CROSS-REFERENCE TO RELATED APPLICATIONS
FIELD OF THE INVENTION
This application is a continuation-in-part application of U.S. patent application Ser. No. 10/338,451, filed Jan. 8, 2003, entire contents of which are incorporated herein by reference.
- BACKGROUND OF THE INVENTION
The invention is related to a method using a self-contained biochip that is preloaded with necessary reagents, and utilizes microfluidic and micro-pressure actuator mechanisms to perform biological reactions and assays, particularly for gene expression profiling or screening of candidate genes for a genetic study. The biochip analysis apparatus is configured rapidly and automatically measuring the quantities of chemical and biological species in a sample.
Current hospital and clinical laboratories are facilitated with highly sophisticate and automated systems with the capability to run up to several thousand samples per day. These high throughput systems have automatic robotic arms, pumps, tubes, reservoirs, and conveying belts to sequentially move tubes to proper position, deliver the reagents from storage reservoirs to test tubes, perform mixing, pump out the solutions to waste bottles, and transport the tubes on a conveyer to various modules. Typically three to five bottles of about 1 gallon per bottle of reagent solutions are required. While the systems are well proved and accepted in a laboratory, they are either located far from the patients or only operated once numerous samples have been collected. Thus, it often takes hours or even days for a patient to know their test results. These systems are very expensive to acquire and operate and too large to be used in point-of-care testing setting.
The biochips offer the possibility to rapidly and easily perform multiple biological and chemical tests using very small volume of reagents in a very small platform. In the biochip platform, there are a couple of ways to deliver reagent solutions to reaction sites. The first approach is to use external pumps and tubes to transfer reagents from external reservoirs. The method provides high throughput capability, but connecting external macroscopic tubes to microscopic microchannel of a biochip is challenging and troublesome. The other approach is to use on-chip or off-chip electromechanical mechanisms to transfer self-contained or preloaded reagents on the chips to sensing sites. While on-chip electromechanical device is very attractive, fabricating micro components on a chip is still very costly, especially for disposable chips. On the other hand, the off-chip electromechanical components, facilitated in an analysis apparatus, that are able to operate continuously for a long period of time is most suited for disposable biochip applications.
U.S. patent application publication 2002/0124879, entire contents of which are incorporated herein by reference, discloses a device for moving a fluid in a fluidics system. The device may include one or more controllably openable closed chambers. The pressure within the closed chambers is lower than the ambient pressure outside the fluidics system or lower than the pressure within another channel of the fluidics system. The closed chamber is configured for being controllably opened. The chamber is configured such that when a chamber is opened the chamber is in fluidic communication with a flow channel included within the fluidics system. The fluid may be moved into the flow channel or may be moved within the flow channel. The fluid may be a liquid, a gas, a mixture of gases or an aerosol. The fluidics system may include a controller for controlling the opening of a selected chamber or chambers.
U.S. patent application publication 2002/0187560, entire contents of which are incorporated herein by reference, discloses a microfluidic device capable of combining discrete fluid volumes generally including channels for supplying different fluids toward a sample chamber and means for establishing fluid communication between the fluids within the chamber. Certain embodiments utilize adjacent chambers or subchambers divided by a rupture region such as a frangible seal. Further embodiments utilize one or more deformable membranes and/or porous regions to direct fluid flow. Certain devices may be pneumatically or magnetically actuated.
European Patent No. 1203959, entire contents of which are incorporated herein by reference, discloses an analyzing cartridge for use in analysis of a trace amount of sample, enabling analysis and detection to be carried out conveniently, and a method for producing the same. The invention also provides an analysis method using the analyzing cartridge and a liquid feed control device that is attached to the analyzing cartridge, and controls the feeding of liquid in the analyzing cartridge.
The microfluidics-based biochips have broad application in fields of biotechnology, molecular biology, and clinical diagnostics. The self-contained biochip, configured and adapted for insertion into an analysis apparatus, provides the advantages of compact integration, ready for use, simple operation, and rapid testing. For microfluidic biochip manufacturers, however, there are two daunting challenge. One of the challenges is to store reagents without losing their volumes over product shelf life. The storage cavity should have a highly reliable sealing means to ensure no leak of reagent liquid and vapor.
Although many microscale gates and valves are commercially available to control the flow and prohibit liquid leakage before use, they are usually not hermetic seal for the vaporized gas molecules. Vapor can diffuse from cavity into microchannel network, and lead to reagent loss and cross contamination. The second challenge is to deliver a very small amount of reagents to a reaction site for quantitative assay. The common problems associated are air bubbles and dead volume in the microchannel system. An air bubble forms when a small channel is merged with a large channel or large reaction area, or vice versa. Pressure drops cause bubble formation. The air bubble or dead volume in the microfluidic channel can easily result in unacceptable error for biological assay or clinical diagnosis.
Several prior art devices have been described for the performance of a number of microfluidics-based biochip and analytical systems. U.S. Pat. No. 5,096,669 discloses a disposable sensing device with special sample collection means for real time fluid analysis. The cartridge is designed for one-step electrical conductivity measurement with a pair of electrodes, and is not designed for multi-step reaction applications. U.S. Pat. No. 6,238,538 to Caliper Technologies Corp. discloses a method of using electro-osmotic force to control fluid movement. The microfabricated substrates are not used for reagent storage. U.S. Pat. No. 6,429,025 discloses a biochip body structure comprising at least two intersecting microchannels, which source is coupled to the least one of the two microchannels via a capillary or microchannel. Although many prior art patents are related to microfluidic platform, none of them discloses liquid sealed mechanism for self-contained biochips. They are generally not designed for multi-step reactions application.
