb 1, 73, (3), 444-52).
- sulfoalkylbetaine N-dimethyl-N-methacryloyloxyethyl-N- (3-sulfopropyl) ammonium betaine
- the basic proteins are separated in the fractionation micro-columns according to different methods with primary eluent (eluent A: water; eluent B: water, 10 mM sodium phosphate).
- the fractionation products are separated in secondary fractionation micro-columns located downstream of capture means. Secondary elutions are modulated by thiocyanate ions (primary eluent + 10 mM thiocyanate) or by perchlorate ions (primary eluent + 10 mMperchlorate).
- the series of successive fingerprints of the first sample is compared with the series of successive fingerprints of the second sample, the series of fingerprints then being archived in a computer database. Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
- the fingerprints of peptides and membrane proteins from two samples can be compared specifically.
- each sample is analyzed on two supports of the type shown in FIG. 8, having a format of the type (FC, 4, 1000, 20, 12-20).
- the integrated supports have fractionation micro-columns grafted with C4 alkyl chains.
- the membrane peptides are dissolved in dichloromethane (CH2C12) -hexafluoro-2-propanol (HFIP) (4: 1) containing traces of pyridine, then separated in micro-columns of fractionation using different primary elutions in different successive fingerprints.
- CH2C12 dichloromethane
- HFIP hexafluoro-2-propanol
- the primary elutions are based on mixtures of eluent A (formic acid-water (2: 3)) on the one hand and eluent B (formic acid -2-propanol (4: 1)) on the other go.
- the primary elutions successively present the following composition variants: (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (At 40%,
- the membrane proteins after extraction with Triton X-114, precipitation with 90% ethanol and redissolution in 65% formic acid, are separated in the micro-columns fractionation using different primary elutions in different successive fingerprints.
- the primary elutions are based on mixtures of eluent A (formic acid-water (65:35)) and eluent B (acetonitrile-water (65:35)).
- the primary elutions successively present the following composition variants: (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (A 40%, B 60%); (A 20%, B 80%); (A 0%, B 100%).
- the series of successive fingerprints of the first sample is then compared to the series of successive fingerprints of the second sample, the series of detection fingerprints then being archived in a computer database. Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
- the data acquired during analyzes carried out using a device according to examples 1 to 3 described above can be used to configure a rapid test on miniaturized consumable. For such a test, it is possible to use a device provided with supports provided only with fractionating microcolumns where numerous comparisons have shown differences in reproducible fingerprints for a given pathology.
- a healthy individual shows an A imprint, the individual suffering from pathology showing a B imprint.
- a first support (sA) is dedicated to the recognition of the A imprint, a second support (sB) being dedicated to the recognition of the 'imprint B.
- microcolumns In this test targeted at said pathology, the number of microcolumns, microchannels and microlevers is very small.
- the fractionation micro-columns chosen, more specifically the selectivity of the separation methods in relation to the composition of the solid and liquid phases used, are adapted to demonstrate the presence in a sample of proteins of which the presence or absence is characteristic. of the targeted pathology.
- An analysis device is suitable for the comparative chemical or biochemical analysis of two samples of a chemical nature, or else two samples of a biochemical nature such as cell extracts which are raw or derived from a prior extraction or which have undergone enzymatic digestion.
- a biological sample is characterized in particular by its composition in each of the proteins, glycoproteins, phosphoproteins, lipoproteins, lipids, polysaccharides, hormones, vitamins synthesized permanently or occasionally depending on the tissue or the physiological or pathological condition envisaged.
- An analysis device allows the detection of these constituents by separation and by obtaining fingerprints.
- An analysis device can rely on a length gradient of a large number of micro-channels or micro-separation columns, for example by micro-electrophoresis, micro-chromatography or micro-electrochromatography.
- a set of fractionation micro-columns can be associated with a second or even a third set of micro-channels or micro-columns, each or each of the micro-channels or microcolumns of separation of the first set being individually coupled to each or each of the micro-channels or micro-columns for separating said second set of micro-channels or micro-columns.
- a detection by selective micro-levers has been described which makes it possible to adapt the selectivity of the micro-levers to the selectivity of the fractionation micro-columns with which they are associated. Additional detection can be performed by mass spectrometry.
- the analysis device can also use other detection means known to those skilled in the art such as fluorescence, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), electrochemistry, spectrophotometry, this list not being not limiting. Additional detection can be performed by mass spectrometry.
- the analysis device can also use other means of detection known to those skilled in the art such as fluorescence, surface plasmon resonance.
