CA2144529A1 - Single use separation cartridge for a capillary electrophoresis instrument - Google Patents
Single use separation cartridge for a capillary electrophoresis instrumentInfo
- Publication number
- CA2144529A1 CA2144529A1 CA002144529A CA2144529A CA2144529A1 CA 2144529 A1 CA2144529 A1 CA 2144529A1 CA 002144529 A CA002144529 A CA 002144529A CA 2144529 A CA2144529 A CA 2144529A CA 2144529 A1 CA2144529 A1 CA 2144529A1
- Authority
- CA
- Canada
- Prior art keywords
- cartridge
- capillary tube
- capillary
- reservoir
- pathway
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- 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
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
Abstract
A disposable cartridge (11) for a capillary electrophoresis in which short capillary tube segments (14) are used for simultaneous multiple lane separations. The cartridge contains all separation components (16, 18) of the instrument which come in contact with the sample and is capable of automatically loading (35) a quantitative portion of a bulk sample into the capillary tube segments.
Electrophoresis occurs without bulk flow through the capillaries, which are scanned in situ by the instrument(100).
Electrophoresis occurs without bulk flow through the capillaries, which are scanned in situ by the instrument(100).
Description
2144~29 094/19514 1 PCT~S94/02013 Description Single Use Separation Cartridge for a Capillary Electrophoresis Instrument Field of the Invention This invention relates to a disposable car-tridge for use in an electrophoresis instrument, and in particular to a disposable multi-lane separation car-tridge which contains all the separation components of anautomated instrument which come in contact with the sample and includes the capability to quantitatively load a portion of the sample into the electrophoretic pathway.
Background of the Invention Electrophoretic separation techniques are based upon the differential mobilities of the components of a mobile phase passing through a stationary separation medium under the influence of an applied electric field.
The components are distinguished by their migration times past a fixed point in the electrophoretic pathway or by their positions within the pathway at a fixed time.
Capillary electrophoresis (CE) is one example of the former while capillary isoelectric focusing (IEF) is an example of the latter. The separation medium in free solution CE is the buffer filled capillary tube itself.
Instruments for performing capillary electro-phoresis are frequently designed as flow-through systems.
In IEF the separated components are commonly mobilized past a fixed detector following separation. The capilla-ries must also be washed between sample runs. In CE
complicated hydraulic systems are required to accurately control sample introduction. A component's velocity is the vector sum of the bulk flow velocity, due to electroosmotic force, and the component's electrophoretic velocity. In capillary IEF, tubes are typically coated on their interior surface to eliminate electroosmosis and buffer reservoirs of high pH at the anode and low pH at wo94/19514214~ 5 2 9 PCT~S94/02013 the cathode are located at either end of the capillary tube. Components are focused within a stationary pH
gradient to their isoelectric points and then mobilized by a variety of methods past a detector. CE capillaries vary in length from 70 mm to lOOO mm, with longer lengths used to improve resolution at the price of increased run time. IEF capillaries are typically 20-lOO mm in length.
Flow-through systems typically employ one capillary and can only separate one sample at a time. Additional controls, calibrators, and samples must be run sequentially. The use of long capillaries in CE, and the requirement for mobilization past a detector in IEF, greatly increase analysis time per sample.
Automated systems designed to perform multiple runs on different samples require wash cycles between runs. This significantly increases the volume of liquid waste produced. Reservoirs are required for wash solutions, waste and reagent which must be monitored and serviced by trained personnel. If the sample contains biohazardous material then waste disposal and instrument contamination become additional problems. Cross contami-nation resulting from electrode contamination is a parti-cular problem. Auto-samplers which quantitatively load a sample into the system are typically designed to operate sequentially on samples and generally incorporate a wash cycle between samples.
Cartridges containing a capillary tube for insertion into a capillary electrophoresis instrument are known. U.S. Pat. No. 4,985,129 to Burd discloses a planar cartridge containing a looped capillary and a pair of aligned windows between which a segment of the capillary tube passes for zone detection. The cartridge is de-signed to connect the capillary tube ends to external reservoirs. U.S. Pat. No. 5,037,523 to Weinberger et al.
discloses a similar cartridge further including air cool-ing slots in the cartridge body and annular electrodes surrounding the capillary tube ends. The cartridge is ~094/19~14 214 4 ~ 2 ~ PCT~S9~/02013 also designed to connect the capillary tube ends to external reservoirs.
U.S. Pat. No. 4,816,123 to Ogan et al. dis-closes a method for forming capillary electrophoresis channels using a wire or capillary tube as a template strand. Detectors are positioned next to the template strand prior to molding a plastic material around the template strand and detectors.
U.S. Pat. No. 4,908,112 to Pace discloses a capillary sized conduit constructed by covering an etched channel in a silicon wafer with a glass plate. Reser-voirs are located at either end of the channel which is intersected by a second channel used for sample intro-duction. Electrodes are located throughout the system so that liquids may be moved by electroosmosis. Multiple channels, which are filled with a gel preparation fluid by capillary action from overhead reservoirs containing electrodes, are also disclosed. The reservoirs are then filled with buffer and a sample is injected into the reservoir with a volumetric syringe. The electroosmotic channels are less than 100 ~m in cross-sectional dimen-sion while the gel-filled channels are greater than 100 ~m in cross-sectional dimension. None of the prior art devices contain only those portions of the electrophoret-ic separation system which contact the sample.
Devices for sample loading in capillary elec-trophoresis are known. U.S. Pat. No. 4,911,807 to Burd discloses a cassette having short capillary segments which are sequentially introduced into an electrophoretic pathway for sample loading or fraction collecting. U.S.
Pat. Nos. 4,906,344 and 5,073,239 to Hjerten disclose thermal and electroendosmotic pumping means respectively for quantitative sample injection in capillaries.
Mechanical pumps are also frequently employed. All of these devices require external manipulation of the system by some means to produce a quantitative sample load.
Use of one or more absorbent materials to provide motive force to fluid samples in disposable assay W094/19514 2 1 ~ PCT~S94/02013 devices is disclosed in U.S. Pat. Nos. 5,006,309 to Khalil et al. and 5,006,474 to Horstman et al. Khalil et al. disclose an immunoassay device in which an absorbent material pulls fluid through an immobilizing fiber matrix where the results of the assay can be read. Horstman et al. disclose a device where two absorbent materials cause lateral bi-directional flow through a chromatographic separation material. Neither device uses a differential rate of flow to quantitatively load a sample into a fixed volume.
Most capillary electrophoresis instruments are flow-through devices which generate large volumes of waste relative to the effective separation volume of the capillaries. A significant portion of this waste volume arises from the need to wash those portions of the system which come in contact with the sample. These include the sample loaders, electrodes, buffer reservoirs and capil-laries. These devices are generally incapable of running simultaneous multiple lane separations.
An object of the invention is to provide a single use separation cartridge containing all of those portions of an electrophoretic separation system which contact the sample.
Another object of the invention is to provide a single use separation cartridge which is capable of automatic quantitative sample loading.
A further object of the invention is to provide a single use separation cartridge that uses capillary forces to quantitatively introduce sample and buffers into the capillary.
A further object of the invention is to provide a single use separation cartridge that replaces large sample reservoirs with hemispherical drops of sample and buffer.
A further object of the invention is to provide a single use separation cartridge that contains all necessary reagents, wash solutions and waste receptacles.
214~2g WO94/19514 PCT~S94/02013 Summary of the Invention The above objects have been achieved in a car-tridge containing short capillary tube segments suspended by a planar support structure. The capillary tube ends are located adjacent to electrodes formed on the support.
When liquid is placed in a gap between the electrode and one of the capillary tube ends the capillary tube segment is filled with liquid by capillary action. A selectively absorbent material located on the support structure which is in fluid communication with the gap slowly removes any excess liquid from the gap. A viscous, electrically conductive substance which minimizes hydrodynamic flow is then placed in each gap electrically bridging the capillary tube to the electrodes. The viscous substance, which is not absorbed by the selectively absorbent material, inhibits hydrodynamic flow in the capillary tube segment.