Furthermore, since the advent of microarrays in the mid 1980s, investigators have continued to manually perform individual steps in microarray experiments, including DNA/RNA probe synthesis, labeling, hybridization, post-hybridization washes, drying, and scanning. Microarrays are generally printed on 25 mm×75 mm microscopic glass or plastic slides. Hybridization is performed using a glass cover slide or dedicated chambers, whereas subsequent washing and drying of the slides takes place in separate dishes. Unfortunately current microarray systems suffer from the consumption of massive amounts of material and time, and from run-to-run variations in data as a result of error prone manual steps (about 15-20 steps). Manual steps may include pipetting of reagents into different tubes and transfer of reagents between tubes and steps, which also increases the risk of contamination. Moreover, inconsistent data might not be corrected by sophisticated normalization algorithms, thus leading to misinterpretations of the underlying biology.
DNA microarrays, especially for expression profiling, have proven to be a very useful tool in molecular biology. There is no doubt about the impact of this method on implementing more accurate and faster diagnostics of complex genetic diseases, like cancer and immunological disorders, and fostering the development of more specific drugs and therapies that are tailored to patient subtypes. Clinical diagnostics is extremely dependent on the confidence of data replication. The disadvantages of the prior microarray systems may include the following, such as low confidence as a research or diagnostic tool due to the high risk of contamination, inconsistency of data due to a large number of error prone manual handling steps and solution transfers, inconvenient and time consuming manual processing steps, large modular or bench top instruments that are not easily accessible or portable, complicated operational procedures that demand highly trained personnel, or requiring the use of a relatively expensive platform.
U.S. Pat. No. 6,618,679, entire contents of which are incorporated herein by reference, discloses methods, compositions and kits for gene expression analysis and gene expression profiling. The method for analyzing gene expression comprises: a) obtaining a plurality of target sequences, wherein the plurality of target sequences comprises cDNA; b) multiplex amplifying the plurality of target sequences, wherein multiplex amplifying comprises combining the plurality of target sequences, a plurality of target-specific primers, and one or more universal primers, and wherein the universal primer is provided in an excess concentration relative to the target-specific primer, thereby producing a plurality of amplification products; c) separating one or more members of the plurality of amplification products; d) detecting one or more members of the plurality of amplification products, thereby generating a set of gene expression data; e) storing the set of gene expression data in a database; and f) performing a comparative analysis on the set of gene expression data, thereby analyzing the gene expression.
U.S. Pat. No. 6,816,790, entire contents of which are incorporated herein by reference, discloses a method for determining a concentration level of a target nucleic acid, the target nucleic acid comprising at least one target oligonucleotide. The method determines (i) a measure of affinity value of the target oligonucleotide with a probe oligonucleotide; and (ii) a hybridization intensity value for the target oligonucleotide and the probe oligonucleotide at a probe spot. The measure of affinity value and the hybridization intensity value are used to determine the concentration level of the target nucleic acid.
- SUMMARY OF THE INVENTION
It is one object of the present invention to provide a method for gene expression profiling via a miniaturized rapid and automated Microarray Expression Profiling Chip (mXP-CHIP) and system.
In accordance with preferred embodiments of the present invention, a self-contained microfluidic disposable biochip is provided for performing a variety of chemical and biological analyses. The disposable biochip is constructed with the ability of easy implementation and storage of necessary reagents over the reagent product shelf life without loss of volume.
Another object of this invention is to provide a ready to use, highly sensitive and reliable biochip. Loading a sample and inserting it into a reading apparatus are the only required procedures. All the commercially available point of care testing (POCT) analyzers have poor sensitivity and reliability in comparison with the large laboratory systems. The key problem associated with a POCT is the variation in each step of reagent delivery during multiple-step reactions. Especially, the problems are occurred in closed confinement. For example, a common sandwiched immunoassay, three to six reaction steps are required depending on the assay protocol and washing process. Each reaction requires accurate and repeatable fluids volume delivery.
Another object of this invention is to provide the ability of a biochip with the flexibility for performing a variety of multi-step chemical and biological measurements. The disposable biochip is configured and constructed to have the number of reagent cavities matching the number of assay reagents, and the analysis apparatus performs multiple reactions, one by one, according to the assay protocol.
Another object of this invention is to provide a biochip that can perform multi-analyte and multi-sample tests simultaneously. A network of microfluidic channel offers the ability to process multiple samples or multiple analytes in parallel.
Another object of this invention is to mitigate the problems associated with air bubble and dead volume in the microchannel. The air bubble or dead volume in the microfluidic channel easily results in unacceptable error for biological assay or clinical diagnosis. This invention is based on a microfluidic system with a reaction well, which has an open volume structure and eliminates the common microfluidic problems.
The present invention with preloaded biochips has the advantages of simple and easy operation. The resulting analysis apparatus provides accurate and repeatable results. It should be understood, however, that the detail description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not of limitation. Further, as is will become apparent to those skilled in the area, the teaching of the present invention can be applied to devices for measuring the concentration of a variety of liquid samples.
Some aspects of the invention relate to a method for analyzing gene expression profiling comprising steps of: a) procurement means for simultaneous nucleic acid synthesis; b) treatment means for nucleic acid amplification and labeling of at least one nucleic acid element; and c) analyzing means for at least single-color hybridization to an integrated array of spotted bio-molecules (such as nucleic acids, peptides, protein-nucleic acids, carbohydrates and lipids).
In a further embodiment, all steps are performed in an apparatus comprising a self-contained disposable microfluidic biochip for performing multi-step reactions, the biochip comprising: a body structure comprising a plurality of reagent cavities and at least one reaction well connected via microfluidic channels, the reagent cavities each storing a reagent and each comprising a breakable seal allowing the reagent to be selectively released into the reaction well upon being punctured. In one embodiment, all steps are performed in the apparatus for out-lab use in a remote area.
In a further embodiment, the microfluidic channels of the biochip have a dimension between about 0.1 μm and 2 mm in cross section, preferably a dimension between about 1 μm and 50 μm in cross section.
In a further embodiment, the surface of the microfluidic channels of the biochip is treated with a surface tension reducing agent.
In a further embodiment, the reaction well is facilitated with at least one biological probe that is selected from a group consisting of proteins, nucleic acids, receptors, and cells. In one embodiment, the biochip comprises a plurality of reaction wells, each in flow communication with the plurality of reagent cavities.