- an analysis device is obtained allowing separation of the constituents of a sample, according to different selectivities, and detection of the constituents.
- An analysis device allows an exhaustive and rapid analysis of a sample and its comparison with another sample.
- Detection by micro-levers linked to analysis means allows storage and comparison of recorded data.
- a device according to the invention is suitable for miniaturization.
- the invention is not limited to the embodiments and to the variants described above. Modifications can be made without departing from the scope of the invention.
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02745510A EP1393059A1 (fr) | 2001-06-08 | 2002-06-10 | Dispositif d'analyse d'echantillon chimique ou biochimique, ensemble d'analyse comparative, et procede d'analyse associe |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR01/07537 | 2001-06-08 | ||
FR0107537A FR2825649B1 (fr) | 2001-06-08 | 2001-06-08 | Support pour analyse comparatives d'echantillons sur micro-colonnes de fractionnement avec gradients de longueur, phases stationnaires alternees, et elutions digitalisees |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2002101382A1 true WO2002101382A1 (fr) | 2002-12-19 |
Family
ID=8864113
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2002/001978 WO2002101382A1 (fr) | 2001-06-08 | 2002-06-10 | Dispositif d'analyse d'echantillon chimique ou biochimique, ensemble d'analyse comparative, et procede d'analyse associe |
Country Status (4)
Country | Link |
---|---|
US (1) | US20030027354A1 (fr) |
EP (1) | EP1393059A1 (fr) |
FR (1) | FR2825649B1 (fr) |
WO (1) | WO2002101382A1 (fr) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11355331B2 (en) | 2018-05-31 | 2022-06-07 | Micromass Uk Limited | Mass spectrometer |
US11367607B2 (en) | 2018-05-31 | 2022-06-21 | Micromass Uk Limited | Mass spectrometer |
US11373849B2 (en) | 2018-05-31 | 2022-06-28 | Micromass Uk Limited | Mass spectrometer having fragmentation region |
US11437226B2 (en) | 2018-05-31 | 2022-09-06 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
US11476103B2 (en) | 2018-05-31 | 2022-10-18 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
US11538676B2 (en) | 2018-05-31 | 2022-12-27 | Micromass Uk Limited | Mass spectrometer |
US11621154B2 (en) | 2018-05-31 | 2023-04-04 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
US11879470B2 (en) | 2018-05-31 | 2024-01-23 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
Families Citing this family (58)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6339217B1 (en) * | 1995-07-28 | 2002-01-15 | General Nanotechnology Llc | Scanning probe microscope assembly and method for making spectrophotometric, near-field, and scanning probe measurements |
US6337479B1 (en) * | 1994-07-28 | 2002-01-08 | Victor B. Kley | Object inspection and/or modification system and method |
US5751683A (en) * | 1995-07-24 | 1998-05-12 | General Nanotechnology, L.L.C. | Nanometer scale data storage device and associated positioning system |
US20080315092A1 (en) * | 1994-07-28 | 2008-12-25 | General Nanotechnology Llc | Scanning probe microscopy inspection and modification system |
US7196328B1 (en) | 2001-03-08 | 2007-03-27 | General Nanotechnology Llc | Nanomachining method and apparatus |
US6802646B1 (en) * | 2001-04-30 | 2004-10-12 | General Nanotechnology Llc | Low-friction moving interfaces in micromachines and nanomachines |
EP1196939A4 (fr) * | 1999-07-01 | 2002-09-18 | Gen Nanotechnology Llc | Systeme et procede servant a inspecter et/ou modifier un objet |
US6406604B1 (en) * | 1999-11-08 | 2002-06-18 | Norberto A. Guzman | Multi-dimensional electrophoresis apparatus |
US7329388B2 (en) * | 1999-11-08 | 2008-02-12 | Princeton Biochemicals, Inc. | Electrophoresis apparatus having staggered passage configuration |
US6813937B2 (en) * | 2001-11-28 | 2004-11-09 | General Nanotechnology Llc | Method and apparatus for micromachines, microstructures, nanomachines and nanostructures |
