This invention relates to methods and apparatus for performing automatic, correlated cell-by-cell quantitative measurement of proteins and nucleic acids from a high throughput sample with the option to sort and collect genomic material.
Fluorescent Activated Cell Sorting (FACS) is widely used in research and clinical applications. These instruments are capable of very fast, multi-parameter analysis and sorting but generally require large sample volumes, a trained operator for operation and maintenance, and are difficult to sterilize. FACS instruments are able to analyze as few as 10,000 and as many as tens of millions of cells. In sorting applications, however, the ability to perform sorting diminishes for sample sizes smaller than 100,000 cells. In all cases, the cells must be labeled in advance. Most often, an antigen or similar membrane bound protein is labeled using antibodies conjugated to fluorescent molecules (e.g., fluorescein isothiocynate a.k.a. FITC), but nuclear stains, intracellular dyes or cell directed synthesis of fluorescent proteins (e.g., green fluorescent protein a.k.a. GFP) may also be detected by flow cytometry. Molecular assays may also be adapted for flow cytometry. For instance, fluorescent beads with multiple colors and/or intensities may be used as solid supports for antibody capture assays of protein or peptide analytes. Similarly, nucleic acids may be hybridized to beads and fluorescent labels for rapid readout using a flow cytometry. In both cases, the biochemistry is performed in advance of the flow cytometry measurement and the instrument is used as a rapid bead reader. In some cases, scanning cytometry approaches may be equally effective for a readout. FACS instruments support multiplexing of information up to the number of independent fluorescent channels supported by the instrument, but the information multiplexed is of a single type (i.e., phenotype-only).
Real-time polymerase chain reactions (a.k.a qPCR) are a technique used to quantitatively measure DNA and RNA extracted from cells of interest. Most qPCR reactions are done in bulk using the pooled genomic equivalent of 10,000 to 100,000 cells. Increasingly, researchers are interested in measuring the genetic contents of individual cells, but this effort is impeded by the high cost of reagents and the labor intensive manual approaches available today. As a result, most single cell PCR studies have been conducted on fewer than 100 cells. Even state of the art robotics and 1536 micro-well plates use volumes in the range of 1-10 μL per well still become costly beyond a few hundred wells. In cases where rare events that may occur in less than 1% of the cell population of interest, it may be desirable to examine up to 50,000 cells one-by-one. Current technologies cannot achieve this level of throughput without significant costs in time and money.
In studies of signal transduction or the pursuit of systems biology, it is often desirable to correlate disparate information. Data that links cell surface receptors or reporters and intracellular signaling is increasingly valuable to these activities. To date, this information has been correlated based on bulk populations of cells. Typically, thousands to millions of cells are assayed for either surface proteins or for RNA expression and the information is statistically linked. Any heterogeneity in the sample is averaged out during the measurement. Powerful techniques such as siRNA gene silencing, which inherently introduce heterogeneity into the sample, are limited by this averaging of information.
The value of quantitatively correlating proteins (or other signatures of the phenotype) and nucleic acids of the genotype on a cell-by-cell basis has been recognized by many researchers. Microfluidics has been identified as one technology that would enable instruments capable of such measurements, but examples of methods and apparatuses have not been produced.
Microfabricated cytometers have the potential to analyze and sort as few as 1,000 cells while concomitantly consuming smaller amounts of reagents in an easy to use, closed system. The former is important when working with high-cost reagents such replication enzymes. The latter is important because, unlike conventional FACS instruments, aerosols are not created, reducing the risks of contamination of the sorted cells and of working with biohazardous materials. Several microfabricated cell analyzers and sorters have been described, but mostly as “proof of concept”.
By combining elements of microfabricated cytometers, techniques for single cell encapsulation and appropriate signal processing, an instrument capable of cell-by-cell correlation of protein expression and gene expression may be achieved.
As described below, these elements are combined to realize a correlated assay in a microfluidic network. Cells are labeled for phenotypic measurement in advance. In one embodiment, a continuous flow microfluidic network integrates all functionality. In the first part of the microfluidic network, cells are measured for the fluorescent signal produced by the phenotypic label. The second part of the microfluidic network individually encapsulates cells in microreactors along with a pre-determined mix of reagents suitable for measurement of gene expression. In the next part of the microfluidic network, the stream of encapsulated cells is encoded with a reference signal that may be included in the microreactor contents or separate from it. In the fourth part of the microfluidic network, the cells are lysed and gene expression is measured by a suitable technique (e.g., real-time polymerase chain reaction a.k.a. real-time PCR or qPCR). The stream of microreactors is then decoded in the next stage and a signal processing algorithm correlates the phenotype and genotype measurements. As a final optional step, the microreactors may be sorted to enrich for specific genomes. In a related embodiment, cells are encapsulated first, so that phenotyping and encoding may take place simultaneously. This is followed by lysing of cells and then measurement of gene expression and decoding simultaneously. This reduces the number of points of interrogation on the microfluidic network.