In a further embodiment, the apparatus further comprises a vacuum suction for removing waste from the reaction well or a sampling port.
In a further embodiment, at least one of the reagent cavities comprises a second reagent stored in a chamber with a second breakable seal, whereby the second reagent flows and interacts with any other reagent in a reagent cavity when the second breakable seal is punctured. In one embodiment, the seal comprises a thin film located at the bottom of each reagent cavity for preventing reagent escape, and wherein each reagent cavity comprises a microcap assembly located at the top of each reagent cavity, a pin being provided at adjacent the film configured for puncturing the film. In another embodiment, the apparatus further comprises a microactuator, wherein the microactuator and the biochip are supported for motion relative to each other adapted for positioning the microactuator at each of the microcap assembly, wherein the microactuator is structured and configured to deliver a downward pressure to the microcap assembly.
Some aspects of the invention relate to a method of screening of candidate genes for a genetic study comprising steps of: a) procurement means for simultaneous nucleic acid synthesis; b) treatment means for nucleic acid amplification and labeling of at least one nucleic acid element; and c) analyzing means for at least single-color hybridization to an integrated array of spotted bio-molecules (such as nucleic acids, peptides, protein-nucleic acids, carbohydrates and lipids), wherein all three means are performed in a self-contained disposable microfluidic biochip apparatus, the apparatus comprising: a body structure comprising a plurality of reagent cavities and at least one reaction well connected via microfluidic channels, the reagent cavities each storing a reagent and each comprising a breakable seal allowing the reagent to be selectively released into the reaction well upon being punctured.
BRIEF DESCRIPTION OF THE DRAWINGS
In a further embodiment, the genetic study is selected from a group consisting of a research of immune and infectious diseases, a research of drug target identification and validation, and a research of identification of a threat agent selected from a group consisting of bacteria, viruses, germs, enzymes, fungi, and combination thereof.
FIG. 1 shows a top view of a self-contained biochip with microfluidic channel connecting reagent cavities and reaction wells.
FIG. 2. shows an exploded top view of the three separate layers of the biochip, showing: (a) a reagent layer, (b) a microchannel layer, and (c) a reaction well layer.
FIG. 3 shows a cross section view of the biochip with microcap assembly and microfuidic channel, taken along line 3-3 in FIG. 1, showing the following sequence of operations: (a) before and (b) after the reagent is released from the reagent cavity and into microfluidic channels and reaction wells driven by a microactuator; the microcap assembly with a stopper and a pin is designed to reliably pierce the sealing thin film and open the cavity; and (c) the residual reagent in the reaction well is withdrawn via the waste port by a vacuum line.
FIG. 4 shows a section view of the self-contained biochip with a four-layer structure for dry reagent, showing the following sequence of operations: (a) the buffer solution and dry reagents are sealed in the separate cavities; (b) the first thin film is pierced, and the reagent buffer is moved into the dry reagent cavity and dissolves the dry reagent; and (c) the second thin film is pierced, and the reagent solution is released from the cavity into the microfluidic channels, and reaction wells.
FIG. 5(a) and (b) show the schematic diagrams of biochip based analytical apparatus including a pressure microactuator, vacuum line, and optical detector.
FIG. 6 shows an example of self-contained chip for chemiluminescence-based sandwich immunoassay protocol, showing the following states of the flow and reaction processes: (A) before and (B) after deliver the sample to the reaction wells; (C) wash away the unbound, and deliver the label conjugates; (D) wash away the unbound, and deliver the luminescent substrate.
FIG. 7 shows an mXP-CHIP (Microarray Expression Profiling Chip) microfluidic system for gene expression profiling.
FIG. 8 shows a laborious process of conventional microarrays (prior art).
FIG. 9 shows a schematic illustration of the processing steps for gene expression analysis using DNA microarrays.
FIG. 10 shows a comparison of differential expression of genes in human kidney and the universal human reference RNA (Stratagene).
FIG. 11 shows a topical view of the mXP-CHIP device.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
FIG. 12 shows the pressure-driven microfluidics for total automation.
This invention is described in various embodiment in the following description with reference to the figures. While this invention is described in terms of the best mode for achieving this invention's objectives, it will be appreciated by those skilled in the art that variations may be accomplished in view of these teachings without deviating from the spirit or scope of the invention. This description is made for the purpose of illustrating the general principles of the invention and should not be taken in a limiting sense. The scope of the invention is best determined by reference to the appended claims.
The pattern of the self-contained microfluidic biochip is designed according to the need of the assay and protocol. For example, the chip (FIG. 1) consists of 6 sets of microfluidic pattern; it depends on the number of analyte and on-chip controls. Each set includes multiple (for example 6) reagent cavities 11, a reaction well 13, a waste port 14, and a network of microfluidic channel 12. The sample can be delivered into individual reaction wells directly or via a main sample port 15 for equal distribution to the reaction wells 13, for example under centrifugal forces by spinning the biochip in an analytical apparatus as discussed below in connection with FIG. 5. The biochip body structure comprises a plurality of reagent cavities and reaction wells via microchannels. The chip has a three-layer composition: (shown in FIG. 2) (a) the top layer is a reagent layer 30, (b) the middle layer is a microchannel layer 31, and (c) the bottom layer is a reaction well layer 32.
The reagent cavities 11 formed in the reagent layer 30 allow for the storage of various reagents or buffer solutions. The microchannel layer contains a network of microfluidic channels 36 are patterned on the bottom side of the layer. The microchannel layer and the reaction well layer form microfluidic channels, which connect the reagent cavities to reaction wells and to the waste port. The reaction well layer has a number of microwells, which are able to hold sufficient volume of samples or reagents for reactions. Reagent sealing means (shown in FIG. 3), which includes a thin film 33 located at the bottom of the reagent cavity and a microcap assembly 20 located at the top of the cavity, confines the reagent 25 in the reagent cavity. The thin film is breakable and is adhered to the reagent layer and the microchannel layer. The microchannel layer and reaction well layer are bonded by either chemical or physical methods. By way of illustrations, the various plastic layers may be bonded by applying ultrasonic energy, causing micro-welding at the adjoining interfaces.