US7282329B2 (en) * | 2002-08-22 | 2007-10-16 | Massachusetts Institute Of Technology | Suspended microchannel detectors |
US7094345B2 (en) * | 2002-09-09 | 2006-08-22 | Cytonome, Inc. | Implementation of microfluidic components, including molecular fractionation devices, in a microfluidic system |
JP2005538855A (ja) * | 2002-09-09 | 2005-12-22 | ジェネラル ナノテクノロジー エルエルシー | 走査型プローブ顕微鏡の流体送達 |
US6989134B2 (en) * | 2002-11-27 | 2006-01-24 | Velocys Inc. | Microchannel apparatus, methods of making microchannel apparatus, and processes of conducting unit operations |
US20050014134A1 (en) * | 2003-03-06 | 2005-01-20 | West Jason Andrew Appleton | Viral identification by generation and detection of protein signatures |
EP1616189A4 (fr) * | 2003-03-31 | 2007-10-17 | Cytonome Inc | Mise en place de composants microfluidiques, y compris de dispositifs de fractionnement moleculaire, dans un systeme microfluidique |
US20040203126A1 (en) * | 2003-04-08 | 2004-10-14 | Yokogawa Electric Corporation | Method and apparatus for separating and purifying biopolymers |
US20050051489A1 (en) * | 2003-08-20 | 2005-03-10 | California Institute Of Technology | IC-processed polymer nano-liquid chromatography system on-a-chip and method of making it |
US7422910B2 (en) | 2003-10-27 | 2008-09-09 | Velocys | Manifold designs, and flow control in multichannel microchannel devices |
US20050095602A1 (en) * | 2003-11-04 | 2005-05-05 | West Jason A. | Microfluidic integrated microarrays for biological detection |
US8030092B2 (en) * | 2003-11-07 | 2011-10-04 | Princeton Biochemicals, Inc. | Controlled electrophoresis method |
EP1706735B1 (fr) * | 2003-11-07 | 2017-01-04 | Princeton Biochemicals, Inc. | Appareil d'electrophorese multidimensionnelle |
TWI253352B (en) * | 2003-12-26 | 2006-04-21 | Ind Tech Res Inst | Solid-phase micro extraction device |
US8592219B2 (en) * | 2005-01-17 | 2013-11-26 | Gyros Patent Ab | Protecting agent |
JP4403853B2 (ja) * | 2004-03-26 | 2010-01-27 | いすゞ自動車株式会社 | 多環芳香族炭化水素分析のための分析前処理方法及び装置 |
US7305850B2 (en) * | 2004-07-23 | 2007-12-11 | Velocys, Inc. | Distillation process using microchannel technology |
US20060051265A1 (en) * | 2004-09-08 | 2006-03-09 | Health Research, Inc. | Apparatus and method for sorting microstructures in a fluid medium |
US7524672B2 (en) * | 2004-09-22 | 2009-04-28 | Sandia Corporation | Microfluidic microarray systems and methods thereof |
US7592139B2 (en) | 2004-09-24 | 2009-09-22 | Sandia National Laboratories | High temperature flow-through device for rapid solubilization and analysis |
US7449116B2 (en) * | 2004-10-01 | 2008-11-11 | Agilent Technologies, Inc. | Methods and systems for protein separation |
US7943046B2 (en) | 2004-10-01 | 2011-05-17 | Agilent Technologies, Inc | Methods and systems for on-column protein delipidation |
JP2008534914A (ja) * | 2005-01-17 | 2008-08-28 | ユィロス・パテント・アクチボラグ | 多目的流路 |
US20060183166A1 (en) * | 2005-02-11 | 2006-08-17 | Michael Mayer | Arrays of supported biomembranes and uses thereof |
EP2543433A1 (fr) * | 2005-04-08 | 2013-01-09 | Velocys Inc. | Regulation de flux a travers des conduits de raccordement paralleles vers / en provenance d'un collecteur |
WO2006113527A2 (fr) | 2005-04-14 | 2006-10-26 | California Institute Of Technology | Dispositifs et systemes de chromatographie integree pour controler des substances a analyser en temps reel et procedes pour les produire |
CA2612685C (fr) * | 2005-06-22 | 2012-04-03 | Adhesives Research, Inc. | Polymere a empreinte moleculaire et son utilisation dans des dispositifs de diagnostic |
US20070028668A1 (en) * | 2005-07-20 | 2007-02-08 | National Institute Of Advanced Industrial Science And Technology | Molecule detection sensor, detection sensor, and gas transferring pump |
JP2007248323A (ja) * | 2006-03-17 | 2007-09-27 | National Institute Of Advanced Industrial & Technology | 分子検出センサ |
US20070048795A1 (en) * | 2005-08-26 | 2007-03-01 | Xiangming Fang | Immunoaffinity separation and analysis compositions and methods |