In a second embodiment, a microfluidic network is combined with arrays of micro-wells to isolate individual cells and correlate genetic information with phenotype clusters pre-selected by the user. In the first part, a microfluidic network is used to measure cells for a fluorescent signal produced by the phenotypic label. In the second part, a multi-path switching network is used to sort cells into individual nanofluidic micro-wells. The wells may be arranged such that each cell is located in a uniquely identified coordinate or the wells may be arranged to cluster cells in two or more arrays. In the latter case, the user selects the desired destination of the cell by selecting one or more gates based on the phenotypic information obtained. The wells are sealed to individually encapsulate the cells with a pre-determined mix of reagents suitable for gene expression measurements. The cells are lysed and gene expression is measured by a suitable technique by readout from the array. The information from the reaction in the micro-wells may be analyzed to examine statistical distributions of the genetic signal and the information may be discretely correlated back to the selected phenotype. As a final optional step, the contents of the individual micro-wells may be collected for further genetic analysis.
In a third embodiment, a scanning cytometry approach using an array of nanofluidic micro-wells is used to isolate individual cells and capture correlated phenotype and genotype information. Again, cells are labeled for phenotypic measurement in advance. The cells are deposited in the micro-wells and the wells are sealed to individually encapsulate the cells with a pre-determined mix of reagents suitable for gene expression measurements. A scanning cytometry analysis of the cells collects phenotype information indexed to the coordinate of the well, the cells are lysed and then gene expression is measured by a suitable technique for readout from the array. The signals are concatenated into a single correlated data set. As a final optional step, the contents of the individual micro-wells may be collected for further genetic analysis.
In all embodiments, one or more phenotypic measurements may be made. Typically, up to four or five independent fluorescent signals are detectable simulataneously. The measurements of gene expression may be measurements of a single gene, or preferably a multiplex of two or more genes within each reaction in order to obtain normalized, quantitative information about the expression levels of selected genes.
The present invention provides methods and apparatus for performing cell by cell analysis of phenotypic (e.g., cell surface proteins or intracellular proteins labeled with fluorescently conjugated antibodies or constitutively expressed fluorescent reporter molecules such as green fluorescent protein) and genotypic (e.g., single-cell, multiplexed quantitative PCR or RT-PCR for detection of single nucleotide polymorphisms (SNPs), DNA copy number, or RNA gene expression) information on a scalable basis for samples numbering from the hundreds of cells to thousands of cells to the tens to hundreds of thousands cells. The larger sample sizes advantageously allow multi-dimensional scatter plots to reveal biological patterns and relationships between genes and proteins. As shown in
In some embodiments, the cells may be sorted based on a parameter selected by the user, such as the measured phenotypic characteristic. For example, the presence of a target cell with the desired characteristic can be detected in the fluid stream by fluorescence, forward scattering or any suitable imaging or detection modality. The target cell can then be directed to a target flow channel, using, dielectrophoresis, a pneumatic switch or a bi-directional fluid or optical switch described in co-pending patent application Ser. No. 11/781,848, entitled “Cell Sorting Systems and Methods” incorporated by reference in its entirety, for encapsulation and/or encoding. Non-target cells can be directed to a waste flow channel attached to a waste reservoir. In some embodiments, the cells can be sorted prior to phenotype measurement. Alternatively, the cells can be sorted based on the measured phenotypic characteristic. In an alternative embodiment, two oils may be used to form a three-phase system where one oil acts as a carrier fluid and the second oil acts as a space between drops. This inhibits coalescence of adjacent aqueous drops. An example of microvessels in a three phase system before and after PCR amplification is shown in
In step 104, the microvessels are encoded with a reference signal to index the phenotype information of the cells in the microvessels for later correlation with the gene expression of the cells in the microvessel. The reference signal can be included in the microvessel or separate from the microvessel. The reference signal can be generated using a pseudorandom pattern of any physical parameter that has a unique signature and can be optically measured. One or more images of the encoded microvessels are then recorded and stored for later comparison and correlation with images of the microvessels following the genotypic measurement with the included in the microreactor or separate from the microreactor. For example, in some embodiments, fluorescent microbeads or dyes may be injected into the microvessels in a pseudorandom pattern which is then imaged. Alternatively, the size of the microvessels, length of the microvessels and/or spacing between microvessels can be varied to create a pseudorandom pattern capable of being reconstructed. The interval of the reference signals in the encoding pattern can be varied depending on the tracking accuracy required. For example, in some embodiments, a reference signal can be associated with every microvessel, alternatively a reference signal can be associated with every 10 microvessels, alternatively with every 100 microvessels, alternatively with every 200 microvessels, alternatively with every 500 microvessels, alternatively with every 1000 microvessels.