The microfluidic biochip can be fabricated by soft lithography with polydimethyl siloxane (PDMS) or micro machining on plastic materials. PDMS-based chips, due to small lithographic depths, have volume limitations (<5 μl). When clinical reagents on the order of 5 μl to 500 μl, the layers are fabricated by micro machining plastic materials. The dimension of the reagent cavity could be easily scaled upward to hold sufficient volumes of clinical samples or reagents. Soft lithography is best suited for microfabrication with a high density of microfluidic channels. But its softness properties and long-term stability remain a problem for clinical products. Therefore, the chip is preferably fabricated by micro machining on plastic materials. The linear dimension of a microfluidic channel is on the order of about 0.1 μm-2 mm, preferably between about 1 μm and 50 μm in cross section. In one embodiment, the plastic chips may be made of multi-layer polystyrene and polyacrylic. The biochip apparatus may further comprise a surface of the microfluidic channels that is treated with a surface tension reducing agent, such as fluorinated material, hydrophilic material, wetting agent or the like. In a preferred embodiment, the surface tension reducing agent does not interfere with the microfluidic operations. Micro machining chips can scale up the cavity dimension easily. It can be mass-produced by injection mold as a disposable chip.
Referring also to FIG. 5 b, the chip is placed on a rotational stage such as supported on a turntable (not shown) or on a spindle drive (not shown) connected to a motor (not shown), which positions a specific reagent cavity under a microactuator 42. All reagents are pre-sealed or pre-capped in reagent cavities. The microcap assembly is fabricated inside the reagent cavity to perform both capping and piercing. A pressure-driven microactuator controls the microfluidic kinetics. The microcap assembly has two plastic pieces: a pin 21 and a stopper 22. In the operation, the actuator engages with the assembly and pushes the element downward. The pin pierces through the thin film and opens the cavity. Then, the stopper is depressed downward to the bottom of the well. The stopper stays at the bottom of the well to prevent backflow. By this method, the microcap assembly opens the cavity as a valve 29 and let the reagent flow into the microfluidic channel. The configuration also prevents causing internal pressure build-up. The microactuator works like a plastic micro plunger or syringe, is simple, rugged, and reliable. The movement of fluid is physically constrained to exit only through the microchannel and to the reaction well. A single actuator can manage a whole circle of reagent cavities.
After delivering the sample into the sample port or into one of the reaction well, the system sequentially delivers reagents one at a time into the reaction well and incubates for a certain time. The reaction well may be provided with a rubber cap 27 to prevent contamination by the environment, and the sample may be delivered directly into the reaction well by a probe piercing through the rubber cap 27, or via the sample port 15 at the center of the biochip. There is a large volume of air space 28 above the reaction well. With this design, air is allowed into the microfluidic system. No bubble is trapped in the microfluidic channel system. In practice, the actuator can also utilize the spare air in the reagent cavity to displace all of the residual liquid left in the microchannel into the reaction well, where there is plenty of air space. Therefore, the common problems associated with microfluidic systems, such as air bubbles, dead volumes, inhomogeneous distribution, and residual liquid left in the microfluidic channel, will not occur or affect the outcome of the test results. After the reaction, the residual reagent is removed away to an on-chip or off-chip waste reservoir. For example, a vacuum line 45 is situated atop the waste port 14 via a vented hole 46 to withdraw small volume of liquid from the reaction well.
The pre-loaded biochip is prepared and ready for use after shipment to the user. Therefore, the reagents, such as enzyme labeled antibody, should be stable for a long period (for example, 1 to 2 years or longer at room temperature). In their liquid form, many biological reagents are unstable, biologically and chemically active, temperature sensitive, or chemically reactive with one another. Because of these characteristics, the chemicals may have a short shelf life, may need to be refrigerated, or may degrade unless stabilized. Therefore some reagents are preferred to be stored in the dried form. One of dry reagent preparation methods is lyophilization, which has been used to stabilize many types of chemical components used in in-vitro diagnostics.
Lyophilization gives unstable chemical solutions a long shelf life when they are stored at room temperature. The process gives product excellent solubility characteristics, allowing for rapid liquid reconstitution. The lyophilization process involved five stages: liquid, frozen state, drying, dry, and seal. The technology that allows lyophilized beads to be processed and packaged inside a variety of containers or cavities. In the case when dry reagents are involved, the chip (shown in FIG. 4) has a four-layer composition: a reagent buffer layer 51, a dry reagent layer 52, a microchannel layer 31, and a reaction well layer 32. The reagent buffer layer with its patterned microwells allows for the storage of liquid form of reagents buffer 50 in individual wells. Buffer solutions are stable for a long period time.
The dry reagent layer contains dry reagent 54 in the dry reagent cavity 55 for rapid liquid reconstitution. When the actuator engages with the microcap assembly, it pushes the pin downward. The pin pierces through the first thin film 53, dissolves the dry reagent into buffer solution. Then the second thin film 56 is pierced, and the stopper is continuously depressed downward to the bottom of the cavity and forces the reagent mixture into the microchannel. Reactions take place in the reaction wells (not shown in FIG. 4) which are similar in structure to that shown in FIG. 3. The waste reagents may be removed by vacuum suction in a similar manner as the previous embodiment. While FIG. 4 illustrates a particular embodiment in which a second, dry reagent is deployed, it is well within the scope and spirit of the present invention to deploy a second, wet reagent in place of the dry reagent. Further, it is contemplated that there could be provision for more than two reagents, comprising a combination of dry and/or wet reagents.
While the embodiments are described in reference to one level of reaction using reagents delivered from multiple reagent cavities to a single reaction well, it is within the scope and spirit of the present invention that the biochip may be configured to perform two or more tiers of reactions in two or more reaction wells coupled in series by micro-channels. The reaction products from one or more reaction wells are feed into another reaction well (e.g., by pressurization using a plunger means (not shown) at the first reaction well or by centrifuging by spinning the biochip to cause the reaction products to move from one reaction well to another reaction well in series), where further reactions (i.e., a second tier of reactions) may take place using additional reagents from additional reagent reservoirs.