CN101472835B (zh) * | 2006-04-19 | 2012-01-25 | 康奈尔研究基金会 | 用于对象识别和鉴定的方法与系统 |
US20070246106A1 (en) | 2006-04-25 | 2007-10-25 | Velocys Inc. | Flow Distribution Channels To Control Flow in Process Channels |
US7709264B2 (en) * | 2006-09-21 | 2010-05-04 | Philip Morris Usa Inc. | Handheld microcantilever-based sensor for detecting tobacco-specific nitrosamines |
JP4984849B2 (ja) * | 2006-11-27 | 2012-07-25 | パナソニック株式会社 | 成分分離デバイスと、この成分分離デバイスを用いた化学分析デバイス |
CN103487490B (zh) * | 2007-04-27 | 2015-10-28 | 爱科来株式会社 | 分析芯片以及分析装置 |
CN101663578B (zh) * | 2007-04-27 | 2013-11-13 | 爱科来株式会社 | 电泳芯片和电泳装置 |
US8778059B2 (en) | 2007-12-03 | 2014-07-15 | Schlumberger Technology Corporation | Differential acceleration chromatography |
US8047829B1 (en) * | 2009-01-26 | 2011-11-01 | Sandia Corporation | Method for forming polymerized microfluidic devices |
US8974651B2 (en) | 2010-04-17 | 2015-03-10 | C.C. Imex | Illuminator for visualization of fluorophores |
US9176012B2 (en) * | 2012-04-16 | 2015-11-03 | David Samuel Lieberman | Methods and systems for improved membrane based calorimeters |
ES2921604T3 (es) * | 2013-03-15 | 2022-08-30 | Haemonetics Corp | Cartucho para pruebas de hemostasia |
KR20150052898A (ko) * | 2013-11-06 | 2015-05-15 | 연세대학교 산학협력단 | 말디톱 질량분석기에 이용가능한 시료 플레이트 및 상기 시료 플레이트의 제조방법 |
US9835587B2 (en) | 2014-04-01 | 2017-12-05 | C.C. Imex | Electrophoresis running tank assembly |
US10768176B2 (en) * | 2014-06-17 | 2020-09-08 | Anteo Technologies Pty Ltd | Hetero functional binding systems |
EP3472610B1 (fr) * | 2016-06-17 | 2023-12-20 | Koninklijke Philips N.V. | Dispositif d'analyse de gaz compact et procédé |
US10746696B2 (en) | 2016-12-19 | 2020-08-18 | Analog Devices, Inc. | Self-calibrated heavy metal detector |
CN108732271B (zh) * | 2018-05-22 | 2023-03-31 | 福州大学 | 三磷酸腺苷及其磷酸化代谢产物的在线固相萃取检测方法 |
CN114152655B (zh) * | 2021-12-31 | 2023-11-14 | 常州大学 | 用于谷氨酸对映体手性识别的聚苯乙烯与L-Phe复合物修饰电极及其制备方法 |
WO2023178222A1 (fr) * | 2022-03-16 | 2023-09-21 | Inso Biosciences Inc. | Dispositifs, procédés et systèmes pour l'enrichissement d'échantillons cellulaires en fonction de leurs propriétés physiques |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4891120A (en) * | 1986-06-06 | 1990-01-02 | Sethi Rajinder S | Chromatographic separation device |
US4908112A (en) * | 1988-06-16 | 1990-03-13 | E. I. Du Pont De Nemours & Co. | Silicon semiconductor wafer for analyzing micronic biological samples |
US4928513A (en) * | 1986-07-29 | 1990-05-29 | Sharp Kabushiki Kaisha | Sensor |
US4935040A (en) * | 1989-03-29 | 1990-06-19 | The Perkin-Elmer Corporation | Miniature devices useful for gas chromatography |
WO1998050154A1 (fr) * | 1997-05-08 | 1998-11-12 | University Of Minnesota | Systeme de test genetique a micropuce integree |
US6120666A (en) * | 1996-09-26 | 2000-09-19 | Ut-Battelle, Llc | Microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same |
-
2001
- 2001-06-08 FR FR0107537A patent/FR2825649B1/fr not_active Expired - Fee Related
-
2002
- 2002-06-10 WO PCT/FR2002/001978 patent/WO2002101382A1/fr not_active Application Discontinuation
- 2002-06-10 US US10/164,423 patent/US20030027354A1/en not_active Abandoned
- 2002-06-10 EP EP02745510A patent/EP1393059A1/fr not_active Withdrawn
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4891120A (en) * | 1986-06-06 | 1990-01-02 | Sethi Rajinder S | Chromatographic separation device |
US4928513A (en) * | 1986-07-29 | 1990-05-29 | Sharp Kabushiki Kaisha | Sensor |
US4908112A (en) * | 1988-06-16 | 1990-03-13 | E. I. Du Pont De Nemours & Co. | Silicon semiconductor wafer for analyzing micronic biological samples |
US4935040A (en) * | 1989-03-29 | 1990-06-19 | The Perkin-Elmer Corporation | Miniature devices useful for gas chromatography |
US6120666A (en) * | 1996-09-26 | 2000-09-19 | Ut-Battelle, Llc | Microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same |