In one embodiment the capillary tube seqments are horizontally positioned in a coplanar array on raised portions of the support. The capillary tube ends are located above thin film electrodes formed on the support.
Capillary action fills the capillary tube segment when a drop of liquid is placed on the electrode surface. The electrode material and solution properties are chosen to produce a non-wetting condition such that the drop edqe has a non-zero contact angle and the drop is confined to the application point. A small hole in the electrode fluidly communicates with an absorbent material located between the electrodes and the underlying support. After application of a drop of a viscous electrically conductive substance at each tube end, the cartridge is positioned in an electrophoresis instrument where the electrodes are connected to an external voltage supply in such a way to avoid contamination of the electrode contacts. In the preferred embodiments, fluorescently labeled substances in transparent capillary tube segments are optically detected by the instrument in situ following electrophoresis.
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WO94/19514 PCT~S94/02013 An advantage of the single use separation cartridge of the present invention is that it contains all of those separation portions of an electrophoretic system which contact the sample.
Another advantage is that the separation cartridge is capable of automatic quantitative sample loading.
A further advantage is that the single use separation cartridge contains all necessary reagents, wash solutions and waste receptacles.
A further advantage is that the single use separation cartridge uses capillary forces to quantita-tively introduce sample and buffers into the capillary.
A further advantage is that the single use separation cartridge replaces large sample reservoirs with hemispherical drops of sample and buffer.
Brief Description of the Drawings Fig. 1 is a side plan view of a horizontal embodiment of the present invention in which the capillary tube ends are located above the electrodes;
Fig. 2a is a top view of a multi-lane laminated cartridge of the present invention;
Fig. 2b is a longitudinal cross-sectional view along line 2b of Fig. 2a;
Fig. 2c is a transverse cross-sectional view along line 2c of Fig. 2a;
Fig. 3 is a side plan view of a horizontal em-bodiment of the present invention with axially aligned electrodes;
Fig. 4 is a top view of a multi-lane cartridge of the present invention;
Fig. 5a is a longitudinal cross-sectional view of one lane of the cartridge along line 5a of Fig. 4;
Fig. 5b is a transverse cross-sectional view along line 5b of Fig. 5a showing one end of the capillary tube;
~094/19514 214 4 ~ 2 9 PCT~S94/02013 Fig. 5c is a transverse cross-sectional view along line 5c of Fig. 5a showing a grooved capillary support;
Fig. 6 is a schematic representation of the auto-loading system;
Fig. 7 is a plan view of an electrophoresis instrument optically scanning the cartridge of Fig. l;
Fig. 8 shows the isoelectric focusing of Cy5 labeled HSA utilizing the auto-loading system; and Fig. 9 shows the isoelectric focusing of Cy5 labeled Fab and Cy5 labeled Fab complexed with CKMB2 in a capillary segment.
Detailed Description of the Invention With reference to Fig. 1, a plan view of a horizontal embodiment in which the capillary tube ends are located above electrodes 16 and 18 is shown. Support structure 12 has a pair of pedestals 22 which position capillary tube 14 above planar electrodes 16 and 18 with the majority of its exterior surface surrounded by air.
In preferred embodiments, support structure 12 is a generally planar rectangular block of electrically insulating material. In one preferred embodiment support structure 12 has raised edges or pedestals on its upper surface from which capillary tube 14 is suspended. In other embodiments of the invention support structure 12 is a laminated structure composed of coplanar film sheets.
Capillary tube 14 may be made of any clear, low reflection, low absorbance material such as plastic, glass or silica and may be coated on the interior surface - to prevent electroosmosis. Borosilicate glass is used in one preferred embodiment. Capillary tube 14 is usually from about one to about ten cm in length, although longer length may be used. Three to six cm lengths and 0.025 to O.l mm i.d.s are preferred. In preferred embodiments, capillary tube 14 has a round or rectangular cross-sectional configuration. If capillary tube 14 is W094/19514 214 ~ ~ 2 ~ PCT~S94/02013 rectangular and is to be optically scanned, a large aspect ratio is preferred. Any cross-sectional configurations having a large enough surface area to volume ratio to dissipate the heat generated during electrophoresis may also be used. The cross-sectional configuration of capillary tube 14 may be symmetrical or asymmetrical in shape and/or material. In some embodi-ments, the capillary is formed by ultrasonically welding two injection molded plastic parts together.
Electrodes 16 and 18 may be electrically conductive wires, if positioned coaxially to capillary tube 14, or planar when located beneath the tube ends.
Electrodes 16 and 18 are metalized, or homogenously conductive, plastic films in preferred embodiments, although any planar conductive material may be used. The electrodes may also be plated directly on support structure 12.
In operation capillary tube 14 is filled with a conductive solution by capillary action. The conductive solution is usually a buffer which may contain additives, such as a surfactant, to inhibit electroosmosis. In the preferred embodiment capillary tube 14 is filled by capillary action after being attached to support structure 12 by placing a drop of solution onto the surface of electrode 16 or 18. The contact angle between the drop and the electrode material must be >Oo to confine the drop edge and not wet the electrode surface.
Alternatively, capillary tube 14 may be filled before being placed on support structure 12 by placing one end into a solution.
When capillary tube 14 is used for capillary electrophoresis (CE) it is first filled with a buffer solution by placing a drop of buffer at one end. In preferred embodiments a drop of sample solution is then placed at the other end and sample is loaded using electrokinetic injection. When capillary tube 14 is used for isoelectric focusing (IEF) it is filled with a mixture containing the sample and carrier ampholytes in WO94/19514 214 4 ~ 2 g PCT~S94/02013 buffer. In both cases, following removal of excess solution, a drop of a viscous conductive substance which inhibits hydrodynamic flow is then placed onto the surface of electrodes 16 and 18 at each end of capillary tube 14. The viscosity of the drops in preferred embodiments is 220 centipoise although viscosities ~ 1 cp will decrease the hydrodynamic flow rate. In a 3 cm capillary tube having a 100 micron interior diameter the hydrodynamic flow due to surface tension and gravita-tional forces in 220 cp drops is reduced to 0.03 cm/min.
Addition of a surfactant will lower the surface tension and thereby lower the internal pressure of the drops which is a driving force for hydrodynamic flow.
Electrodes 16 and 18 are connected to an exter-nal voltage supply when cartridge 11 is placed in theelectrophoresis instrument. In IEF, after separation is complete, capillary tube 14 is scanned by the instrument.
In CE separated components migrate past a fixed detector.
Optical detection of fluorescently labeled components is used in a preferred embodiment. Any other well known detection method, such as UV absorbance, may also be used.
With reference to Figs. 2a-c, the plan view of a second horizontal embodiment in which cartridge 11 is constructed by laminating planar films is shown. In this preferred embodiment, support structure 12 is a lami-nated, generally rectangular, block incorporating five coplanar sheets of a variety of films. A base sheet of acrylic film 21, 0.030" in thickness, is covered with a 0.003" thick adhesive sheet of double stick polyethene 23a. A second adhesive sheet of double stick polyethene 23b, 0.012" thick having a length equal to or less than the length of capillary tube 14 is centrally placed. An absorbent sheet of cellulose filter paper 30, 0.005" in thickness, is placed on adhesive sheet 23a at either end of adhesive sheet 23b. A conductive polymer sheet, 0.004" in thickness, is placed atop absorbent sheet 30 at either end of capillary 14 forming electrodes 16 and 18.
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WO94/19514 PCT~S94102013 A hole 28 in the conductive polymer sheet may be located at one or both ends of capillary 14. A drop of solution 31 is shown at either end of capillary tube 14. The soluton may be buffer, sample, or a viscous electrically conductive substance. The diameter of hole 28 is much smaller than the base diaméter of drop 31. Capillary tube 14 is adhesively attached to the laminate by adhesive sheet 23b.
With reference to Fig. 3, a plan view of a 10 horizontal embodiment of the present invention is shown.