The analytical apparatus (as shown in FIG. 5 a and FIG. 5 b) includes a pressure-driven microactuator 42, vacuum line 45, detector 48, electronics, and microprocessor 72 for protocol control and data processing. The biochip may be supported on a turntable (not shown), or on a drive spindle (not shown) connected to a motor (not shown). Such details have been omitted from the schematic diagram in FIG. 5 b, so as not to obscure the present invention, but are well within the ability of a person in the art, given the present disclosure of the functions and features of the present invention. The microactuator 42 and vacuum line 45 may be actuated using linear actuators built with a motor operated lead screw that provides for linear movement force. The microactuator has a 5˜10 mm travel distance to press the microcap assembly to break the sealing film and push liquid into the microfluidic channel.
For certain applications, such as the enzyme-linked immunosorbent assay (ELISA) or fluorescence assay, a light source 47 can be implemented. No external light source is required for chemiluminescence or bioluminescence detection. However, other detection schemes may require a light source 47. The detector is one of the key elements that define the detection limit of the system. Depending on the sensitivity requirement, many detectors can be selected to be used. For example, the optical detector 48 may comprise a photodiode or photomultiplier tube (PMT), that measures the change of absorption, fluorescence, light scattering, and chemiluminescence 70 due to the probe-target reactions.
The photon counting photomultiplier tube has a very high amplification factor. This detector incorporates an internal current-to-voltage conversion circuit, and is interfaced with a microprocessor unit that controls the integration time. This detector has a very low dark count and low noise. The detector is packaged as part of a light tight compartment and is located either at the bottom or top of the transparent reaction well. One detector is sufficient to scan all reaction wells on the rotational stage. A collecting lens can be used to improve light collection efficiency. Arrangement of the reaction wells should minimize cross talk signals. A narrow band optical filter ensures detection of luminescence. The output of the detector is interfaced to a signal processor, which may be implement within the apparatus shown in FIG. 5 b, or externally in a notebook computer or a digital meter. The optical signal corresponds to an analyte concentration, for example. Depending on the types of reactions undertaken, other types of detection schemes may be implemented without departing from the scope and spirit of the present invention. For example, electro-conductivity detection may be implemented using probes (not shown) inserted into the reaction mixture in the reaction well. The analytical apparatus may also include a probe (not shown) that can be positioned for injecting a sample into the sample port 15 on the biochip.
The control sequence for the various device components of the analytical apparatus may be configured in accordance with the desired reaction and reagent requirements.
- 2. Dispense washing solution from a reagent cavity to the reaction well; then remove the unattached analyte or residual sample from the reaction well to the waste reservoir.
- 3. Move the label conjugate from the reagent cavity to the reaction well and incubate it; then remove the unattached conjugate to the waste reservoir.
- 4. Wash the reaction sites two or three times with washing solutions from reagent cavities to remove unbound conjugates; then remove the unattached conjugate to the waste reservoir.
- 5. Deliver chemiluminescence substrate solution 64 to the reaction well.
- 6. The reaction site would start to emit light only if the probe-target-label conjugate complex 69 formed. The signal intensity is recorded. The detector scans each reaction well with an integration time of 1 second per reading.
Chemiluminescence occurs only when the sandwich immuno-complex 69 ((e.g. Ab—Ag—Ab*), positive identification) is formed. The labeling enzyme amplifies the substrate reaction to generate bright luminescence 70 for highly sensitive detection and identification.
Microarray Genomic Profiling
Some aspects of the present invention relate to rapid and automated Microarray Expression Profiling Chip (mXP-CHIP), system, and methods for gene expression profiling. Microarray genomic profiling is a powerful tool in molecular characterization of diseases, but remained restricted to research, having not yet made a breakthrough as a diagnostic tool in disease identification and drug development. This is due to current time and labor consuming macro-scale platforms and the inconsistencies in generating results. In one embodiment, the mXP-CHIP system provides a miniaturized and automated system to integrate all steps of probe synthesis, labeling, and hybridization, configured and adapted for reducing the risk of contamination to a minimum and significantly decreasing assay costs due to the consumption of lower reagent volumes and shorter assay times. Microarray assays would then be performed with higher confidence and cost-efficiency for the screening of target genes in the academic research of genetic, immune and infectious diseases, and for relevant drug target identification and validation in the pharmaceutical industry.
Microfluidic technology, by nature, shows excellent advantages for integrating the multiple steps of the DNA microarray process. FIG. 7 shows an mXP-CHIP microfluidic system for gene expression profiling, that utilizes parallel processing, microfabrication and microfluidic technologies in order to provide biochemistry automation in an about 3″ compact disc package. In one embodiment, the integrated probe generation and hybridization within microarrays may be up to 30,000 spots into one single sterilized device. The need for a network of tubing connected to external reservoirs and pumps of a prior system is eliminated. The mXP-CHIP system utilizes a simple pressure driven microactuator, vacuum line, and heating elements in order to control fluid dynamics and regulate biochemical reactions configured and adapted for reducing multistep reactions to a single step. The mXP-CHIP and system integrates all the steps of probe synthesis, labeling and purification, probe hybridization to a microarray, post-hybridization washes and drying so that the array is ready for scanning.
FIG. 7 shows a system 80 comprising a minituarilized compact, automated, self-contained microarray expression profiling chip 81 (mXP-CHIP) for automated inline RNA probe processing from synthesis to hybridization. The mXP-CHIP is equipped with a centered hybridization well 82 (HYB) that contains a whole human genome microarray 83 of DNA spots (20,000-30,000 genes). The system integrates all the required reagents in the surrounding microwells and the steps of probe synthesis, labeling, post-labeling purification, dual-color hybridization (magnified section 84 of the array in the right upper corner), and post-hybridization processing to the point that the arrays would be ready for scanning. The mXP-Chip utilizes parallel processing, microfabrication and integrated microfluidic technologies to provide biochemistry automation in a small disc plastics package.