In step 106, the genotype of the cells is measured. The cells are lysed and subjected to thermal conditions necessary for amplification of the target DNA sequence, for example by isothermal amplification, endpoint PCR, reverse transcription PCR or quantitative PCR. In embodiments performing amplification via PCR, the temperature is cycled to produce suitable temperatures for the desired number of PCR cycles. This may be accomplished with a standard thermal cycler using a heat block or Peltier device, or it may be accomplished with alternative technologies such as an oven, hot and cold air, flowing a heated liquid with good thermal conductivity, transferring the device between instrument components held at different temperatures or any other suitable heating elements known in the art (this same list will be understood to be applicable to other embodiments described herein). In some embodiments, the microvessels can be continuously flowed through a serpentine flow channel that repeatedly passes through fixed temperature zones to achieve a polymerase chain reaction. Alternatively, the microvessels can be loaded into a flow channel and remain more or less static while the temperature of the flow channel is repeatedly cycled through the temperature profile needed to achieve a polymerase chain reaction. The microvessels are subjected to the desired number of PCR cycles for amplification of the target DNA and the amplified product is measured The reagent mix encapsulated with the cell in the microreactor includes non-specific fluorescent detecting molecules such as intercalating dyes or sequence specific fluorescent probes such as molecular beacons, TaqMan® probes or any other suitable probe or marker known in the art that will fluoresce at a level proportional to the quantity of the amplified product when excited. These probes may bind to double stranded DNA products, single stranded DNA products or non-product oligonucleotides created by the DNA amplification process that are present in an amount proportional to the DNA product. An imaging system uses light of a desired wavelength to excite the fluorescent probes to measure the amplified product directly or indirectly.
In step 108, the genotype measurement is decoded. An optical detector such as a photomultiplier tube, a CCD camera, photodiodes or photodiode arrays or other optical detector measures the intensity of the fluorescent signal from the probes or markers in each microvessel and determines the quantity of amplified product at least once during each PCR cycle. The image(s) from the optical detector are sent to a computer for image processing and comparison to the recorded encoding pattern of the microvessels. In step 110, image processing algorithms known in the art can be used to compare the index images with the images from the genotype measurements and the genotype measurements can then be correlated to the phenotype measurements on a cell-by-cell basis.
In some embodiments, in step 112, the microvessels can be sorted based on the measured gene expression to capture target nucleic acids for further analysis. An optical switch, as discussed above, can be used to direct the microvessels into a target flow channel or a waste flow channel based upon the genotype measurement.
In an alternative embodiment, as shown in
The individual cells are lysed and then the microvessels are subjected to the thermal conditions necessary for amplification of the target DNA sequence, for example by real-time or quantitative PCR. The microvessels are subjected to the desired number of PCR cycles for amplification of the target DNA. The amplified product is measured in between the PCR cycles by exciting the flouoprobes included in the PCR reagent mix and detecting the intensity of the fluorescence with an optical detector. The optical detector can be a scanning detector, for use with a continuous flow system, or alternatively a stationary detector The images are sent to a computer for image processing and comparison to the recorded encoding pattern of the microvessels. The genotype measurements can then be correlated to the phenotype measurements on a cell-by-cell basis.
The sheath buffer 201 focuses the cells 200 into single file line of cells 200 which are then interrogated at an analysis region 205 to measure one or more desired phenotypic characteristics of the cells. A source of illumination, such as fixed or scanning lasers, UV lamps, light emitting diodes or an other collimated light source, causes the labels, such as fluorescing dyes, antibodies, GFP or other flourochromes, attached to the cells 200 to fluoresce and scatters off cells and vessels to provide information on the physical properties of each cell and vessel (e.g., size, morphology, or boundaries). One or more optical detectors, such as CCD imaging, PMTs, or photodiode arrays, measure the resulting signal(s). Other types of optical measurements, such as light scattering, may also be performed at this analysis region 205 to measure phenotypic characteristics of the cells 200. In some embodiments, multiple phenotype measurements can be performed using for example four color flow cytometry or by measuring fluorescence and/or scattering. For example, by labeling cells with multiple fluorophore and using additional fluorescence detection channels that are sensitive to fluorescence emissions at different wavelength, typically using a single excitation wavelength, such as, but not limited to, 488 nm, multiple phenotypic measurements can be made. Here, each detection channel would incorporate a PMT with an appropriate dichroic mirror and emission filter for the fluorescence emission wavelength of the additional fluorophore. From two to four fluorescence detection channels are readily accommodated in this manner.