Cartridge 11 consists of a support structure 12 which horizontally positions a capillary tube 14 between a pair of electrodes 16 and 18. Electrodes 16 and 18 are each separated from the adjacent capillary tube end by a gap 15 20. Support structure 12 is an acrylic block and the ends of capillary tube 14 and wire electrodes 16 and 18 are located in V-shaped channels in the upper surface of the opposed raised edges of the block.
With reference to Fig. 4, a top view of a 20 multi-lane cartridge 11 is shown. Three capillary tubes 14 are horizontally disposed in a coplanar array by support pedestals 22 of support structure 12. Comparison of lane to lane results is facilitated when capillary tubes 14 have identical lengths and cross-sectional di-25 mensions. Thin-film electrodes 18 and 16 are located beneath opposite ends of each capillary tube 14. Support structure 12 has a pair of opposed elevated edges 24.
Each edge contains a pair of spaced apart apertures 26 which may be used as detents to index the location of 30 cartridge 10 in the electrophoretic instrument. Each thin-film electrode 16 and 18 has a hole 28 passing through it to provide fluid communication between the surface of thin-film electrodes 16 and 18 and an absorb-ent material located beneath the electrodes, not shown.
Referring now to Fig. 5a a cross-sectional view along line 5a of Fig. 4 is shown. An absorbent material 30 is located beneath electrodes 16 and 18 and in fluid communication through hole 28 with the surface of elec-2141~
W O 94/19514 PCTrUS94/02013 trodes 16 and 18 and gaps 20. When performing IEF only the electrode located at the sample injection end needs to be in fluid communication with absorbent material 30.
Fig. 5b is a transverse cross-sectional view along line 5b of Fig. 5a showing the relationship between the components located at one end of capillary tube 14. Fig.
5c is a transverse cross-sectional view along line 5c of Fig. 5a showing capillary tube 14 lying in a V-shaped groove in support pedestal 22. The position of capillary tubes 14 in relation to apertures 26 allows the electrophoretic instrument to optically scan the length of capillary tube 14. Precise positioning of the axis of capillary tube 14 is not critical as the optical system has the ability to track in the x, y and z axis.
Referring now to Fig. 6, a schematic represen-tation of the auto-loading system is shown. When a bulk sample is placed in first reservoir 32 a quantitative portion is rapidly loaded into pathway 34 by auto-loading means 35. The remainder of the sample is slowly trans-ported to a second reservoir 36 by draining means 37 where it is held leaving first reservoir 32 empty and a quantitative portion of the sample loaded into pathway 34. Autoloading means 35 is filling of a fixed volume by capillary action or electrokinetic pulse injection in the preferred embodiments. Other well known means by which a discrete quantitative portion of the sample is rapidly moved from first reservoir 32 into pathway 34, such as hydrodynamic or thermal injection, may also be used.
With reference to Fig. 7, an electrophoresis instrument 100 optically scanning a horizontally disposed cartridge 11 is shown. A strongly emitting light source, such as light emitting diode or laser 123 is used to generate a beam 125. LED 123 has an output power of - about 50 milliwatts and a wavelength band which will excite fluorescence in the fluorescent labeling material.
Such excitation radiation is known as actinic radiation.
The beam is intercepted by a focusing lens 127 which directs the beam through a slit aperture and barrier 129.
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WO94/19514 PCT~S94/02013 -Light emerging from the slit is divergent and is intercepted by a collimating lens 131. The beam is then directed onto a reflecting surface 133 which is part of a dichroic mirror 135.
Dichroic mirror 135 is chosen to selectively reflect light at the waveléngths emitted by light source 123 while transmitting light at the wavelengths emitted by the fluorescent label. The reflected beam is directed toward a focusing lens 137. Light passing through the focusing lens carries an image of the slit 129 which is directed onto capillary 14. The image of slit 29 can be scanned along the longitudinal axis of capillary 14 by moving separation cartridge 11 relative to lens 137.
Fluorescent light emitted by a label, and some reflected light from the capillary, travel in a retrobeam to focusing lens 137. Note that the focusing lens is used by light traveling in each direction. From there, the retrobeam is directed to reflecting surface 133 which is a part of dichroic mirror 135. Light reflected from the capillary is reflected toward light source 123 while fluorescent light is passed through. The fluorescent light is then directed by a mirror 141 through a filter 143 which rejects any light other than the desired wavelength from the fluorescent label. Light transmitted through the filter is directed toward a focusing lens 145. From there the beam is directed to a light detector, such as photomultiplier tube 147 with a slit located at the image plane of the separation medium. The focused locations of the label are measured relative to one end of the capillary.
The output of photomultiplier tube 147 is maintained in a buffer memory 149. A data reader 150 is connected to the buffer memory 149 for receiving recorded signals which represent the fluorescent peaks. The data reader is a computer which correlates the various peaks.
In multi-lane embodiments, one end of each of capillary tubes 14 may be located above a common thin-film electrode 16. In this embodiment each capillary 2144~i29 WO94/19514 PCT~S94/02013 tube 14 may be subjected to a separate voltage difference between each of electrodes 18 and the common voltage applied to electrode 16. Alternatively each capillary tube 14 may be positioned between a single pair of common electrodes and subjected to the same voltage difference.
The cartridge of the present invention can be used for capillary electrophoresis or capillary IEF. The conductive substance in the capillary tubes may be a liq-uid solution such as a buffer or biological fluid in capillary electrophoresis, or a mixture containing sample and ampholytes in IEF. The conductive substance may also be a gel. One advantage of the present invention is that the conductive substance is confined to a short capillary tube segment which is scanned in situ by the electropho-resis instrument when separation is complete. Adequateseparations have been accomplished in 30 mm long segments shortening analysis time. After scanning, the disposable cartridge containing all parts of the separation system which have come in contact with the sample may be discarded. Liquid waste generated by wash cycles is completely eliminated and the total volume of waste is drastically reduced.
Another advantage of the present invention is its ability to auto-load a quantitative sample volume when a bulk sample is applied to the cartridge. Slow competi-tive removal of material from the sample reservoir by a selectively absorbent material eliminates the need for a complicated auto-sampling system. This reduces the cost of the instrument as well as reducing the waste generat-ed.
Example 1 IEF of Cy5-HSA Utilizinq Differential Sample Loading Differential sample loading was accomplished by placing an absorbent material between the conductive film electrode and the underlying plastic support. Excess sample is removed from the conductive film after the 21~4529 WO94/19514 PCT~S94102013 sample has been quantitatively loaded into the capillary tube through a small hole in the conductive film. In IEF
the capillary tube is filled with a mixture of sample and ampholytes by capillary action. In CE a sample plug is electrokinetically injected into a buffer filled capillary tube.
Human serum albumin (HSA), fraction V, was obtained from Sigma Chemical Company (St. Louis, Missouri). Cy5 labeled HSA was synthesized by the coupling of Cy5 fluorescent dye (Biological Detection Systems, Inc., Pittsburgh, Pennsylvania) to HSA. Cy5-HSA
was added to a 2 percent solution of ampholytes having a pH range of 3 to 10 (Biorad Inc., Hercules, California) to a final Cy5-HSA concentration of 10 mg/ml. A capilla-ry (Polymicro Technologies Inc., Phoenix, Arizona), having an inside diameter of 100 microns and an outside diameter of 365 microns, was coated to prevent electroendosmosis (Capillary Electrophoresis, Academic Press, Inc., San Diego, California (1992), pp. 191-214).
The capillary was placed in the fixture described in Figs. 4 and 5. A double coated pressure sensitive adhesive film (RX264-S, Coating Sciences Inc., Bloomfield, Connecticut) was used to bond 601-25 cellulose blotting paper (Intermountain Scientific Corporation, Bountiful, Utah) to the underlying cartridge support structure 12. The conductive plastic film with adhesive on one side (AR clad 8010, Adhesive Research Inc., Glenrock, Pennsylvania) was bonded to the cellulose blotting paper. A hole with a diameter of 3/64 inch was punched through the conductive plastic film allowing direct access to the cellulose blotting paper beneath.