FIG. 8 shows a prior art illustration of a laborious process of conventional microarrays. The prior art microarrays may include steps of: labeled probe is generated by (step 1) mixing the reaction components together in a tube and (step 2) incubating in a programmable thermocycler; (step 3) after cRNA synthesis and labeling, the solution is transferred to a silicagel-column, which purifies labeled cRNA from enzymes and unincorporated dye molecules via centrifugation; (step 4) the collected purified cRNA is then heat-fragmented in the thermocycler and (step 5) then applied to the array in the hybridization chamber; (step 6) hybridization takes place in an oven at 60° C. under gentle rotation; (step 7) post-hybridization washes are performed consecutively in 2-3 jars with washing solution of increasing stringency, including transfer of the slides between the jars; and (step 8) the slides are then blow-dried with a nitrogen gun or spin-dried in a centrifuge before being scanned.
Many firms and research institutes are actively engaged in developing microfluidic technologies. Currently there are commercialized stations (for example, Genomic Solutions, by Perkin Elmer, Boston, Mass. and Gene Chips™ by Affymetrix, Santa Clara, Calif.) that only perform hybridization/washing. These devices are expensive to procure & maintain, and do not integrate the important steps of probe generation (i.e. cRNA synthesis and labeling as well as cRNA purification and array hybridization) into the automated process. Thus gene expression profiling remains laborious and susceptible to inconsistent manual operations with these commercial stations.
During the mXP-CHIP assay procedure, the actuator sequentially aliquots a pocket of solution from one microwell to another, initiates enzymatic and non-enzymatic chemical reactions, rinses off unbound reagents, and removes excess solution into a waste reservoir with the assistance of the vacuum line. FIG. 12 shows a topical view of the mXP-CHIP, wherein the mXP-CHIP's unique design is not affected by common microfluidic problems, such as dead volumes, air bubbles, or residual liquid inside the microfluidic system. These problems have hampered the technology from becoming commercially viable for clinical diagnostics in the past. The application of a geometrically defined system, in combination with defined input volumes, decreases reagent handling variations within the steps of probe generation and hybridization. Even more importantly, this system is sized and configured for gene expression profiling, whereby the chip can process multiple probes at the same time and subsequently perform dual-color hybridization.
- EXAMPLE NO. 1
FIG. 9 shows a schematic illustration of the processing steps for gene expression analysis using DNA microarrays. The procedure starts from minute amounts of extracted and purified total RNA (5-10 ng) as sources for probe generation, i.e. cDNA synthesis 86, followed by cDNA amplification/aminoallyl labeling 87, fluorescent dye coupling 88, hybridization 89 to an array of DNA target elements, subsequent washing 90 of unbound material, drying of the array & scanning 91 and data acquisition by image analysis.
The system as shown in FIG. 7 is designed and configured to simultaneously synthesize and label probes from two samples and hybridize the two probes to an integrated array of 400 spotted DNA elements. These elements would represent 400 well characterized unique genes and are selected so that according to preliminary data (FIG. 10), ⅔ of the genes are known to be highly expressed between normal human kidney (Asterand, Detroit, Mich.) and the Universal Human Reference RNA™ (Stratagene, La Jolla, Calif.). While ⅓ are known to show a low to no differential expression (between 0.5- and 1.5-fold) between the two specimens. The expression profiles have been generated using the Agilent Human 1A Oligo Microarray™ (22K array) and the Agilent platform for probe synthesis, hybridization and scanning. The slight bias of the data points towards the Reference RNA is caused by the fact that the Universal RNA is a pool of total RNAs of 10 different human tissues. Thus for the majority of the genes displayed, the sum of gene expression in the pool is higher than in kidney alone.
- EXAMPLE NO. 2
In one embodiment, gene expression profiling using the mXP-CHIP system is comprised of three major processing steps: 1) cDNA synthesis, 2) fluorescent dye coupling, and 3) hybridization. The whole process starts with probe synthesis from 1-5 ng of purified total RNA from normal human kidney (Asterand) and the Universal Human Reference RNA™ (Stratagene). The probe generation starts with (A) a reverse transcription from minute amounts of total RNA (5-10 ng) using a chimeric RNA/DNA-primer (using the Ovation Nanosample Amplification™ kit of Nugen (San Carlos, Calif.)), (B) the formation of double-stranded cDNA, and (C) an isothermal step, in which cDNA is linearly amplified while incorporating aminoallyl-dUTP. The rapid accumulation of cDNA (5-10 μg) is the result of a cascade of subsequent reactions taking place: primer annealing, cDNA strand synthesis by DNA polymerase, strand displacement and RNA cleavage of the chimeric primer by RNase H. The aminoallyl-labeled cDNA can then be coupled to amine-reactive Cy3 or Cy5 dyes for fluorescent labeling in a following step (not shown).
- EXAMPLE NO. 3
cDNA Synthesis & Amplification: cDNA synthesis includes three different reaction steps: (A) a reverse transcription (RT) step; (B) the formation of double-stranded cDNA step; and (C) the isothermal amplification (IA) step. In the RT, the mRNA (poly A+) portion within the total RNA is transcribed into single-stranded cDNA using a chimeric RNA-DNA primer. The primer binds with its DNA part to the poly(A) tail of the mRNA but the RNA part stays single-stranded. In this way the resulting RNA-cDNA complex has a unique RNA target sequence at the 5′ end of the cDNA. After fragmentation of the RNA in the RNA-cDNA complex, DNA polymerase is used to synthesize a second strand including DNA complementary to the unique 5′ RNA sequence of the first strand. The result is a double-stranded cDNA with a unique RNA/DNA heteroduplex at the 5′ end. In the IA part of the unique 5′ RNA sequence is removed by added RNase H. The exposed cDNA sequence is then available for binding a second chimeric RNA-DNA primer. The DNA polymerase subsequently extends the strand while incorporating aminoally-dUTP into the de novo synthesized sequence starting at the 3′ end of the primer and displacing the existing forward strand. The process of chimeric primer binding, DNA synthesis, strand displacement, and RNA cleavage of the primer is repeated multiple times, resulting in a rapid accumulation of single-stranded aminoallyl labeled cDNA, that is complementary to the original mRNA (antisense cDNA). The cDNA is stopped by heat-inactivation and the product, i.e. aminoallyl-labeled cDNA, is purified from enzymes and unincorporated nucleotides with magnetic beads (Dynal Biotech, Lake Success, N.Y.).