In the embodiment shown, the PCR mix including reagents necessary for measurement of gene expression and fluorescent DNA detecting molecules or probes is injected into the flow stream via lateral reagent flow channels 206a,b placed between the sheath flow channels 203, 204 and the encapsulation flow channels 207a,b. In an alternative embodiment, the PCR mix can be introduced into the cell stream by the sheath buffer flow channels 203, 204.
In the next step 102, encapsulation is performed by introducing a hydrophobic encapsulation media into the flow channel 209 via lateral encapsulation flow channels 207a,b. Silicon oil, mineral oil, fluorocarbon oils or other hydrophobic liquids may be used to facilitate creation of discrete aqueous drops. The encapsulation media pinches the stream of PCR mix and cells into individual water-based nano-liter microvessels, or microreactors, 208. The nano-liter microvessels 208 preferably have diameters larger than the cells 200 but not significantly larger than the microfluidic channel 209. Flow conditions in the encapsulation flow channels 207a,b are selected to ensure that zero or one cell per microvessel are preferably encapsulated. For example, microfluidic channels compatible with flow cytometry typically have cross sections of 50-100 μm by 150-300 μm and Teflon capillary tubing is available with diameters as small as 400 μm, so the microvessel 208 diameters would be in the range of 30-400 μm.
In the next step 104, encoding and decoding the sequence of microreactors 208 is performed in order to facilitate correlation of phenotype data with genotype data. As shown in
In the illustrated embodiment, a second, redundant encoding signal is provided by varying the flow conditions in the flow channel 209 to vary the spacing between the microvessels 208. As shown in
Next, in step 106, the encapsulated cells 200 are lysed and analyzed for gene expression by realtime or quantitative PCR. Several means known in the art can be employed to lyse the cells including photolysing with a laser, ultrasound-based lysing, or chemical lysing. The microvessels 208 then flow through a serpentine microfluidic channel 210 that passes the microvessels 208 through one or more thermal zones necessary for amplification and real-time detection of amplified gene products. In the illustrated embodiment, the serpentine channel 210 repeatedly flows through three temperature zones 211, 212 and 213. The temperature zones are maintained at suitable temperatures for conducting PCR cycles. For example as shown here, a first temperature zone 211 is approximately 60°, a second temperature zone 212 is approximately 72°, and a third temperature zone 213 is approximately 96°. A temperature control system adjusts the flow conditions to ensure that the microvessels are held at each temperature zone for the appropriate time for a PCR cycle. The microvessels 208 are passed through the temperature zones 211, 212, 213 multiple times and measured after each passage to determine the quantity of amplified product. As discussed above, the PCR mix includes probes that fluoresce at a level proportional to the quantity of amplified product. The fluorophores may be excited by a light source 214, such as fixed and scanning lasers, UV lamps, light emitting diodes, at a specific time or temperature during each PCR cycle and detected by a detector 215 such as photomultiplier tube, CCD camera, photodiodes or photodiode arrays, or other optical detector. The detector 215 measures the intensity of the fluorescence to determine the quantity of amplified product in the microreactors 208.
Next, in step 108, the previously recorded phenotype measurements are decoded. As shown in
In some embodiments, it may be desired to recover genetic material from a selected portion of the cells measured, or alternatively to sort of cells based on a genetic signature without correlating to a phenotype. For example, as shown in
The chip is bonded with UV adhesive to the optical window region of the cartridge 320, and inlet ports from the chip interface with their respective reservoir volumes on the disposable cartridge 320. An inlet port in sample flow channel 309 on the chip is fluidically connected to the cell sample reservoir 323 on the disposable cartridge to introduce the cells into the sample flow channel 309. The cell sample reservoir 323 is typically conical in shape, tapering towards the inlet port. In the preferred embodiment, the inlet reservoir contains a polypropylene insert to minimize cell adhesion and consequently maximize cell yield. Microfluidic channels 305, 307 on the chip fluidically connect outlets in reservoirs 321, 324 to the sample flow channel 309. Lateral flow channels 305a,b are configured to add the PCR mix to the sample flow channel 309 and then lateral flow channel 307 introduces the oil into the sample flow channel 309 to encapsulate the sample cells within the oil. The cartridge 320 is positioned within the instrument 300 such that a light source such as 488 nm laser 303 and a fluorescence detector 312 are positioned adjacent the sample flow channel 309. The cartridge is preferably manufactured from optically clear acrylic plastic. Optical windows further allow visible interrogation of selected points in the microfluidic network and enable projection of the excitation sources and optical detectors through the cartridge and into the microfluidic chip. Other optically clear plastics or suitable materials may be substituted for acrylic if appropriate. The microfluidic channels are likewise produced in optically transparent substrates to enable projection of cell detection optics into the sample flow channel 309. This substrate is typically, but not limited to, glass, quartz, plastics, e.g., polymethylmethacrylate (PMMA), etc., and other castable or workable polymers (e.g. polydimethylsiloxane, PDMS or SU8).