WO1998050154A1 (fr) * | 1997-05-08 | 1998-11-12 | University Of Minnesota | Systeme de test genetique a micropuce integree |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11355331B2 (en) | 2018-05-31 | 2022-06-07 | Micromass Uk Limited | Mass spectrometer |
US11367607B2 (en) | 2018-05-31 | 2022-06-21 | Micromass Uk Limited | Mass spectrometer |
US11373849B2 (en) | 2018-05-31 | 2022-06-28 | Micromass Uk Limited | Mass spectrometer having fragmentation region |
US11437226B2 (en) | 2018-05-31 | 2022-09-06 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
US11476103B2 (en) | 2018-05-31 | 2022-10-18 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
US11538676B2 (en) | 2018-05-31 | 2022-12-27 | Micromass Uk Limited | Mass spectrometer |
US11621154B2 (en) | 2018-05-31 | 2023-04-04 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
US11879470B2 (en) | 2018-05-31 | 2024-01-23 | Micromass Uk Limited | Bench-top time of flight mass spectrometer |
Also Published As
Publication number | Publication date |
---|---|
FR2825649B1 (fr) | 2003-10-17 |
FR2825649A1 (fr) | 2002-12-13 |
EP1393059A1 (fr) | 2004-03-03 |
US20030027354A1 (en) | 2003-02-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1393059A1 (fr) | Dispositif d'analyse d'echantillon chimique ou biochimique, ensemble d'analyse comparative, et procede d'analyse associe | |
Ali et al. | Nanochromatography and nanocapillary electrophoresis: pharmaceutical and environmental analyses | |
Yuan et al. | Advances in microchip liquid chromatography | |
Lazar et al. | Microfabricated devices: A new sample introduction approach to mass spectrometry | |
Freire et al. | Proteome-on-a-chip: mirage, or on the horizon? | |
Breadmore et al. | Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2012–2014) | |
Yu et al. | Monolithic porous polymer for on-chip solid-phase extraction and preconcentration prepared by photoinitiated in situ polymerization within a microfluidic device | |
Lee et al. | Microfluidic chips for mass spectrometry‐based proteomics | |
Heegaard et al. | Affinity capillary electrophoresis: important application areas and some recent developments | |
Lion et al. | Microfluidic systems in proteomics | |
de Mello | FOCUS On-chip chromatography: the last twenty years | |
Huikko et al. | Introduction to micro-analytical systems: bioanalytical and pharmaceutical applications | |
US9718676B2 (en) | Polymeric nanopillars and nanotubes, their manufacture and uses | |
Batz et al. | Chemical vapor deposition of aminopropyl silanes in microfluidic channels for highly efficient microchip capillary electrophoresis-electrospray ionization-mass spectrometry | |
Song et al. | Free-flow zone electrophoresis of peptides and proteins in PDMS microchip for narrow pI range sample prefractionation coupled with mass spectrometry | |
Novo et al. | Current advances and challenges in microfluidic free-flow electrophoresis—A critical review | |
Gao et al. | Recent (2018–2020) development in capillary electrophoresis | |
JP5220598B2 (ja) | キャピラリーゾーン電気泳動の感度を向上させるための方法及び装置 | |
Kist et al. | Separation of biomolecules using electrophoresis and nanostructures | |
Anciaux et al. | Reduced surface adsorption in 3D printed acrylonitrile butadiene styrene micro free‐flow electrophoresis devices | |
Fernández‐Abedul et al. | Improving the separation in microchip electrophoresis by surface modification | |
Mikuš et al. | Column coupling electrophoresis in biomedical analysis | |
Fogarty et al. | Microchip capillary electrophoresis: application to peptide analysis | |
Mangelings et al. | High-throughput screening and optimization approaches for chiral compounds by means of microfluidic devices | |
Houbart et al. | Advances in Low Volume Sample Analysis Using Microfluidic Separation Techniques |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2002745510 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2002745510 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
NENP | Non-entry into the national phase |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2002745510 Country of ref document: EP |