- EXAMPLE NO. 4
Fluorescent Dye Coupling: The fluorescence labeling of the cDNA is achieved in a simple chemical by coupling of NHS-ester cyanine 3 (Cy3) or cyanine 5 (Cy5) to aminoallyl groups of the cDNA. The uncoupled dye is removed by purification with magnetic beads process (Dynal).
Hybridization: The two differently labeled cDNAs from sample A and B are pooled together with competitor DNA & control RNA and subjected to dual-color hybridization onto the integrated array of 400 printed DNA elements. Hybridization would be performed for 16 hours following the protocols for conventional hybridization with microarrays. The hybridized chip would be subjected to post-hybridization washes and scanning for image analysis and the collection of gene expression data.
In co-pending applications, U.S. Ser. No. 10/338,451 and PCT WO2004/062804, entire contents of which are incorporated herein by reference, disclose certain microfluidic chip platform for configuring the mXP-CHIP structure. The principle behind mXP-CHIP operations is that the system sequentially delivers reagents one at a time from a reagent well to a reaction well, and then to the hybridization well. The mXP-CHIP has a 3D structure that makes it easier to transfer the fluid to different locations. A pressure driven microactuator (external) is used to initiate fluid transport from well to well, while a vacuum port 92 and vacuum line (external) with a vent hole is associated with each reaction well for fluid removal. Depending on the assay protocol and the number of samples, the mXP-CHIP or cartridge can be fabricated with an array of patterned microchannels and microwells. FIG. 12 shows a topical view of the mXP-CHIP. The diagram shows the chip with sets of microwells for reagents (R1-R22), reactions like probe synthesis/labeling (RA-1 & RA-2), and hybridization 82 (HYB).
In one embodiment, the chip 81 has a circular format (d=3 inch). It sits on a rotational stage for accurate microwell positioning. FIG. 13 shows the pressure-driven microfluidics for total automation. The stopper element 93, with a stopper 22 and a pin 21, is designed to reliably pierce the thin film gasket 94 and open the microwell 96. The sequence of microfluidics is as follows: (FIG. 13 a) the reagent solution is sealed in the separate cavity of the microwell; (FIG. 13 b) the reagent solution is released from the microwell into the microchannels 95, and reaction wells 13; and (FIG. 13 c) the reactive solution in the reaction well is withdrawn to the external waste reservoir by a vacuum line 45. Repeat the above processes to complete a sequence of reactions. In one embodiment, the mXP-CHIP consists of a three-layer microwell structure (as shown in FIG. 13): a reagent well layer 30 (top), a microchannel layer 31 and a reaction well layer/hybridization well (bottom) layer. The reagent microwells have a pattern allowing for the storage of reagent solutions. Each microwell has a sufficient amount of liquid for a reaction (up to 500 μl). Between layers, there are gasket thin films. These gaskets not only separate the layers, but also contain a network of microfluidic channels (100 μm) patterned on the bottom side of the gasket.
These microfluidic channels connect the reagent wells to the reaction wells and to the hybridization well or waste port. In some embodiments, two types of thin film gaskets are used, one made of PDMS and one from a polyacrylic composite. In one embodiment, the fluidic chip bodies are made from plastic materials (e.g. polystyrene, polyacrylic, etc.), and can be mass-produced by injection molding. The microactuator works like a plastic micro syringe, is simple, and reliable. It is operated by a micro stopper element that can be plugged inside the microwell-capping the reagent in a microwell and piercing the gasket at the bottom of the well. FIG. 13 shows the micro stopper element with two partially connected plastic pieces (a stopper and a pin).
When the external actuator, similar to a linear screwdriver, engages with the micro stopper element, it pushes the pin element (right on top of the film) downward without causing internal pressure build-up. The pin pierces through the thin film gasket, and stops at the bottom of the microchannel. The downward force separates the plastic pieces and the stopper is depressed downward to the bottom of the well. The stopper stays at the bottom of the well to prevent backflow. By this method the stopper element opens the microwell as a one-way valve. The movement of fluid is physically constrained to exit through a microchannel to the reaction well. To move all the residual liquid (1-3 μl) left in the microchannel into the reaction well, spare airs in the reagent wells are intentionally provided to displace all of the fluid left in the microchannel.
- EXAMPLE NO. 5
A reaction/hybridization well, capped with a rubber seal with a small vent hole, has a large liquid and air volume. With this design, air is allowed into the microfluidic system. Therefore, the common problems associated with microfluidic systems, such as air bubbles, trapped air, dead volumes, inhomogeneous distribution, and backflow into the channels, will not affect the outcome. Furthermore, the microwell/rubber seal structure provides fluid accessibility. Therefore, every step of reaction in the protocol can be monitored for yields and qualities. A vacuum line, with a port, is associated with each reaction and hybridization well to provide fluid removal from each reaction well to a waste storage and transport fluid from the reaction wells to the hybridization wells. The make-up air from the vent hole would replace the volume of fluid been removed. The hybridization slide (about 1 cm×1 cm) can be placed in or taken away from a hybridization well for laser scanning.
- EXAMPLE NO. 6
The bioassay process includes four major steps: simultaneous cDNA synthesis; cDNA linear amplification & aminoallyl labeling from two samples (as described in Example No. 1); fluorescent dye coupling of the cDNAs; and dual-color hybridization to the integrated array of spotted DNA elements (as shown in FIG. 9). Both samples are purified, once after aminoallyl and once after fluorescent dye coupling, with magnetic beads (Dynal Biotech, Lake Success, N.Y.), pooled together with Competitor DNA (for specific hybridization) and Control RNA (for signal normalization) and applied to a microarray for hybridization. In the first step of the process the mixture of total RNA and chimeric RNA-DNA primer is manually loaded into the reaction wells RA-1 and RA-2. The rest of the steps are performed in an automated fashion (FIG. 12).