Light from the 488 nm laser 303 is projected through the cartridge 320 into the sample flow channel 309 just upstream of the encapsulation region to interrogate the cells. The 488 nm laser 303 with a selected phenotypic characteristic of the sample cells. The fluorescence detector 312 measures the fluorescence to measure the phenotypic information for the cells. An encapsulation medium, such as oil, is then introduced into the sample flow cell 309 under conditions suitable to encapsulate single cells into individual nanoliter microvessels. The microvessel are then sorted based on one or more measured phenotypic characteristics of their encapsulated cell(s) though the sample flow channel 309 for further genotyping or into a waste flow channel 330 for transportation to a waste reservoir 334. As the microvessels are flowed through the sample flow channel 309, an index decoding laser 308 excites the index beads previously associated with the cells in a pseudorandom sequence. A decoding sensor 306 detects, images and stores the sequence of index beads attached to the cells in the order they are flowed through the sample flow channel 309 to create an index of the microvessels for later correlation with images of the genotypic measurements. Once the microvessels have been encoded, they are loaded into a serpentine microfluidic channel on the microfluidic chip for PCR amplification. A thermal module 302 on the instrument is positioned adjacent the preloaded microfluidic channel cycle the microvessels in the microfluidic chip through the temperature zones necessary to achieve PCR amplification. The thermal module 302 comprises a thermal control element and a standard thermal cycler using a heat block or Peltier device, or it may be accomplished with alternative technologies such as an one or more integrated heating wires, an oven, hot and cold air, flowing a heated liquid with good thermal conductivity, transferring the device between instrument components held at different temperatures or any other suitable heating elements known in the art. In some embodiments, multiple heating elements can be used to create spatial thermal zones which the chip is physically passed using a motorized element. Alternatively, a single heating element such as a cold/hot air heater can be used to alternately heat and cool the microfluidic channel to cycle it through the temperature profile for PCR amplification. The thermal control element controls the temperature and cycle the serpentine channel through the PCR temperature profile multiple times for the desired number of PCR cycles.
The microvessels are analyzed after each PCR cycle to determine the quantity of amplified product. As discussed above, the PCR mix includes probes that fluoresce at a level proportional to the quantity of amplified product. The probes may be excited by a light source, such as fixed and scanning lasers, UV lamps, light emitting diodes, at a specific time or temperature during each PCR cycle. The fluorescent signal is measured by a PCR image sensor 304 positioned adjacent to the microfluidic chip such as photomultiplier tube, a CCD camera, photodiodes or photodiode arrays, or other optical detector. The image sensor 304 measures the intensity of the fluorescence to measure the quantity of amplified product in the microreactors 208 and measure the genotype of the cells in each microvessel. The index decoding laser 308 also excites the index beads associated with each microvessel and the encoding signal is imaged by decoding sensor 306. The encoding signal and the genotype data for each microvessel are then sent to a processor for indexing and correlation of the phenotype measurement with the genotype data from the individual microvessels 208. In some embodiments, the microvessels are imaged and tracked cycle-by-cycle, for example using the drop size and the position in each image. In alternative embodiments, the microvessels are imaged and the encoding signals are read after the desired number of PCR cycles has been completed.
As discussed above, the devices and methods for performing the phenotype-genotype correlation are scalable for samples numbering in the hundreds of cells to thousands of cells to the tens and hundreds of thousands of cells. The instrument platform 300 and the disposable sample loading cartridge 320 can be used with several different capillary tubes or microfluidic network chips configured to process samples of up to a hundred cells, alternatively up to a thousand cells, alternatively up to ten thousand cells, alternatively up to one hundred thousand cells.