A drop (5 microliter) of the Cy5-HSA solution was placed upon the conductive film, concentric with the hole, and in contact with the end of the capillary.
Within two seconds the solution filled the capillary.
After thirty seconds, excess solution had completely wicked into the cellulose blotting paper. A drop (5 microliter) of 0.02 M sodium hydroxide, thickened to a WO94/19514 214 ~ 5 2 9 PCT~S94102013 viscosity of lO00 cp, was then placed upon the conductive film concentric~with the hole and in direct contact with the end of the capillary. A drop of 0.02 M phosphoric acid, thickened to a viscosity of lO00 cp, was applied to the other end of the capillary in similar fashion. These drops bridged the capillary to the cathode and the anode.
Electrophoresis was performed at l kV constant voltage for ten minutes with a CZE lO00 R high voltage supply (Spellman Corp., Plainville, New Jersey). Current was allowed to drop from 30 to 2 microamps as focusing took place. The position of the fluorescent proteins was determined at this point by scanning the capillary with a He-Ne laser optic system. A translation stage moved the cartridge relative to the optic system while the focusing field remained on. Reflected fluorescence was detected with a R928 PMT and the data was collected using data acquisition software on an IBM (trademark) personal computer.
Separation in this capillary system is based on the isoelectric points of the proteins. Results are shown as a plot of fluorescence versus distance on the capillary in Fig. 8. A single peak 41 corresponding to Cy5-HSA is focused. The excess solution in the cellulose blotting paper does not adversely affect the focusing of Cy5-HSA.
Exam~le 2 Detection of Proteins Present in Human Blood Creatine kinase is an enzyme present in various mammalian tissue. It occurs in three different forms - known as isoenzymes: CK-MM (skeletal), CK-MB (cardiac) and CK-BB (brain). After release from tissue and on cir-culation in blood the MM and MB forms themselves break-down to smaller fragments known as isoforms or subforms.
In the event of myocardial infarction, the MB isoenzyme, present in cardiac muscle, is released in the plasma.
Hence, it serves as a specific diagnostic molecular W0 94/195142 1 4~ 2 ~ PCT~S94102013 marker for cardiac ischemia or necrosis. The early and rapid detection of this isoenzyme and its isoforms are very crucial for the diagnosis of myocardial infarction and for initiating thrombotic therapy.
Separation of Cy5 labeled CK-MB antibody from its immune complex was performed using a capillary isoelectric focusing system. CK-MB2 (human heart) and monoclonal anti CK-MB were obtained from Biospecific (Emeryville, California). Fab fragments were prepared by digesting the monoclonal anti CK-MB with the enzyme papain. Cy5 labeled Fab was synthesized by the coupling of CyS fluorescent dye (Biological Detection Systems, Pittsburgh, Pennsylvania) to Fab and purified by conventional gel permeation and ion exchange methods.
This fluorescent substance is the labeled binding agent.
Differential separation assay (DSA) was done as follows: Cy5 labeled Fab (binding agent) was incubated with CK-MB2 (target) at a final concentration of 5 0 ~g/ml Cy5-Fab and 1 mg/ml CK-MB2 in 1 mM phosphate 15 mM NaCl pH 7 . 2 . A control sample consisted of Cy5-Fab alone at 5 0 ~g/ml without added CK-MB2. Reactions were performed in 1. 5 ml Eppendorf tubes in a total reaction volume of 10 ~1 . After incubating the samples at room temperature (2 0C) for 3 0 minutes, HSA was added as a carrier at a final concentration of 2 mg/ml. The reaction mixture was then diluted 3 0-fold with a 2% solution of ampholytes having a pH range of 3 to 10 (Biorad Inc., Hercules, California) in deionized water. Capillary action was used to fill a 50 x 0.3 x 0. 03 mm borosilicate glass 3o rectangular capillary (R&S Medical, Mountain Lakes, New Jersey), coated to suppress electroendosmosis (CaPillary ElectroPhoresis, Academic Press, Inc., San Diego, Cali-fornia (199 2), pp. 19 1-2 14), by dipping its end in the diluted reaction mixture. The capillary was then placed horizontally on an acrylic platform and platinum elec-trodes were bonded to the acrylic adjacent to the ends of the capillary. A drop of 0. 02 M sodium hydroxide, thickened to a viscosity of 100 cp, was applied to one WO94/19514 21~ ~ 5 2 3 PCT~S94/02013 -end of the capillary to bridge it with the cathodic electrode and a drop of 0.02 M phosphoric acid was applied to the other end of the capillary to bridge it with the anode. Electrophoresis was performed at 2 kV
constant voltage for lO minutes with a CZE lOOOR high voltage supply (Spellman, Plainville, New Jersey). The current was allowed to drop from 30 to 2 ~amps as the focusing took place.
The positions of the fluorescent proteins were determined at this point by scanning the capillary with a He-Ne laser optic system. A translation stage moved the capillary while the focusing field remained on. The re-flected fluorescence was detected with a R928 PMT and the data was collected using data acquisition software on an IBM ttrademark) personal computer.
Separation in this capillary system is based on the isoelectric points of the proteins. The results are shown as a plot of fluorescence versus distance on the capillary in Fig. 9. When Cy5-Fab alone is run a Cy5-Fab control peak 152 is focused at 18 mm. When CK-MB2 is present a second peak 154 corresponding to the immune complex consisting of Cy5-Fab/CK-MB2 is focused at 23 mm while a peak 156 corresponding to the residual uncomplexed labeled Cy5-Fab is focused at 15 mm.
Background of the Invention Electrophoretic separation techniques are based upon the differential mobilities of the components of a mobile phase passing through a stationary separation medium under the influence of an applied electric field.
The components are distinguished by their migration times past a fixed point in the electrophoretic pathway or by their positions within the pathway at a fixed time.
Capillary electrophoresis (CE) is one example of the former while capillary isoelectric focusing (IEF) is an example of the latter. The separation medium in free solution CE is the buffer filled capillary tube itself.
Instruments for performing capillary electro-phoresis are frequently designed as flow-through systems.
In IEF the separated components are commonly mobilized past a fixed detector following separation. The capilla-ries must also be washed between sample runs. In CE
complicated hydraulic systems are required to accurately control sample introduction. A component's velocity is the vector sum of the bulk flow velocity, due to electroosmotic force, and the component's electrophoretic velocity. In capillary IEF, tubes are typically coated on their interior surface to eliminate electroosmosis and buffer reservoirs of high pH at the anode and low pH at wo94/19514214~ 5 2 9 PCT~S94/02013 the cathode are located at either end of the capillary tube. Components are focused within a stationary pH
gradient to their isoelectric points and then mobilized by a variety of methods past a detector. CE capillaries vary in length from 70 mm to lOOO mm, with longer lengths used to improve resolution at the price of increased run time. IEF capillaries are typically 20-lOO mm in length.
Flow-through systems typically employ one capillary and can only separate one sample at a time. Additional controls, calibrators, and samples must be run sequentially. The use of long capillaries in CE, and the requirement for mobilization past a detector in IEF, greatly increase analysis time per sample.
Automated systems designed to perform multiple runs on different samples require wash cycles between runs. This significantly increases the volume of liquid waste produced. Reservoirs are required for wash solutions, waste and reagent which must be monitored and serviced by trained personnel. If the sample contains biohazardous material then waste disposal and instrument contamination become additional problems. Cross contami-nation resulting from electrode contamination is a parti-cular problem. Auto-samplers which quantitatively load a sample into the system are typically designed to operate sequentially on samples and generally incorporate a wash cycle between samples.
Cartridges containing a capillary tube for insertion into a capillary electrophoresis instrument are known. U.S. Pat. No. 4,985,129 to Burd discloses a planar cartridge containing a looped capillary and a pair of aligned windows between which a segment of the capillary tube passes for zone detection. The cartridge is de-signed to connect the capillary tube ends to external reservoirs. U.S. Pat. No. 5,037,523 to Weinberger et al.
discloses a similar cartridge further including air cool-ing slots in the cartridge body and annular electrodes surrounding the capillary tube ends. The cartridge is ~094/19~14 214 4 ~ 2 ~ PCT~S9~/02013 also designed to connect the capillary tube ends to external reservoirs.