- EXAMPLE NO. 7
- 1. Load a 7 μl mixture of total RNA from sample A and First Strand Primer Mix as well as a 7 μl mixture of sample B and First Strand Primer Mix into RA-1 and RA-2, respectively. Incubate at 65° C. for 5 minutes (denaturing step).
- 2. Cool RA-1 & RA-2 to 48° C. and forward 13 μl of First Strand Master Mix from reagent wells R1 and R22 to RA-1 and RA-2, respectively. Mix by rotating disc back and forth for 1 minute and incubate for 1 hour at 48° C. (cDNA first strand synthesis).
- 3. Cool RA-1 & RA-2 to 37° C. and forward 20 μl of Second Strand Master Mix from R2 and R21 to RA-1 and RA-2, respectively. Mix by rotating disc back and forth for 1 minute and incubate at 37° C. for 30 minutes (cDNA second strand synthesis).
- EXAMPLE NO. 8
- 1. Forward 120 μl of SPIA™ Master Mix from R3 and R20 to RA-1 and RA-2, respectively. Mix by rotating disc back and forth for 1 minute. Heat RA-1 and RA-2 to 50° C. and incubate for 90 minutes.
- 2. Heat RA-1 and RA-2 to 95° C. and incubate for 5 minutes (enzyme inactivation).
- 3. Cool reaction the chamber to 22° C. and forward the magnetic bead solution from R4 and R19 to RA-1 and RA-2, respectively. Incubate at room temperature for 5 minutes (cDNA is captured by the beads).
- 4. Immobilize the magnetic beads with an external magnet and vacuum the reactant into the waste/vacuum line. Forward 100 μl wash buffer from R5 and R18 to RA-1 and RA-2, respectively. Incubate for 1 minute and then remove wash buffer.
- EXAMPLE NO. 9
Fluorescent Dye Coupling:
- 1. Forward 10 μl of Cy3 Dye Mix and Cy5 Dye Mix from R6 and R17 to RA-1 and RA-2, respectively. Incubate for 60 minutes at 22° C.
- 2. Forward 100 μl of wash Buffer from R7 and R16 to RA-1 & RA-2, respectively and incubate 1 minute. Hold the magnetic beads at the bottom of RA-1 and RA-2 and remove wash buffer.
- 3. Forward 50 μl elution buffer from R8 and R15 to RA-1 and RA-2, respectively. Incubate for 5 minutes and move the reactant (including the purified cDNA) from RA-1 and RA-2 into the hybridization chamber HYB (containing the microarray).
Hybridization & Post-Hybridization Processing:
- 1. Forward 50 μl of Hybridization Mix from R9 to HYB and rotate for 30 seconds. Heat HYB to 95° C. and incubate for 3 minutes. Cool HYB to 60° C. and incubate for 16 hours.
- 2. Replace the hybridization solution with 100 μl of Wash Buffer A from R10. Cool the array chamber down to 25° C. and incubate for 3 minutes. Repeated washes with another 100 μl of Wash Buffer A (R11), Buffer B (R12), and then Buffer C (R14), and for 3 minutes while rotating the disc at 25° C. The microarray would be dried and subject to scanning.
Microarrays are critical in the process of determining the function of the human genome, speeding up the discovery process many times over. Microarray biochips in combination with microfluidics are revolutionizing drug discovery, laboratory testing and device development leading to a new paradigm for the big pharmaceutical companies. Point of care diagnostics is poised to make a leap forward as microarrays and microfluidics chips become less expensive. Home diagnosis with disposable chips may become a reality within 10 years. The worldwide market for microarrays and microfluidics is expected to grow to $1 Billion by 2005. This expected growth would mean an annual growth rate of 21.8% during the 5-year forecast period since 2000 (Business Communications Co., Inc).
The chip system is not restricted to serve as a reliable tool for gene expression profiling. The chip design and the control software can be flexibly modified to also address any application that involves nucleic acid probe labeling and array/target hybridization, including different types of genotyping, such as comparative genomic hybridization (CGH), detection of single nucleotide polymorphisms (SNPs) or identification of thread agents like bacteria and viruses. The self-contained design of the chip makes the device especially suitable as a portable system for out-lab use in remote areas. Microarrays in combination with microfluidics have the potential to also significantly decrease assay costs due to the consumption of lower reagent volumes and shorter assay times. Thereby microarray assays can be used with much higher confidence and cost-efficiency for: (1) screening of candidate genes in the research of genetic, immune & infectious diseases; (2) drug target identification and validation by the pharmaceutical industry, and (3) identification of threat agents (bacteria and viruses).
While the present invention has been particularly shown and described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit, scope, and teaching of the invention. For example, while the present invention has been described in reference to a biochip having circular array of reagent cavities and reaction wells, the present invention can well be implemented in a biochip having a rectangular array, or an array of other geometries. Furthermore, the present invention may be implemented on a biochip having a footprint or format compatible to a 96-well micro-titer plate, so that compatible apparatus may be used to handle the biochip, such as laboratory robotic equipments. Still further, while the invention has been described in reference to a process using a biochip analysis apparatus that includes a detector, the present invention may be implemented in a process using an apparatus that allows the reactions to complete in the biochip, and then the biochip is transferred to another apparatus that is dedicated to detection of the final reaction product.
The biochip system and methods of the present invention are not restricted to serve as a reliable tool for gene expression profiling. The chip design and the control software can be flexibly modified to also address any application that involves nucleic acid probe labeling and array/target hybridization, including different types of genotyping, such as comparative genomic hybridization (CGH), detection of single nucleotide polymorphisms (SNPs) or identification of thread agents like bacteria and viruses. The self-contained design of the chip makes the device especially suitable as a portable system for out-lab use in remote areas. Accordingly, the disclosed embodiments are to be considered merely as illustrative and the present invention is limited in scope only as specified in the appended claims.