The stream of microvessels 408 is then loaded into a microfluidic capillary tube 420 for amplification of genetic material in the microvessels and measurement of the gene expression. As discussed above, the flow conditions in the sample flow channel 409 and the encapsulation flow channel 307 can be further controlled to vary the size of the nanoliter microvessels and/or the spacing between the nanoliter microvessels as discussed above to provide a unique sequence of microvessels for encoding and indexing the relative position the microvessels. In the illustrated embodiment, the distance (d) between microvessels in the capillary tube is between about 500 to 1000 μm and the total length (L) of the microfluidic capillary tube 420 is between about 500 to 1000 mm to provide the capacity for about 1000 microvessels. In alternative embodiments, the drop spacing and or the total length of the microfluidic capillary tube can be adjusted to accommodate a larger or smaller sample. In use, the capillary tube is formed into a serpentine arrangement comprising a plurality of adjacent parallel segments connected by couplings on each end. For example, in some embodiments, the capillary tube is partitioned into between 10-20 parallel segments. The couplings are cut off leaving a plurality of adjacent parallel segments each approximately 40-50 mm in length. The capillary tube is partitioned into the plurality of segments prior to the amplification process to minimize the ripple effect of thermal expansion during the PCR amplification cycle on the position of the microvessels within the capillary tube and thereby improve the ability to track the location of the individual microvessels so that the measurements of the amplified product can be correlated to prior phenotype measurements for each individual microvessel.
As discussed above, in use, the microfluidic chip 500 is attached to a disposable sample loading cartridge containing the sample, PCR mix and encapsulation media. The chip 500 has a cell inlet 513, PCR mix inlet 512 and oil inlet 511 configured connect to the cell reservoir, PCR mix reservoir and oil reservoir on the disposable cartridge when the chip is positioned on the optical window of the disposable cartridge. Cell inlet port 515 is fluidically connected to sample flow channel 509 so that in use the cells contained in sample reservoir of the disposable cartridge will be transported into sample flow channel 509. PCR mix inlet 411 is fluidically connected to PCR flow channels 505a,b which intersect sample flow channel 509 to introduce the PCR mix into the stream of cells flowing through sample flow channel 409. In some embodiments, the flow conditions of the PCR mix are controlled such that introduction of the PCR mix also focuses the sample flow into a single file stream of cells. Alternatively, the diameter of the sample flow cell 509 can be configured to focus the sample into a single file stream of cells when the cells are introduced at the cell input 513. Oil inlet 512 is fluidically connected to encapsulation flow channel 507 which intersects sample flow channel 409 at an L-junction downstream of the PCR mix intersection. The oil flow is injected into the stream of cells and PCR mix at the “L” junction between the sample flow channel 409 and the encapsulation flow channel 407 under flow conditions suitable to pinch off droplets of cells and PCR mix into a stream of nanoliter microvessels preferably containing a single cell surrounded by the oil encapsulation media. In some embodiments, a sorting region may be provided to sort the sample prior to encapsulation in order to limit the number of encapsulated cells to a pre-selected subpopulation that can still be correlated back to those phenotype measurements. For example, sorting can be performed to reject red blood cells to focus only on nucleated cells. The nanoliter microvessels preferably have a cross sectional diameter larger than the sample cells and smaller than the cross sectional diameter of the flow channel. For example, the microvessels preferably have a cross-sectional diameter between 30 μm-400 μm depending on the flow channel size.
As discussed above, the flow conditions in the encapsulation flow channel 507 are controlled to ensure that a majority of the microvessels preferably contain either one cell or zero cells, for example, in some embodiments at least 90% of the microvessels contain one or zero cells, alternatively at least 80% of the microvessels contain one or zero cells, alternatively at least 70% of the microvessels contain one or zero cells, alternatively at least 60% of the microvessels contain one or zero cells, In addition, in some embodiments, the flow conditions of the encapsulation flow channel 507 and/or the sample flow channel 509 can be further controlled to periodically vary the microvessel size and/or the spacing between microvessels to create an encoding signal for indexing and tracking the individual microvessels. The setting and control of the flow conditions in the microfluidic channel network can be achieved by direct drive pumping, pneumatic pumping, electro-kinetics, capillary action, gravity, or other means to generate fluidic flow.