U.S. Pat. No. 4,816,123 to Ogan et al. dis-closes a method for forming capillary electrophoresis channels using a wire or capillary tube as a template strand. Detectors are positioned next to the template strand prior to molding a plastic material around the template strand and detectors.
U.S. Pat. No. 4,908,112 to Pace discloses a capillary sized conduit constructed by covering an etched channel in a silicon wafer with a glass plate. Reser-voirs are located at either end of the channel which is intersected by a second channel used for sample intro-duction. Electrodes are located throughout the system so that liquids may be moved by electroosmosis. Multiple channels, which are filled with a gel preparation fluid by capillary action from overhead reservoirs containing electrodes, are also disclosed. The reservoirs are then filled with buffer and a sample is injected into the reservoir with a volumetric syringe. The electroosmotic channels are less than 100 ~m in cross-sectional dimen-sion while the gel-filled channels are greater than 100 ~m in cross-sectional dimension. None of the prior art devices contain only those portions of the electrophoret-ic separation system which contact the sample.
Devices for sample loading in capillary elec-trophoresis are known. U.S. Pat. No. 4,911,807 to Burd discloses a cassette having short capillary segments which are sequentially introduced into an electrophoretic pathway for sample loading or fraction collecting. U.S.
Pat. Nos. 4,906,344 and 5,073,239 to Hjerten disclose thermal and electroendosmotic pumping means respectively for quantitative sample injection in capillaries.
Mechanical pumps are also frequently employed. All of these devices require external manipulation of the system by some means to produce a quantitative sample load.
Use of one or more absorbent materials to provide motive force to fluid samples in disposable assay W094/19514 2 1 ~ PCT~S94/02013 devices is disclosed in U.S. Pat. Nos. 5,006,309 to Khalil et al. and 5,006,474 to Horstman et al. Khalil et al. disclose an immunoassay device in which an absorbent material pulls fluid through an immobilizing fiber matrix where the results of the assay can be read. Horstman et al. disclose a device where two absorbent materials cause lateral bi-directional flow through a chromatographic separation material. Neither device uses a differential rate of flow to quantitatively load a sample into a fixed volume.
Most capillary electrophoresis instruments are flow-through devices which generate large volumes of waste relative to the effective separation volume of the capillaries. A significant portion of this waste volume arises from the need to wash those portions of the system which come in contact with the sample. These include the sample loaders, electrodes, buffer reservoirs and capil-laries. These devices are generally incapable of running simultaneous multiple lane separations.
An object of the invention is to provide a single use separation cartridge containing all of those portions of an electrophoretic separation system which contact the sample.
Another object of the invention is to provide a single use separation cartridge which is capable of automatic quantitative sample loading.
A further object of the invention is to provide a single use separation cartridge that uses capillary forces to quantitatively introduce sample and buffers into the capillary.
A further object of the invention is to provide a single use separation cartridge that replaces large sample reservoirs with hemispherical drops of sample and buffer.
A further object of the invention is to provide a single use separation cartridge that contains all necessary reagents, wash solutions and waste receptacles.
214~2g WO94/19514 PCT~S94/02013 Summary of the Invention The above objects have been achieved in a car-tridge containing short capillary tube segments suspended by a planar support structure. The capillary tube ends are located adjacent to electrodes formed on the support.
When liquid is placed in a gap between the electrode and one of the capillary tube ends the capillary tube segment is filled with liquid by capillary action. A selectively absorbent material located on the support structure which is in fluid communication with the gap slowly removes any excess liquid from the gap. A viscous, electrically conductive substance which minimizes hydrodynamic flow is then placed in each gap electrically bridging the capillary tube to the electrodes. The viscous substance, which is not absorbed by the selectively absorbent material, inhibits hydrodynamic flow in the capillary tube segment.
In one embodiment the capillary tube seqments are horizontally positioned in a coplanar array on raised portions of the support. The capillary tube ends are located above thin film electrodes formed on the support.
Capillary action fills the capillary tube segment when a drop of liquid is placed on the electrode surface. The electrode material and solution properties are chosen to produce a non-wetting condition such that the drop edqe has a non-zero contact angle and the drop is confined to the application point. A small hole in the electrode fluidly communicates with an absorbent material located between the electrodes and the underlying support. After application of a drop of a viscous electrically conductive substance at each tube end, the cartridge is positioned in an electrophoresis instrument where the electrodes are connected to an external voltage supply in such a way to avoid contamination of the electrode contacts. In the preferred embodiments, fluorescently labeled substances in transparent capillary tube segments are optically detected by the instrument in situ following electrophoresis.
21~4S~
WO94/19514 PCT~S94/02013 An advantage of the single use separation cartridge of the present invention is that it contains all of those separation portions of an electrophoretic system which contact the sample.
Another advantage is that the separation cartridge is capable of automatic quantitative sample loading.
A further advantage is that the single use separation cartridge contains all necessary reagents, wash solutions and waste receptacles.
A further advantage is that the single use separation cartridge uses capillary forces to quantita-tively introduce sample and buffers into the capillary.
A further advantage is that the single use separation cartridge replaces large sample reservoirs with hemispherical drops of sample and buffer.
Brief Description of the Drawings Fig. 1 is a side plan view of a horizontal embodiment of the present invention in which the capillary tube ends are located above the electrodes;
Fig. 2a is a top view of a multi-lane laminated cartridge of the present invention;
Fig. 2b is a longitudinal cross-sectional view along line 2b of Fig. 2a;
Fig. 2c is a transverse cross-sectional view along line 2c of Fig. 2a;
Fig. 3 is a side plan view of a horizontal em-bodiment of the present invention with axially aligned electrodes;
Fig. 4 is a top view of a multi-lane cartridge of the present invention;
Fig. 5a is a longitudinal cross-sectional view of one lane of the cartridge along line 5a of Fig. 4;
Fig. 5b is a transverse cross-sectional view along line 5b of Fig. 5a showing one end of the capillary tube;
~094/19514 214 4 ~ 2 9 PCT~S94/02013 Fig. 5c is a transverse cross-sectional view along line 5c of Fig. 5a showing a grooved capillary support;
Fig. 6 is a schematic representation of the auto-loading system;
Fig. 7 is a plan view of an electrophoresis instrument optically scanning the cartridge of Fig. l;
Fig. 8 shows the isoelectric focusing of Cy5 labeled HSA utilizing the auto-loading system; and Fig. 9 shows the isoelectric focusing of Cy5 labeled Fab and Cy5 labeled Fab complexed with CKMB2 in a capillary segment.
Detailed Description of the Invention With reference to Fig. 1, a plan view of a horizontal embodiment in which the capillary tube ends are located above electrodes 16 and 18 is shown. Support structure 12 has a pair of pedestals 22 which position capillary tube 14 above planar electrodes 16 and 18 with the majority of its exterior surface surrounded by air.
In preferred embodiments, support structure 12 is a generally planar rectangular block of electrically insulating material. In one preferred embodiment support structure 12 has raised edges or pedestals on its upper surface from which capillary tube 14 is suspended. In other embodiments of the invention support structure 12 is a laminated structure composed of coplanar film sheets.
Capillary tube 14 may be made of any clear, low reflection, low absorbance material such as plastic, glass or silica and may be coated on the interior surface - to prevent electroosmosis. Borosilicate glass is used in one preferred embodiment. Capillary tube 14 is usually from about one to about ten cm in length, although longer length may be used. Three to six cm lengths and 0.025 to O.l mm i.d.s are preferred. In preferred embodiments, capillary tube 14 has a round or rectangular cross-sectional configuration. If capillary tube 14 is W094/19514 214 ~ ~ 2 ~ PCT~S94/02013 rectangular and is to be optically scanned, a large aspect ratio is preferred. Any cross-sectional configurations having a large enough surface area to volume ratio to dissipate the heat generated during electrophoresis may also be used. The cross-sectional configuration of capillary tube 14 may be symmetrical or asymmetrical in shape and/or material. In some embodi-ments, the capillary is formed by ultrasonically welding two injection molded plastic parts together.