The stream of microvessels are then loaded into a serpentine channel 520 fluidically connected to the sample flow channel 509 for amplification of a target sequence in the sample cells. The serpentine analysis channel 509 is partitioned into a plurality of parallel segments 521a-j with adjacent segments 521a-j connected by a plurality of turn segments 531a-i. The serpentine channel is preferably partitioned into between 50-100 parallel segments, each 50-100 mm long depending upon the sample size and the desired spacing between microvessels. Venting vias 540a-j are fluidically connected to each end of each parallel segment 521a-j of the serpentine channel 520. The venting vias 540a-j are configured to accommodate the thermal expansion and contraction of the stream of microvessels in each segment 521a-j between temperature cycles in the amplification process. Partitioning the serpentine channel 520 into a plurality of parallel segments connected to venting vias isolates the movement of the microvessels due to thermal expansion to each small segment 521a-j, thereby eliminating the ripple effect of the thermal expansion along the entire serpentine channel 520. Thus, the movement of the microvessels within the serpentine channel 520 during temperature cycling can be greatly reduced, improving the ability to track individual microvessels in a large volume sample such as contained within the serpentine channel 520. Minimizing the movement of the microvessels during temperature cycling also minimizes the risk of contamination from the microvessels moving back and forth over space occupied by adjacent microvessels and reduces the likelihood of coalescence of adjacent microvessels.
In use, the venting vias 541a-j are sealed off during loading of the serpentine channel 520 with a high pressure seal to maintain positive pressure on the serpentine channel 520 and ensure loading of the microvessels in the serpentine channel 520. In some embodiments, the first, high pressure seal can comprise a mechanical membrane and one or more rubber gaskets to seal off each venting via 540a-j during sample loading. Alternatively, the venting vias can be covered with photoresist material that can be etched off after loading or a hydrophobic valve that can be overcome by the thermal expansion pressure. Once the serpentine channel 520 has been loaded, the first high-pressure seal can be disengaged or removed. A second seal is then used to isolate the venting vias 540a-j from the outside environment to prevent contamination of the microvessels. The second seal is preferably mechanically compliant to accommodate the thermal expansion and contraction of the microvessel stream in each segment 521a-j during the thermocycling. For example, in some embodiments, the second seal can comprise a thin film or flexible membrane affixed to the venting vias 540a-j. Alternatively, the venting vias 540a-j can be fluidically connected to one or more common venting reservoir(s) on the disposable cartridge which is isolated from the outside environment by a flexible membrane or a lid with a filter.
For example, in some embodiments, the disposable cartridge can have shared reservoirs for the encapsulation media and the PCR mix that are either alternately connected to each chip for example using a motorized stage as described above, or multiplexed to each chip or loading zone. Alternatively, the disposable cartridge can have a plurality of independent oil and PCR mix reservoirs, each attached to a single loading zone. Likewise, in some embodiments, the cartridge can have a single sample reservoir that is multiplexed to each of the cell inputs using one or more valves and fluidic channels. Alternatively, in some embodiments, the cartridge may have multiple sample reservoirs, each independently connected to a single cell input and analysis zone. The cartridge is further configured to allow optical access to each loading zone for performing phenotype analysis.
As previously described in reference to microfluidic chip 500, each loading and analysis zone 610a-d in the 100,000 PCR chip 600 comprises a serpentine channel 620a-d fluidically connected to the sample flow channel 609a-d for amplification of a target sequence in the sample cells in each microvessel. The serpentine analysis channels 620 are partitioned into a plurality of parallel segments with connected by a plurality of turn segments. Each serpentine channel 620a-d is preferably partitioned into between 50-100 parallel segments between about 50-100 mm long depending upon the sample size and the desired spacing between microvessels. As described above, venting vias 640 are fluidically connected to each end of each parallel segment of the serpentine channels 620a-d to seal off the serpentine channels 620a-d during the loading process and to allow for thermal expansion and contraction while isolating the serpentine channel for m the outside environment during the amplification process.
In an alternative embodiment, the phenotype-genotype analysis and correlation can be performed with a sample preloaded into a nanowell array chip.
In step 702, the cells may be sorted based on a parameter selected by the user, such as the measured phenotypic characteristic. The target cell can then be directed to a target flow channel for delivery to the nanowell array. Non-target cells can be directed to a waste flow channel attached to a waste reservoir.
In step 704, the target cells are sequentially placed in the nanowells of one or more arrays. The nanowells are preferably sized to contain one cell each. The nano-wells are preloaded with reagents necessary for gene expression measurements and fluorescent detecting molecules or probes that fluoresce at a level proportional to the quantity of the amplified product when excited. In some embodiments, the number of available wells preferrably exceeds the number of cells being measured to ensure limiting dilution. The individual wells are isolated from each other using a hydrophobic fluidic lid (e.g., oil). The phenotype information for the cells in each nanowell can be indexed and recorded based on the exact location of each cell in the array.