Electrodes 16 and 18 may be electrically conductive wires, if positioned coaxially to capillary tube 14, or planar when located beneath the tube ends.
Electrodes 16 and 18 are metalized, or homogenously conductive, plastic films in preferred embodiments, although any planar conductive material may be used. The electrodes may also be plated directly on support structure 12.
In operation capillary tube 14 is filled with a conductive solution by capillary action. The conductive solution is usually a buffer which may contain additives, such as a surfactant, to inhibit electroosmosis. In the preferred embodiment capillary tube 14 is filled by capillary action after being attached to support structure 12 by placing a drop of solution onto the surface of electrode 16 or 18. The contact angle between the drop and the electrode material must be >Oo to confine the drop edge and not wet the electrode surface.
Alternatively, capillary tube 14 may be filled before being placed on support structure 12 by placing one end into a solution.
When capillary tube 14 is used for capillary electrophoresis (CE) it is first filled with a buffer solution by placing a drop of buffer at one end. In preferred embodiments a drop of sample solution is then placed at the other end and sample is loaded using electrokinetic injection. When capillary tube 14 is used for isoelectric focusing (IEF) it is filled with a mixture containing the sample and carrier ampholytes in WO94/19514 214 4 ~ 2 g PCT~S94/02013 buffer. In both cases, following removal of excess solution, a drop of a viscous conductive substance which inhibits hydrodynamic flow is then placed onto the surface of electrodes 16 and 18 at each end of capillary tube 14. The viscosity of the drops in preferred embodiments is 220 centipoise although viscosities ~ 1 cp will decrease the hydrodynamic flow rate. In a 3 cm capillary tube having a 100 micron interior diameter the hydrodynamic flow due to surface tension and gravita-tional forces in 220 cp drops is reduced to 0.03 cm/min.
Addition of a surfactant will lower the surface tension and thereby lower the internal pressure of the drops which is a driving force for hydrodynamic flow.
Electrodes 16 and 18 are connected to an exter-nal voltage supply when cartridge 11 is placed in theelectrophoresis instrument. In IEF, after separation is complete, capillary tube 14 is scanned by the instrument.
In CE separated components migrate past a fixed detector.
Optical detection of fluorescently labeled components is used in a preferred embodiment. Any other well known detection method, such as UV absorbance, may also be used.
With reference to Figs. 2a-c, the plan view of a second horizontal embodiment in which cartridge 11 is constructed by laminating planar films is shown. In this preferred embodiment, support structure 12 is a lami-nated, generally rectangular, block incorporating five coplanar sheets of a variety of films. A base sheet of acrylic film 21, 0.030" in thickness, is covered with a 0.003" thick adhesive sheet of double stick polyethene 23a. A second adhesive sheet of double stick polyethene 23b, 0.012" thick having a length equal to or less than the length of capillary tube 14 is centrally placed. An absorbent sheet of cellulose filter paper 30, 0.005" in thickness, is placed on adhesive sheet 23a at either end of adhesive sheet 23b. A conductive polymer sheet, 0.004" in thickness, is placed atop absorbent sheet 30 at either end of capillary 14 forming electrodes 16 and 18.
21~2~
WO94/19514 PCT~S94102013 A hole 28 in the conductive polymer sheet may be located at one or both ends of capillary 14. A drop of solution 31 is shown at either end of capillary tube 14. The soluton may be buffer, sample, or a viscous electrically conductive substance. The diameter of hole 28 is much smaller than the base diaméter of drop 31. Capillary tube 14 is adhesively attached to the laminate by adhesive sheet 23b.
With reference to Fig. 3, a plan view of a 10 horizontal embodiment of the present invention is shown.
Cartridge 11 consists of a support structure 12 which horizontally positions a capillary tube 14 between a pair of electrodes 16 and 18. Electrodes 16 and 18 are each separated from the adjacent capillary tube end by a gap 15 20. Support structure 12 is an acrylic block and the ends of capillary tube 14 and wire electrodes 16 and 18 are located in V-shaped channels in the upper surface of the opposed raised edges of the block.
With reference to Fig. 4, a top view of a 20 multi-lane cartridge 11 is shown. Three capillary tubes 14 are horizontally disposed in a coplanar array by support pedestals 22 of support structure 12. Comparison of lane to lane results is facilitated when capillary tubes 14 have identical lengths and cross-sectional di-25 mensions. Thin-film electrodes 18 and 16 are located beneath opposite ends of each capillary tube 14. Support structure 12 has a pair of opposed elevated edges 24.
Each edge contains a pair of spaced apart apertures 26 which may be used as detents to index the location of 30 cartridge 10 in the electrophoretic instrument. Each thin-film electrode 16 and 18 has a hole 28 passing through it to provide fluid communication between the surface of thin-film electrodes 16 and 18 and an absorb-ent material located beneath the electrodes, not shown.
Referring now to Fig. 5a a cross-sectional view along line 5a of Fig. 4 is shown. An absorbent material 30 is located beneath electrodes 16 and 18 and in fluid communication through hole 28 with the surface of elec-2141~
W O 94/19514 PCTrUS94/02013 trodes 16 and 18 and gaps 20. When performing IEF only the electrode located at the sample injection end needs to be in fluid communication with absorbent material 30.
Fig. 5b is a transverse cross-sectional view along line 5b of Fig. 5a showing the relationship between the components located at one end of capillary tube 14. Fig.
5c is a transverse cross-sectional view along line 5c of Fig. 5a showing capillary tube 14 lying in a V-shaped groove in support pedestal 22. The position of capillary tubes 14 in relation to apertures 26 allows the electrophoretic instrument to optically scan the length of capillary tube 14. Precise positioning of the axis of capillary tube 14 is not critical as the optical system has the ability to track in the x, y and z axis.
Referring now to Fig. 6, a schematic represen-tation of the auto-loading system is shown. When a bulk sample is placed in first reservoir 32 a quantitative portion is rapidly loaded into pathway 34 by auto-loading means 35. The remainder of the sample is slowly trans-ported to a second reservoir 36 by draining means 37 where it is held leaving first reservoir 32 empty and a quantitative portion of the sample loaded into pathway 34. Autoloading means 35 is filling of a fixed volume by capillary action or electrokinetic pulse injection in the preferred embodiments. Other well known means by which a discrete quantitative portion of the sample is rapidly moved from first reservoir 32 into pathway 34, such as hydrodynamic or thermal injection, may also be used.
With reference to Fig. 7, an electrophoresis instrument 100 optically scanning a horizontally disposed cartridge 11 is shown. A strongly emitting light source, such as light emitting diode or laser 123 is used to generate a beam 125. LED 123 has an output power of - about 50 milliwatts and a wavelength band which will excite fluorescence in the fluorescent labeling material.
Such excitation radiation is known as actinic radiation.
The beam is intercepted by a focusing lens 127 which directs the beam through a slit aperture and barrier 129.
214~52~
WO94/19514 PCT~S94/02013 -Light emerging from the slit is divergent and is intercepted by a collimating lens 131. The beam is then directed onto a reflecting surface 133 which is part of a dichroic mirror 135.
Dichroic mirror 135 is chosen to selectively reflect light at the waveléngths emitted by light source 123 while transmitting light at the wavelengths emitted by the fluorescent label. The reflected beam is directed toward a focusing lens 137. Light passing through the focusing lens carries an image of the slit 129 which is directed onto capillary 14. The image of slit 29 can be scanned along the longitudinal axis of capillary 14 by moving separation cartridge 11 relative to lens 137.