In step 706, the genotype of the individual cells is measured. The cells are lysed by heat, laser, ultrasound, or chemical lysing or any other suitable technique known in the art. The array is thermally coupled to a block heater and the nanowells are cycled through one or more temperatures necessary to amplify the gene products via isothermal amplification or a polymerase chain reaction (PCR). The array is subjected to the desired number of amplification cycles and the amplified gene product is measured.
In step 708, the genotype measurement is decoded. The fluorescent detecting molecules or probe are excited using a light source, such as fixed and scanning lasers, UV lamps, light emitting diodes, and an optical detector, such as CCD imaging, Photomultiplier tubes, photodiodes or photodiode arrays, images the array to measure the intensity of the fluorescent signal from each nanowell and detect the quantity of amplified product. In some embodiments, the optical detector, such as a CCD camera, images the entire array. Alternatively, each nanowell can be sequentially interrogated by scanning the excitation source or by a scanning detector such as a fiber optic couple to a photomultiplier tube). The images are sent to a processor for image processing and correlation to the phenotype measurements previously recorded for each nanowell to provide correlated phenotype and genotype data 74 for each cell.
As shown in
In step 704, the target flow channel 711 is fluidically connected to a microfluidic channel 803 on the nanowell array chip 800 for delivering the stream of target cells to the individual nanowells 801a-f. The target cells 808 are sequentially deposited in individual nanowells 801a-f. Each nanwell location 801a-f has been preloaded with the necessary PCR reagents and fluorescent detectors or markers that fluoresce at a level proportional to the quantity of amplified product The location each cell in the nanowell array creates an index which can later be used to correlate the phenotype measurement for each cell with the genotype measurement based on that location in the array. Each nanowell 801a-f preferably has a volume of a few nanoliters for holding a single cell and the reagents necessary for amplification of a target DNA sequence. For example, in one embodiment, the wells are 100 μm×100 μm squares with a depth of 70 μm. The nanowells 801a-f can be microfabricated using a variety of materials, including but not limited to, glass, quartz, plastics, e.g., polymethylmethacrylate (PMMA), etc., and other castable or workable polymers (e.g. polydimethylsiloxane, PDMS or SU8). The depth of the microfluidic channels 803 connecting the nanowells 801a-f is typically in, but not limited to, the range 10 μm to 100 μm. The width of the microfluidic channels is typically, but not limited to, 1 to 5 times the depth. Once the cells have been loaded into the individual nanowells 801a-f, oil or any other suitable encapsulation media is flowed through the microfluidic channel 803 to isolate the individual cells along with the PCR reagents in each well.
Next in step 706, the isolated cells are lysed and cycled through a temperature profile needed to achieve a polymerase chain reaction. For example as shown here, the array 800 is cycled through a first temperature around 96° C., a second temperature around 60° C. and a third temperature around 72° C. In some embodiments, the temperature of the nanowell array can be adjusted to produce suitable temperatures for PCR by using one or more heating elements know in the art such as a heating block, integrated heating wires, Peltier heaters, or by circulating hot/cold fluid or hot/cold air. The desired number of PCR cycles are performed and the amplified product is measured. The flouroprobes or markers in each nanwell are excited by a light source, such as fixed and scanning lasers, UV lamps, light emitting diodes, and an optical detector 815, such as CCD imaging, Photomultiplier tubess, photodiodes or photodiode arrays, images the nanowells to measure the intensity of the fluorescent signal. In an alternative embodiment, UV light may be used to measure the absorption of the nucleic acid product. The measurements of the genotype can then be indexed according to their location on the array. Thus, the phenotype and genotype measurements for each individual cell can then be correlated based on the location in the nanowell array.
In an alternative embodiment shown in
As shown in
In some embodiments, as shown in
An additional method of use of the nanowell embodiment shown in
Although the foregoing invention describes methods and devices for correlating flow cytometry phenotype data with nucleic acid amplification, one can easily imagine performing other molecular assays such as DNA methylation, protein abundance, cytokine detection, or other enzymatic or protein assays in the microvessels for correlation with phenotype measurements for each cell. Moreover, while emphasis has been placed on performing single cell measurements and correlation within the microvessels, it should be appreciated that the above described devices and methods can be used for correlating phenotype-genotype data for measurements on multiple cells.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to those of ordinary skill in the art, in light of the teachings of this invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the invention.
This application claims the benefit of provisional application no, 60/954,946 filed Aug. 9, 2007, entitled “Method for Correlated, Multi-Parameter Single Cell Measurements and Recovery of Remnant Biological Material,” which is hereby expressly incorporated by reference in its entirety.
Number | Date | Country | |
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60954946 | Aug 2007 | US |