Fluorescent light emitted by a label, and some reflected light from the capillary, travel in a retrobeam to focusing lens 137. Note that the focusing lens is used by light traveling in each direction. From there, the retrobeam is directed to reflecting surface 133 which is a part of dichroic mirror 135. Light reflected from the capillary is reflected toward light source 123 while fluorescent light is passed through. The fluorescent light is then directed by a mirror 141 through a filter 143 which rejects any light other than the desired wavelength from the fluorescent label. Light transmitted through the filter is directed toward a focusing lens 145. From there the beam is directed to a light detector, such as photomultiplier tube 147 with a slit located at the image plane of the separation medium. The focused locations of the label are measured relative to one end of the capillary.
The output of photomultiplier tube 147 is maintained in a buffer memory 149. A data reader 150 is connected to the buffer memory 149 for receiving recorded signals which represent the fluorescent peaks. The data reader is a computer which correlates the various peaks.
In multi-lane embodiments, one end of each of capillary tubes 14 may be located above a common thin-film electrode 16. In this embodiment each capillary 2144~i29 WO94/19514 PCT~S94/02013 tube 14 may be subjected to a separate voltage difference between each of electrodes 18 and the common voltage applied to electrode 16. Alternatively each capillary tube 14 may be positioned between a single pair of common electrodes and subjected to the same voltage difference.
The cartridge of the present invention can be used for capillary electrophoresis or capillary IEF. The conductive substance in the capillary tubes may be a liq-uid solution such as a buffer or biological fluid in capillary electrophoresis, or a mixture containing sample and ampholytes in IEF. The conductive substance may also be a gel. One advantage of the present invention is that the conductive substance is confined to a short capillary tube segment which is scanned in situ by the electropho-resis instrument when separation is complete. Adequateseparations have been accomplished in 30 mm long segments shortening analysis time. After scanning, the disposable cartridge containing all parts of the separation system which have come in contact with the sample may be discarded. Liquid waste generated by wash cycles is completely eliminated and the total volume of waste is drastically reduced.
Another advantage of the present invention is its ability to auto-load a quantitative sample volume when a bulk sample is applied to the cartridge. Slow competi-tive removal of material from the sample reservoir by a selectively absorbent material eliminates the need for a complicated auto-sampling system. This reduces the cost of the instrument as well as reducing the waste generat-ed.
Example 1 IEF of Cy5-HSA Utilizinq Differential Sample Loading Differential sample loading was accomplished by placing an absorbent material between the conductive film electrode and the underlying plastic support. Excess sample is removed from the conductive film after the 21~4529 WO94/19514 PCT~S94102013 sample has been quantitatively loaded into the capillary tube through a small hole in the conductive film. In IEF
the capillary tube is filled with a mixture of sample and ampholytes by capillary action. In CE a sample plug is electrokinetically injected into a buffer filled capillary tube.
Human serum albumin (HSA), fraction V, was obtained from Sigma Chemical Company (St. Louis, Missouri). Cy5 labeled HSA was synthesized by the coupling of Cy5 fluorescent dye (Biological Detection Systems, Inc., Pittsburgh, Pennsylvania) to HSA. Cy5-HSA
was added to a 2 percent solution of ampholytes having a pH range of 3 to 10 (Biorad Inc., Hercules, California) to a final Cy5-HSA concentration of 10 mg/ml. A capilla-ry (Polymicro Technologies Inc., Phoenix, Arizona), having an inside diameter of 100 microns and an outside diameter of 365 microns, was coated to prevent electroendosmosis (Capillary Electrophoresis, Academic Press, Inc., San Diego, California (1992), pp. 191-214).
The capillary was placed in the fixture described in Figs. 4 and 5. A double coated pressure sensitive adhesive film (RX264-S, Coating Sciences Inc., Bloomfield, Connecticut) was used to bond 601-25 cellulose blotting paper (Intermountain Scientific Corporation, Bountiful, Utah) to the underlying cartridge support structure 12. The conductive plastic film with adhesive on one side (AR clad 8010, Adhesive Research Inc., Glenrock, Pennsylvania) was bonded to the cellulose blotting paper. A hole with a diameter of 3/64 inch was punched through the conductive plastic film allowing direct access to the cellulose blotting paper beneath.
A drop (5 microliter) of the Cy5-HSA solution was placed upon the conductive film, concentric with the hole, and in contact with the end of the capillary.
Within two seconds the solution filled the capillary.
After thirty seconds, excess solution had completely wicked into the cellulose blotting paper. A drop (5 microliter) of 0.02 M sodium hydroxide, thickened to a WO94/19514 214 ~ 5 2 9 PCT~S94102013 viscosity of lO00 cp, was then placed upon the conductive film concentric~with the hole and in direct contact with the end of the capillary. A drop of 0.02 M phosphoric acid, thickened to a viscosity of lO00 cp, was applied to the other end of the capillary in similar fashion. These drops bridged the capillary to the cathode and the anode.
Electrophoresis was performed at l kV constant voltage for ten minutes with a CZE lO00 R high voltage supply (Spellman Corp., Plainville, New Jersey). Current was allowed to drop from 30 to 2 microamps as focusing took place. The position of the fluorescent proteins was determined at this point by scanning the capillary with a He-Ne laser optic system. A translation stage moved the cartridge relative to the optic system while the focusing field remained on. Reflected fluorescence was detected with a R928 PMT and the data was collected using data acquisition software on an IBM (trademark) personal computer.
Separation in this capillary system is based on the isoelectric points of the proteins. Results are shown as a plot of fluorescence versus distance on the capillary in Fig. 8. A single peak 41 corresponding to Cy5-HSA is focused. The excess solution in the cellulose blotting paper does not adversely affect the focusing of Cy5-HSA.
Exam~le 2 Detection of Proteins Present in Human Blood Creatine kinase is an enzyme present in various mammalian tissue. It occurs in three different forms - known as isoenzymes: CK-MM (skeletal), CK-MB (cardiac) and CK-BB (brain). After release from tissue and on cir-culation in blood the MM and MB forms themselves break-down to smaller fragments known as isoforms or subforms.
In the event of myocardial infarction, the MB isoenzyme, present in cardiac muscle, is released in the plasma.
Hence, it serves as a specific diagnostic molecular W0 94/195142 1 4~ 2 ~ PCT~S94102013 marker for cardiac ischemia or necrosis. The early and rapid detection of this isoenzyme and its isoforms are very crucial for the diagnosis of myocardial infarction and for initiating thrombotic therapy.
Separation of Cy5 labeled CK-MB antibody from its immune complex was performed using a capillary isoelectric focusing system. CK-MB2 (human heart) and monoclonal anti CK-MB were obtained from Biospecific (Emeryville, California). Fab fragments were prepared by digesting the monoclonal anti CK-MB with the enzyme papain. Cy5 labeled Fab was synthesized by the coupling of CyS fluorescent dye (Biological Detection Systems, Pittsburgh, Pennsylvania) to Fab and purified by conventional gel permeation and ion exchange methods.
This fluorescent substance is the labeled binding agent.
Differential separation assay (DSA) was done as follows: Cy5 labeled Fab (binding agent) was incubated with CK-MB2 (target) at a final concentration of 5 0 ~g/ml Cy5-Fab and 1 mg/ml CK-MB2 in 1 mM phosphate 15 mM NaCl pH 7 . 2 . A control sample consisted of Cy5-Fab alone at 5 0 ~g/ml without added CK-MB2. Reactions were performed in 1. 5 ml Eppendorf tubes in a total reaction volume of 10 ~1 . After incubating the samples at room temperature (2 0C) for 3 0 minutes, HSA was added as a carrier at a final concentration of 2 mg/ml. The reaction mixture was then diluted 3 0-fold with a 2% solution of ampholytes having a pH range of 3 to 10 (Biorad Inc., Hercules, California) in deionized water. Capillary action was used to fill a 50 x 0.3 x 0. 03 mm borosilicate glass 3o rectangular capillary (R&S Medical, Mountain Lakes, New Jersey), coated to suppress electroendosmosis (CaPillary ElectroPhoresis, Academic Press, Inc., San Diego, Cali-fornia (199 2), pp. 19 1-2 14), by dipping its end in the diluted reaction mixture. The capillary was then placed horizontally on an acrylic platform and platinum elec-trodes were bonded to the acrylic adjacent to the ends of the capillary. A drop of 0. 02 M sodium hydroxide, thickened to a viscosity of 100 cp, was applied to one WO94/19514 21~ ~ 5 2 3 PCT~S94/02013 -end of the capillary to bridge it with the cathodic electrode and a drop of 0.02 M phosphoric acid was applied to the other end of the capillary to bridge it with the anode. Electrophoresis was performed at 2 kV
constant voltage for lO minutes with a CZE lOOOR high voltage supply (Spellman, Plainville, New Jersey). The current was allowed to drop from 30 to 2 ~amps as the focusing took place.
The positions of the fluorescent proteins were determined at this point by scanning the capillary with a He-Ne laser optic system. A translation stage moved the capillary while the focusing field remained on. The re-flected fluorescence was detected with a R928 PMT and the data was collected using data acquisition software on an IBM ttrademark) personal computer.
Separation in this capillary system is based on the isoelectric points of the proteins. The results are shown as a plot of fluorescence versus distance on the capillary in Fig. 9. When Cy5-Fab alone is run a Cy5-Fab control peak 152 is focused at 18 mm. When CK-MB2 is present a second peak 154 corresponding to the immune complex consisting of Cy5-Fab/CK-MB2 is focused at 23 mm while a peak 156 corresponding to the residual uncomplexed labeled Cy5-Fab is focused at 15 mm.
Claims (25)
1. A single use cartridge for an electrophoresis instrument comprising:
a support structure;
at least one capillary tube horizontally disposed by said support structure, said capillary tube having longitudinally opposed ends; and electrodes formed on said structure adjacent to said capillary tube ends, said electrodes being planar electrically conductive films adapted for electrical contact with said capillary ends when said capillary tube is filled with a conductive substance, said conductive substance being statically confined in said capillary tube, whereby an electromotive force is produced along said capillary tube by connecting the pair of electrodes located at opposite ends of said capillary tube to an external voltage supply when said capillary tube is filled with said conductive substance.
a support structure;
at least one capillary tube horizontally disposed by said support structure, said capillary tube having longitudinally opposed ends; and electrodes formed on said structure adjacent to said capillary tube ends, said electrodes being planar electrically conductive films adapted for electrical contact with said capillary ends when said capillary tube is filled with a conductive substance, said conductive substance being statically confined in said capillary tube, whereby an electromotive force is produced along said capillary tube by connecting the pair of electrodes located at opposite ends of said capillary tube to an external voltage supply when said capillary tube is filled with said conductive substance.
2. The cartridge of claim 1 wherein said capillary tube is filled with a liquid solution.
3. The cartridge of claim 1 wherein said electrically conductive film is a homogenously conductive plastic.
4. The cartridge of claim 1 wherein electrical contact between said capillary tube and said electrode comprises a drop of an electrically conductive substance disposed at said capillary tube end on said electrode, said conductive substance having a viscosity sufficient to reduce hydrodynamic flow within said capillary tube.
5. The cartridge of claim 1 wherein said capillary tube is filled by capillary action when a liquid solution is placed on said electrode at said tube end.
6. The cartridge of claim 1 wherein said capillary is formed by ultrasonically welding two injection molded plastic parts together.
7. The cartridge of claim 1 wherein said capillary tube is less than six centimeters in length.
8. The cartridge of claim 1 wherein said capillary tube is permanently attached to said support structure.
9. The cartridge of claim 1 wherein said capillary tube has a high surface area to volume ratio.
10. The cartridge of claim 1 wherein said capillary tube is optically transparent.
11. The cartridge of claim 29 wherein said capillary tube is rectangular in cross-section with a large aspect ratio.
12. The cartridge of claim 1 wherein said capillary tube is formed by joining separate plastic parts together.
13. The cartridge of claim 1 wherein said conductive substance contains a surfactant.
14. A single use disposable cartridge for an electrophoresis instrument comprising:
a support structure;
at least one electrophoretic pathway supported by said structure;
a first reservoir adapted for application of a bulk sample, said first reservoir being in material communication with said pathway;
auto-loading means for quantitatively drawing a selected sample volume into said pathway from said first reservoir at a first rate;
a second reservoir in material communication with said first reservoir; and means for moving material from said first reservoir to said second reservoir at a second rate, said first rate being much faster than said second rate, whereby all remaining material in said first reservoir is moved into said second reservoir after said selected sample volume has been quantitatively loaded into said pathway.
a support structure;
at least one electrophoretic pathway supported by said structure;
a first reservoir adapted for application of a bulk sample, said first reservoir being in material communication with said pathway;
auto-loading means for quantitatively drawing a selected sample volume into said pathway from said first reservoir at a first rate;
a second reservoir in material communication with said first reservoir; and means for moving material from said first reservoir to said second reservoir at a second rate, said first rate being much faster than said second rate, whereby all remaining material in said first reservoir is moved into said second reservoir after said selected sample volume has been quantitatively loaded into said pathway.
15. The cartridge of claim 14 wherein said pathway has longitudinally opposed ends and said first reservoir is located at one of said ends.
16. The cartridge of claim 14 wherein said auto-loading means comprises the filling of a capillary segment by capillary action.
17. The cartridge of claim 14 wherein said second reservoir comprises an absorbent material in fluid communication with said first reservoir.
18. The cartridge of claim 14 further comprising electrodes formed on said structure adjacent said pathway ends.
19. The cartridge of claim 18 wherein said first reservoir is located between said pathway end and said adjacent electrode.
20. The cartridge of claim 19 wherein said adjacent electrode is spaced apart from said pathway end and said first reservoir comprises an open-sided gap between said pathway end and said adjacent electrode.
21. The cartridge of claim 19 wherein said electrically conductive film is a homogenously conductive plastic.
22. The cartridge of claim 19 wherein said pathway is a horizontally disposed capillary tube, said capillary ends being disposed above said electrodes.
23. The cartridge of claim 22 wherein said capillary is optically transparent.
24. The cartridge of claim 22 wherein said capillary tube is rectangular with a large aspect ratio.
25. The cartridge of claim 14 wherein said pathway has longitudinally opposed ends further comprising electrodes formed on said structure, said electrodes being disposed adjacent to said ends and said first reservoir, and wherein said auto-loading means comprises electrokinetic injection.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/023,088 US5338427A (en) | 1993-02-26 | 1993-02-26 | Single use separation cartridge for a capillary electrophoresis instrument |
| US023,088 | 1993-02-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2144529A1 true CA2144529A1 (en) | 1994-09-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002144529A Abandoned CA2144529A1 (en) | 1993-02-26 | 1994-02-16 | Single use separation cartridge for a capillary electrophoresis instrument |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US5338427A (en) |
| EP (1) | EP0664841A4 (en) |
| CA (1) | CA2144529A1 (en) |
| WO (1) | WO1994019514A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001229A (en) * | 1994-08-01 | 1999-12-14 | Lockheed Martin Energy Systems, Inc. | Apparatus and method for performing microfluidic manipulations for chemical analysis |
| US5627041A (en) * | 1994-09-02 | 1997-05-06 | Biometric Imaging, Inc. | Disposable cartridge for an assay of a biological sample |
| US5560811A (en) * | 1995-03-21 | 1996-10-01 | Seurat Analytical Systems Incorporated | Capillary electrophoresis apparatus and method |
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-
1993
- 1993-02-26 US US08/023,088 patent/US5338427A/en not_active Expired - Lifetime
-
1994
- 1994-02-16 WO PCT/US1994/002013 patent/WO1994019514A1/en not_active Application Discontinuation
- 1994-02-16 EP EP94909789A patent/EP0664841A4/en not_active Withdrawn
- 1994-02-16 CA CA002144529A patent/CA2144529A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| US5338427A (en) | 1994-08-16 |
| WO1994019514A1 (en) | 1994-09-01 |
| EP0664841A4 (en) | 1995-09-06 |
| EP0664841A1 (en) | 1995-08-02 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |