HK1206673B - Methods and devices for correlated, multi-parameter single cell measurements and recovery of remnant biological material - Google Patents

Description

Method and apparatus for correlated multiparameter single cell assay and recovery of residual biological material

The application is a divisional application of a same-name application with the application number of 200880107316.3, and the application date of 2008, 8 and 8.

Technical Field

The present invention relates to a method and apparatus for automated, correlated quantitative determination of proteins and nucleic acids from cell to cell in high throughput samples obtained by sorting and collecting genomic material.

RELATED APPLICATIONS

The international application claims the right of provisional application No.60/954,946 entitled "method of correlated multiparameter single cell assay and recovery of residual biological material" filed on 8, 9, 2007, which is incorporated herein by reference in its entirety.

Background

Fluorescence Activated Cell Sorting (FACS) is widely used in research and clinical applications. The instrument has the ability to perform multi-parameter analysis and sorting very quickly, but it typically requires large sample volumes and trained operators for operation and maintenance, and is difficult to sterilize. FACS instruments can analyze as few as 10000 and up to tens of millions of cells. In sorting applications, however, the ability to perform sorting is reduced because the sample size is less than 100,000 cells. In all cases, the cells must be labeled beforehand. In most cases, an antigen or similar membrane bound protein is labeled with an antibody conjugated to a fluorescent molecule (such as fluorescein isothiocyanate, also known as FITC), but nuclear stains, intracellular dyes, or cell-directed synthesis of fluorescent proteins (e.g., green fluorescent protein, also known as GFP) can also be detected by flow cytometry. Flow cytometry is also suitable for molecular detection. For example, fluorescent beads having various colors and/or intensities may be used as solid supports for antibody capture assays of protein or polypeptide analytes. Likewise, nucleic acids can be hybridized to beads and fluorescent labels for rapid reading using flow cytometry. In both cases, biochemical assays are performed prior to using flow cytometry assays, and the instrument can be used as a bead rapid reader. In some cases, scanning cytometry is equally effective in reading. FACS instruments support multiple information to the number of independent fluorescence channels supported by the instrument, but the multiple information is of a single type (e.g., phenotype only).

Real-time polymerase chain reaction (also known as qPCR) is a technique for quantitative determination of DNA and RNA extracted from cells of interest. Most qPCR reactions were performed using pooled genomic quantities corresponding to 10,000-100,000 cells. Researchers are increasingly interested in determining the genetic content of individual cells, but this effort is hampered by the high cost of reagents and labor intensive manipulations currently available. Therefore, most single cell PCR studies are only performed on less than 100 cells. Even the existing robotics and 1536 microplates in 1-10 mul/well are more expensive than hundreds of wells. When the rare case that the ratio of the desired cells to the number of cells is less than 1% occurs, 50000 cells need to be examined one by one. Current technology cannot achieve this level of throughput without significant expenditure of time and money.

In the work on signal transduction studies and system biology, it is often necessary to correlate differential information. Data linking cell surface receptors or reporters with intracellular signal transduction are increasingly valuable for these activities. To date, the information is based on a large number of cellular associations. Typically, thousands to millions of cells are assayed for surface protein or RNA expression and the information statistically correlated. Any heterogeneity in the sample was averaged out during the assay. Powerful techniques such as siRNA gene silencing can introduce heterogeneity into a sample by itself, and are therefore limited by this averaging of information.

Many researchers have obtained values that quantify protein (or other phenotypic characteristic) and nucleic acid associated genotypes on a cell-by-cell basis. Microfluidics (microfluidics) has been identified as a technique that enables the instrument to perform the assay, but has not resulted in methods and instruments as examples.

Microfabricated cytometers have the potential to analyze and sort as few as 1000 cells with the concomitant consumption of less reagents in an easy-to-use closed system. The former is important when working with high cost reagents such as replicase. The latter is important because, unlike conventional FACS instruments, it does not produce aerosols, reducing the risk of contaminating the sorted cells and working with biohazardous materials. Several microfabricated cell analyzers and sorters have been described, but most are "proof of concept".

By combining these elements of microfabricated cytometry, single cell encapsulation technology and appropriate signal processing, an instrument with correlation of protein expression and gene expression for cell-by-cell detection can be produced.

Disclosure of Invention

These elements combine to achieve a relevant assay in a microfluidic network, as described below. Cells were previously labeled for phenotypic determination. In one embodiment, a continuous flow microfluidic network integrates all functions. In a first part of the microfluidic network, cells are assayed for fluorescent signals produced by phenotypic markers. The second part of the microfluidic network independently encapsulates the cells in microreactors with a predetermined mixing reagent suitable for gene expression assays. In the next part of the microfluidic network, the encapsulated cell stream is encoded with a reference signal that can be contained in or separated from the microreactor contents. In the fourth part of the microfluidic network, cells are lysed and gene expression is determined using a suitable technique, such as real-time polymerase chain reaction (aka real-time PCR) or qPCR. The microreactor streams are then decoded at the next stage and signal processing algorithms correlate the phenotypic and genotypic determinations. As a final optional step, the microreactors may be sorted to enrich for a particular genome. In a related embodiment, the cells are first encapsulated so that phenotyping and coding occur simultaneously. The cells are subsequently lysed and then gene expression assays and decoding are performed simultaneously. This reduces the number of interrogation points on the microfluidic network.

In a second embodiment, the microfluidic network is combined with a microwell array to isolate individual cells and correlate user pre-selected genetic information with a phenotypic population. In the first part, the cells are assayed for fluorescent signals generated by phenotypic markers using a microfluidic network. In the second section, cells are sorted into nanofluidic (nanofluidic) microwells using a multi-path switching network. The wells may be arranged so that each cell is located in a uniquely defined coordinate, or the wells may be arranged to divide the cells into more than 2 arrays. In the latter case, the user selects more than one gate to select the desired destination of the cells based on the acquired phenotypic information. Sealing the wells to individually encapsulate the cells with a predetermined mixed reagent suitable for gene expression assays. The cells are lysed and gene expression is determined by reading the array using a suitable technique. Reaction information in the microwells can be analyzed to detect statistical distributions of genetic signals, and this information can be correlated in turn with selected phenotypic discrepancies. As a final optional step, the contents of individual microwells can be collected for further genetic analysis.

In a third embodiment, individual cells are isolated using scanning cytometry using a nanofluidic (nanofluidic) microwell array and associated phenotypic and genotypic information is obtained. Similarly, cells were previously labeled for phenotypic determination. Placing the cells in the microwells and sealing the wells to individually wrap the cells with a predetermined mixed reagent suitable for a gene expression assay. Scanning cytometry analysis of cells collects phenotypic information directed to the well coordinates, lyses the cells, and then determines gene expression by reading the array using a suitable technique. Concatenating the signals to a single correlation data set. As a final optional step, the contents of individual microwells can be collected for further genetic analysis.

In all embodiments, more than one phenotypic assay may be performed. Typically, up to 4 or 5 independent fluorescent signals are detected simultaneously. The measured value of gene expression in each reaction can be a measured value of one gene or preferably a measured value of a complex of two or more genes to obtain standardized quantitative information about the expression level of the selected gene.

Brief Description of Drawings

FIG. 1 is a conceptual diagram showing how phenotypic (e.g., cell surface proteins labeled with fluorescence-coupled antibodies) information and genotypic (e.g., single cell, multiplex quantitative PCR) information can be obtained and correlated individually for analysis in accordance with the present invention.

Fig. 2 is a schematic block diagram of the elements of the present invention in continuous flow form.

FIG. 3 is a schematic block diagram of the components of the present invention in a modified continuous flow format or scanning cytometry format.

Fig. 4 is a schematic view of a microfluidic element used in the continuous flow embodiment of fig. 2.

FIG. 5 is a schematic illustration of an example of how phenotype and genotype determination correlations can be obtained by encoding and decoding periodic events or non-periodic events in the continuous flow embodiment of FIG. 2.

FIG. 6 is a functional block diagram of an embodiment of an integrated system comprising a freestanding disposable cartridge and an instrument for performing phenotype and genotype correlations.

Figure 7 illustrates an embodiment of a sample loading cartridge for use with the microfluidic network shown in figure 6.

FIG. 8 illustrates an embodiment of a sample loader, microfluidic loading chip and capillary for cell-by-cell phenotypic and genotypic analysis and correlation.

FIG. 9A illustrates an embodiment of a thermal control assembly for use with the capillary tube shown in FIG. 8.

FIG. 9B illustrates an embodiment of a capillary tube holder in the thermal control assembly shown in FIG. 9A.

FIG. 9C illustrates another embodiment of a capillary tube holder in the thermal control assembly shown in FIG. 9A.

FIG. 10 illustrates an embodiment of a microfluidic chip for use with the sample loader shown in FIG. 7 for cell-by-cell phenotypic and genotypic analysis and correlation.

FIG. 11 illustrates another embodiment of a microfluidic chip for use with the sample loader shown in FIG. 7 for cell-by-cell phenotypic and genotypic analysis and correlation.

FIG. 12 is a functional block diagram of a discrete correlation embodiment of single cell phenotype and genotype analysis and correlation.

Fig. 13A is a schematic diagram of a microfluidic element and nanohole (nanowell) array for performing the discrete association embodiment shown in fig. 12.

FIG. 13B is a schematic illustration of a step for sorting and indexing individual cells in the embodiment shown in FIG. 13A.

Fig. 14A illustrates another embodiment of a method associated with nanopore dispersion in a multi-branched microfluidic circuit.

FIG. 14B shows phenotype-genotype correlations for samples sorted into 9 phenotype populations using the embodiment shown in FIG. 14A.

FIG. 15A is a schematic representation of the elements of another embodiment of phenotypic and genotypic analysis and correlation of single cells using scanning cytometry in a nanopore (nanowell).

FIG. 15B shows the step of scanning and indexing individual cells in the embodiment shown in FIG. 15A.

FIG. 16 is a schematic diagram of microfluidic elements used in a continuous flow embodiment of a single cell genome analyzer and sorter.

FIG. 17 is a schematic representation of microfluidic elements and nanowell (nanowell) arrays used in discrete embodiments involving genotyping.

FIGS. 18A-D show actual micrographs of an actual device showing a flow pattern.

Fig. 19 is a tracking oscilloscope showing the amplitude as a function of time for cell forward scatter and droplet forward scatter.

FIG. 20 is a micrograph of six parallel capillaries containing encoded drops of different lengths.

FIG. 21 is a micrograph of three capillaries with encoded drops of different lengths and order.

FIGS. 22A and B show the results of detection in the micro-container before and after PCR, respectively.

FIGS. 23A and B show the micro-containers in a three-phase system before and after PCR amplification.

Detailed Description

The present invention provides methods and devices suitable for cell-by-cell analysis of phenotypic (e.g., cell surface or intracellular proteins labeled with fluorescent-conjugated antibodies or constitutively expressed fluorescent reporter molecules (e.g., green fluorescent protein)) and genotypic (e.g., single cell, multiplex quantitative PCR or RT-PCR for detection of Single Nucleotide Polymorphisms (SNPs), DNA copy number or RNA gene expression) information, for example, on samples ranging from hundreds to thousands to hundreds of thousands of cells. Larger sample sizes facilitate the use of multidimensional scatter plots to reveal biological patterns and relationships between genes and proteins. As shown in fig. 1, the main object of the present invention is to obtain selected phenotypic characteristics and selected gene expression by interrogating individual cells one by one and then correlating the information cell by cell. Methods for correlating phenotypic-genotypic information for high throughput samples can be performed on continuous flow samples in microfluidic networks, or on static samples pre-loaded in a nanopore (nanowell) array chip. For example, a static sample preloaded in a nanopore (nanowell) chip may be phenotyped using flow cytometry in a continuous flow embodiment or using scanning cytometry to detect a fluorescent signal indicative of a physical characteristic of a cell in the sample, such as cell morphology, cell size, or localization of a fluorescent label (in the nucleus, cell surface, or cytoplasm). In certain embodiments, the multiplex assay is performed using, for example, two, three, or four color fluorescence detection. Other phenotyping methods include measuring light scattering, i.e., forward and side scattering by the cells. The cells are then encoded to create a phenotyping index for subsequent association. In a nanowell chip, the cell locations on the array provide the encoded signals. In a continuous flow embodiment, cells are packed in nanoliter micro-containers, and dyes, coded beads, or any optically detectable physical parameter can be pseudo-randomly combined with the micro-containers to create an index. The cells are then lysed and the target DNA sequence is amplified (e.g., by isothermal amplification or Polymerase Chain Reaction (PCR) (including end-point PCR, real-time PCR (RT-PCR), or quantitative PCR (qPCR)), or any other method of amplifying nucleic acids) to determine gene expression, genotype, or other desired characteristic of the nucleic acid in the cells. The amplification products (i.e., amplified DNA sequences) in each of the micro-containers are detected and determined with an optical image sensor and this data is sent to a processor to be correlated with the previously determined phenotypic information in each of the micro-containers. The phenotypic data in each micro-container is decoded using indices created from reference signals or nanopore (nanowell) array positions and correlated with the genotype information obtained from amplification of the target DNA or RNA, such as Single Nucleotide Polymorphisms (SNPs), copy number of DNA, or detection of RNA gene expression.

FIG. 2 shows the steps of analyzing individual cells and correlating phenotypic and genotypic data in a continuous flow embodiment. In step 100, the entire population of sample inputs 10 containing a plurality of cells in an aqueous solution is analyzed for selected phenotypic characteristics, such as protein expression, cell morphology, cell size, or location of fluorescent markers (in the nucleus versus on the cell surface or in the cytoplasm). First, the cells are labeled, e.g., with an antibody labeled with a fluorescent molecule (fluorescent dye), green fluorescent protein, or a dye. The sample is then introduced into microfluidic flow cells and the cells are pooled into a single cell stream. The fluorescent signal generated by the cell flow was measured individually by phenotypic markers. In some embodiments, the plurality of phenotypic characteristics is determined using four color flow cytometry. Next, in step 102, the cells are individually packaged in nanoliter micro-containers or droplets containing a predetermined mixture of reagents suitable for amplifying the target DNA sequence in the cells. For example, as discussed further below, the cell and PCR reagent mixture may be introduced into flow channels in a microfluidic network designed to separate and encapsulate individual cells in a water-in-oil trickle flow. The droplet or micro-container preferably has a cross-sectional diameter larger than the sample cells and smaller than the cross-sectional diameter of the flow channel. For example, the micro-containers are preferably between 30 μm and 500 μm, depending on the size of the flow channels. Most micro-containers preferably contain one cell or 0 cells according to poisson distribution based on cell concentration and drop volume. In addition, active control of the encapsulation (e.g., metering cells in a controlled manner using constriction, valves, or gates) can be used to load 0 or one cell in a non-poisson distribution. For example, in some embodiments, at least 90% of the micro-containers contain 1 or 0 cells, or at least 80% of the micro-containers contain 1 or 0 cells, or at least 70% of the micro-containers contain 1 or 0 cells, or at least 60% of the micro-containers contain 1 or 0 cells. The number of cells in each microreactor was determined to normalize the resulting data. For example, in one method, the flow channel is illuminated and a signal (e.g., forward scatter) is detected with an optical detector that indicates the presence or absence of a cell in the droplet. More sophisticated detection uses detection by a forward scatter array to provide better spatial resolution to calculate and locate cells/particles in the droplet. In another approach, dark field illumination and imaging may be added to a single detector scatterometry. The CCD camera can simultaneously capture dark field images of the droplet as the forward scatter signal detects the leading edge of the droplet. The cells/particles will be bright spots in the image, which can be processed and identified for counting.

In some embodiments, the cells may be sorted based on a user-selected parameter (e.g., a measured phenotypic characteristic). For example, the presence of target cells having the desired characteristics may be detected in the fluid stream by fluorescence, forward scatter, or any suitable imaging or detection format. The target cells can then be introduced to the target flow channel for encapsulation and/or encoding using an dielectrophoresis, pneumatic switch, or an ovonic fluidic or optical switch (as described in co-pending patent application serial No. 11/781,848 entitled "cell sorting system and method," which is incorporated herein by reference in its entirety). Non-target cells can be introduced to a waste channel connected to a waste reservoir. In some embodiments, the cells may be sorted prior to the phenotypic assay. Additionally, the cells can be sorted based on the determined phenotypic characteristic. In another embodiment, a three-phase system may be formed using two oils, with one oil acting as the loading fluid and the second oil acting as the inter-droplet spacing. This suppresses coalescence of adjacent water droplets. FIGS. 23A and B show examples of micro-containers in a three-phase system before and after PCR amplification.

In step 104, the micro-containers are encoded with reference signals to index phenotypic information of the cells in the micro-containers for subsequent correlation with gene expression of the cells in the micro-containers. The reference signal may be contained in or separated from the micro-containers. The reference signal may be generated in a pseudo-random pattern using any physical parameter that has a unique signature and that can be optically measured. One or more encoded micro-vessel images are then recorded and stored for subsequent comparison and correlation with the micro-vessel images after genotyping with assays contained in or isolated from the microreactors. For example, in some embodiments, fluorescent microbeads or dyes can be injected into the micro-containers in a pseudo-random pattern, which is then imaged. In addition, the size of the micro-containers, the length of the micro-containers, and/or the spacing between the micro-containers may be varied to create a reconstructable pseudo-random pattern. The reference signal time interval in the encoding mode may vary depending on the required tracking accuracy. For example, in some embodiments, a reference signal may be associated with each micro-container, or a reference signal may be associated with every 10 micro-containers, or with every 100 micro-containers, or with every 200 micro-containers, or with every 500 micro-containers, or with every 1000 micro-containers.

In step 106, the genotype of the cell is determined. The cells are lysed and subjected to the thermal conditions required for amplification (e.g., isothermal amplification, end-point PCR, reverse transcription PCR, or quantitative PCR) of the target DNA sequence. In embodiments where amplification is performed by PCR, the temperature is cycled to generate the appropriate temperature for the number of cycles required for PCR. This may be accomplished by a standard thermal cycler using a thermal block or Peltier device, or may be accomplished by alternate techniques such as an oven, hot and cold air, flowing a heated liquid with good thermal conductivity, transporting the device between instrument components at different temperatures, or any other suitable heating element known in the art (the same list is considered applicable to the other embodiments described herein). In some embodiments, the micro container can be continuously through the fixed temperature zone of the serpentine flow channel, to complete the polymerase chain reaction. Alternatively, the micro-containers may be loaded into the flow channels andmore or less static electricity is retained while the temperature of the flow channel is cycled through the desired temperature profile repeatedly to complete the pcr. The micro-containers are subjected to the required number of PCR cycles to amplify the target DNA and the amplification products are determined. The mixed reagent that coats the cells in the microreactors may include non-specific fluorescent detection molecules (e.g., intercalating dyes), or sequence-specific fluorescent probes (e.g., molecular beacons,probes) or any other suitable probe or label known in the art that emits fluorescence in proportion to the amount of amplification product when excited. The probe can bind to a double-stranded DNA product, a single-stranded DNA product, or a non-product oligonucleotide produced by a DNA amplification process in an amount proportional to the DNA product. The imaging system uses light of the desired wavelength to excite the fluorescent probes to directly or indirectly measure the amplified product.

In step 108, the genotype determination is decoded. An optical detector (e.g., a photomultiplier tube, a CCD camera, a photodiode or photodiode array, or other optical detector) measures the intensity of the fluorescent signal emitted from the probe or label in each of the micro-containers and measures the amount of amplified product at least once during each PCR cycle. The image of the optical detector is sent to a computer for image processing and comparison to record the encoding pattern of the micro-containers. In step 110, the images obtained from the genotyping and the index images may be compared using image processing algorithms known in the art, and the genotyping and phenotyping may then be correlated cell-by-cell.

In some embodiments, in step 112, the micro-containers can be sorted based on the measured gene expression to capture the target nucleic acid for further analysis. The optical switch described above can be used to direct the micro-containers into the target flow channel or waste flow channel based on the genotyping assay.

In another embodiment, as shown in FIG. 3, multiple steps may be performed simultaneously to minimize the number of interrogation spots on the microfluidic network. Here, the cells for phenotypic analysis that have been labeled in the sample input 12 are introduced into the instrument and, in step 102, are individually wrapped in micro-containers or drops containing predetermined mixed reagents suitable for amplifying the target DNA in the cells. The reference signal for encoding may be simultaneously wrapped in a transparent micro-container or otherwise the flow conditions may be controlled to pseudo-randomly alter the physical characteristics of the micro-container to establish the reference signal. In step 103, a phenotype and reference signal may be determined and recorded in a step to encode phenotype information and create an index for association with a genotype determination. Next in step 105, the genotype and decoding signals may also be determined in one step. For example, in the first embodiment described above, the genotyping light source and detector are different from the decoding light source and detector. Here, the light source and/or the detector may be shared.

The individual cells are lysed and the micro-containers are then subjected to the thermal conditions required for amplification (e.g., real-time PCR or quantitative PCR) of the target DNA sequence. The micro-containers are subjected to the desired number of PCR cycles to amplify the target DNA. The amplification product in the PCR cycle is determined by exciting the fluorescent probe contained in the PCR reagent mixture and detecting the fluorescence intensity with an optical detector. The optical detector may be a scanning detector using a continuous flow system, or a stationary detector. The graphics are sent to a computer for processing and comparison of the images to record the encoding pattern of the micro-containers. The genotypic and phenotypical measurements can then be correlated cell by cell.

FIGS. 4-5 show the above steps in further detail and are performed using microfluidic components. In step 100, a phenotypic analysis is performed by introducing cells 200 into a flow channel 209 via microfluidic inlet 202. The cell 200 is clamped by a sheath buffer flow 201 established by two lateral sheath buffer channels 203, 204. The buffer used in the sheath flow can be any buffer that is biologically compatible with the cells being analyzed and is compatible with the optical illumination used in the fluorescence detection (i.e., the buffer has sufficiently low absorbance at the fluorescence excitation/detection wavelength) and optical switching wavelengths. A preferred embodiment of sheath buffer uses PBS/BSA in Phosphate Buffer (PBS) at pH 7.2 containing 1% Bovine Serum Albumin (BSA) fraction 5.

Sheath buffer 201 focuses cells 200 into a single beam of cells 200, which are then interrogated at analysis zone 205 to determine one or more desired phenotypic characteristics of the cells. An illumination source (e.g., a stationary or scanning laser, UV lamp, light emitting diode, or other collimated light source) may cause a label (e.g., a fluorescent dye, antibody, GFP, or other fluorescent pigment) attached to the cells 200 to fluoresce and scatter the cells and containers to provide information (e.g., size, morphology, or border) about the physical properties of each cell and container. More than one optical detector (e.g., CCD imaging, PMT, or photodiode array) measures the resulting signal. Other types of optical measurements (e.g., light scattering) may also be performed at the analysis zone 205 to determine the phenotypic characteristics of the cells 200. In some embodiments, a multi-phenotype assay may be performed using, for example, four color flow cytometry or by measuring fluorescence and/or scattering. For example, a multi-phenotypic assay may be performed by labeling the cells with multiple fluorophores, or using other fluorescence detection channels that are sensitive to fluorescence emitted at different wavelengths, typically using a single excitation wavelength (e.g., but not limited to 488 nm). Here, each detection channel incorporates a PMT with a suitable dichroic mirror and an emission filter for the fluorescence emission wavelength of the additional fluorophore. In this way, channel adaptation is accomplished from 2-4 fluorescence detections.

In the embodiment shown, the PCR mixture containing the reagents required for the gene expression assay and the fluorescent DNA detection molecules or probes is injected into a flow stream (flow stream) through lateral reagent flow channels 206a, b placed between sheath flow channels 203, 204 and encapsulation flow channels 207a, b. In another embodiment, the PCR mixture can be introduced into the cell stream through the sheath buffer flow channels 203, 204.

In a next step 102, the encapsulation is performed by introducing a hydrophobic encapsulation medium into the flow channel 209 via the lateral encapsulation flow channels 207a, b. Silicone oil, mineral oil, fluorocarbon oil, or other hydrophobic liquids may be used to facilitate the creation of dispersed water droplets. The entrapment medium holds the PCR mixture stream and cells into a water-based nanoliter micro-vessel or micro-reactor 208. The nanoliter micro-containers 208 preferably have a diameter larger than the cells 200 but not significantly larger than the microfluidic channels 209. The flow conditions in the encapsulation flow channels 207a, b are preferably chosen to ensure that preferably 0 or one cell/micro-container is encapsulated. Microfluidic channels, for example, that are flow cytometry compatible, typically have a cross-section of 50 μm to 100 μm by 150 μm to 300 μm, and polytetrafluoroethylene capillaries are available with diameters as small as 400 μm, so that the diameter of the micro-containers 208 is in the range of 30 μm to 400 μm.

In a next step 104, sequences of microreactors 208 are encoded and decoded to aid in correlating phenotypes with genotypes. As shown in fig. 5, the encoding is performed between the phenotypic analysis and the oil droplet wrapping by pseudo-randomly attaching more than one encoding bead 220 to some of the cells 200 in the cell stream. In the illustrated embodiment, the encoded beads 220 have a fluorescent signal that can be read and recorded to create an index of the location of individual micro-containers 208 in the stream of micro-containers 208. Alternatively, optically detectable pulses of dye can be linked to individual cells in a pseudo-random pattern. Once the encoded beads 220 are attached to the cells 200, the cells 200 are packed into individual micro-containers 208, the micro-containers 208 then flow through a decoding laser 225 that causes the encoded beads 220 to fluoresce, and one or more detectors 225 measure the resulting signals to encode the micro-containers 208 and create an index of the micro-containers 208. The pattern of encoded beads (or dyes) and cells recorded becomes the only marker that can be used after cell lysis, so that the signal from each reactor is in turn correlated with the determined cell phenotype. After amplification, a second decoding laser 226 is placed adjacent to the serpentine flow channel 210 to image the micro-containers. The second decoding laser 226 uses a light source (e.g., a fixed or scanning laser, UV lamp, or light emitting diode) to cause the encoded beads 206 to fluoresce or scatter, and an optical detector (e.g., CCD imaging, photomultiplier tube, photodiode, or photodiode array) to measure the signal to create an image of the micro-containers 208 that can be compared to the stored encoded image to identify individual micro-containers 208. The sequence of events is typically determined using an error correction algorithm that is used to identify data bits that are missing in the magnetic data memory.

In the illustrated embodiment, the second redundant coded signal is provided by changing the flow conditions in the flow channels 209 to change the spacing between the micro-containers 208. As shown in fig. 5, this creates another optically detectable reference signal (i.e., a liquid edge signal) that can be imaged, stored, and reconstructed to track individual micro-containers 208. In another embodiment, any method for creating a unique, reconstructable pattern of reference signals can be used independently to encode the micro-containers. Furthermore, in both embodiments, the encoded signal produced by increasing or decreasing the frequency of linking the encoded information to individual micro-containers is more or less strongly dependent on the level of acceptable error in cell tracking. For example, in some embodiments, a coded bead may be attached to each micro-container. In addition, random patterns of encoded beads can be applied to each 10 containers, or each 50 containers, or each 100 containers, or each 500 containers, or each 1,000 containers of multiple micro containers. Similarly, if the encoded signal is created by varying the distance between the micro-containers, the distance may be adjusted to be encoded in blocks of 10 containers, or blocks of 50 containers, or blocks of 100 containers, or blocks of 500 containers, or blocks of 1000 containers.

Next, in step 106, the encapsulated cells 200 are lysed and their gene expression is analyzed using real-time PCR or quantitative PCR. The cells can be lysed using a variety of methods known in the art, including photolysis with a laser, sonication-based lysis, or chemical lysis. The micro-containers 208 then flow through the serpentine microfluidic channel 210 such that the micro-containers 208 flow through one or more thermal zones required for amplification and real-time detection of the amplified gene products. In the illustrated embodiment, serpentine channel 210 repeatedly flows through three temperature zones 211, 212, and 213. The warm zone is maintained at an appropriate temperature for the PCP cycle. For example, as shown herein, the first temperature zone 211 is about 60 °, the second temperature zone 212 is about 72 °, and the third temperature zone 213 is about 96 °. The temperature control system adjusts the flow conditions to ensure that the micro-containers remain in each temperature zone for the appropriate PCR cycle time. The micro-container 208 is caused to flow through the temperature zones 211, 212, 213 a plurality of times, and measurement is performed after each pass to measure the amount of the amplified product. As discussed above, the PCR mixture includes probes that emit a level of fluorescence proportional to the amount of amplification product. The fluorophores may be excited by a light source 214 (e.g., fixed and scanning laser, UV lamp, light emitting diode) at a particular time or temperature during each PCR cycle and detected by a detector 215 (e.g., photomultiplier tube, CCD camera, photodiode or photodiode array, or other optical detector). The detector 215 measures the fluorescence intensity to determine the amount of amplification product in the microreactor 208.

Next, in step 108, the previously recorded phenotypical measurements are decoded. As shown in fig. 5, a second decoding laser 225 reads the encoded signal from an individual micro-container 208 as the individual micro-container 208 is amplified in the serpentine channel 210. The encoded signal and gene expression assay results are then sent to a processor for decoding of the encoded signal and correlation of the phenotypic information of the cells to which the encoded signal is linked with the newly determined gene expression.

In some embodiments, it is desirable to recover genetic material from a selected portion of the cells being assayed, or to sort the cells based on genetic markers (without correlating phenotypes). For example, as shown in fig. 12, in some embodiments, at the end of a gene expression assay, micro-containers 208 may be sorted into two pools, such as waste pool 231 and target pool 230. The micro-containers are sorted using a lateral force switch 225 to switch flow of the micro-containers 208 between laminar flows into the target flow channel 220 and the waste flow channel 21 based on the correlated phenotype-genotype determination. The lateral force switch is controlled using optical force, dielectrophoresis, fluidic pulses or similar means. In some embodiments, no phenotypic assay is performed and sorting after PCR amplification is based solely on genotypic assay.

Fig. 6 illustrates an embodiment of an integrated system for cell-by-cell phenotypic and genotypic correlation in a microfluidic network. Instrument 300 contains a reusable platform that holds thermal assembly 302, excitation sources 303, 308, and detectors 312, 304, and 306 as needed to interrogate and analyze the sample contained in cartridge 320. A disposable sample loading cartridge 320 containing a microfluidic network chip is attached to the instrument 300 chip for analysis of the sample. The cartridge 320 has a plurality of wells 321-324 configured to contain the cell sample and all reagents, buffers, coding dyes or coding beads and encapsulation media necessary to encode, isolate and amplify the sample cells. In the embodiment shown, cell sample is contained in well 323, oil-encapsulating medium is contained in well 324, encoded beads are contained in well 322, and sheath buffer is contained in well 321 for generating a single cell stream. Here, the sheath groove 321 also includes a PCR mixture containing a reagent required for amplification and a fluorescent label.

The chip is attached to the optical window area of the cartridge 320 with a UV adhesive and the chip input port is attached to its corresponding slot volume (reservoir volumes) on the disposable cartridge 320. An input port in the sample flow path 309 on the chip is fluidly connected to the cell sample reservoir 323 on the disposable cartridge to introduce the cells into the sample flow path 309. The cell sample chamber 323 has a generally conical shape, tapering toward the input port. In a preferred embodiment, the inlet slot contains a polypropylene insert to minimize cell adhesion and thus maximize cell yield. Microfluidic channels 305, 307 on the chip flow through the outlets of connecting slots 321, 324 to sample flow channel 309. Lateral flow channels 305a, b are configured to add PCR mixture to sample flow channel 309, and then lateral flow channel 307 directs oil into sample flow channel 309 to encapsulate the sample cells in the oil. A cartridge 320 is positioned within the instrument 300 to position a light source (e.g., a 488nm laser 303 and a fluorescence detector 312) adjacent to the sample flow channel 309. The cassette is preferably made of an optically clear acrylic plastic. The optical viewing window also allows for visual interrogation of selected sites in the microfluidic network and projection of the excitation light source and optical detector through the cartridge to the microfluidic chip. Other optically clear plastics or suitable materials may be substituted for the acrylic, if appropriate. The microfluidic channels are typically made of optically transparent material to allow cell detection light to be projected into the sample flow channels 309. The substrate is typically, but not limited to, glass, quartz, plastics (e.g., Polymethylmethacrylate (PMMT), etc.), and other moldable or workable polymers (e.g., polydimethylsiloxane, PDMS, or SU 8).

Light from the 488nm laser 303 is projected through the cartridge 320 into the sample flow channel 309 upstream of the encapsulation zone to interrogate the cells. 488nm laser 303, selected phenotypic characteristics of the sample cells. The fluorescence detector 312 measures fluorescence to determine phenotypic information of the cells. An encapsulation medium (e.g., oil) is then introduced into the sample flow channel 309 under suitable conditions to encapsulate the individual cells into individual nanoliter micro-containers. The micro-containers are then sorted for further genotyping via the sample flow channel 309 based on more than one determined phenotypic characteristic of the encapsulated cells, or into the waste flow channel 330 for transport to the waste tank 334. As the micro-containers flow through the sample flow channel 309, the index decode laser 308 excites the index beads previously attached to the cells in a pseudo-random order. The decoding sensor 306 detects, images and stores the sequence of index beads attached to cells so that they flow through the sample flow channel 309 to create an index of micro-containers for correlation with subsequent genotyping images. Once the micro-containers have been encoded, they are loaded into a serpentine microfluidic channel on a microfluidic chip for PCR amplification. The thermal assembly 302 on the instrument is placed adjacent to the preloaded microfluidic channels and the micro-containers in the microfluidic chip are cycled through the temperature zones required to complete the PCR amplification. Thermal assembly 302 includes thermal control elements and a standard thermal cycler using a thermal block or Peltier device, which can also be accomplished using other techniques, such as one or more integrated heating wires, an oven, hot and cold air, flowing a heated liquid with good thermal conductivity, a transfer device between instrument assemblies at different temperatures, or any other suitable heating element known in the art. In some embodiments, multiple heating elements may be used to create a spatial thermal zone through which the chip physically passes with a motive element. In addition, a single heating element (e.g., a cold/hot air heater) may 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 causes the serpentine channel to cycle through a PCR temperature profile that completes the desired number of PCR cycles.

The micro-containers were analyzed after each PCR cycle to determine the amount of amplified product. As described above, the PCR mixture contains a probe that emits a level of fluorescence proportional to the amount of amplification product. The probes may be excited by a light source (e.g., stationary and scanning lasers, UV lamps, light emitting diodes) at specific times or at specific temperatures during each PCR cycle. The fluorescence signal is measured by a PCR image sensor 304 (such as a photomultiplier tube, a CCD camera, a photodiode or photodiode array, or other optical detector) positioned adjacent to the microfluidic chip. The image sensor 304 measures the fluorescence intensity to determine the amount of amplified product in the microreactor 208 and the genotype of the cells in each microcontainer. The index decode laser 308 also excites the index beads attached to each micro-container and images the encoded signal through the decode sensor 306. The encoding signals and genotype data for each micro-container are then sent to the processor to index and correlate the phenotypic assay with the genotype data from each micro-container 208. In some embodiments, the micro-containers are imaged and tracked cycle by cycle, for example using droplet size and position in each image. In another embodiment, the microwavable is imaged after the desired number of PCR cycles is completed and the encoded signal is read.

FIG. 7 illustrates an embodiment of a disposable sample loading cartridge for use with a microfluidic capillary or microfluidic chip for performing single cell analysis and correlating phenotypic and genotypic information. The cartridge has 6 built-in grooves 321-326, each with an output port individually configured to provide an interface with a microfluidic channel on a connection chip. The slot volumes (reservoir volumes) 321-326 are sealed with snap-on caps 340 having drilled-in ports for connecting the pneumatic controller and each slot 321-326. The ports can apply air pressure to the wells, which can drive fluid and cells through the microfluidic network of the attached microfluidic chip by applying pressure to the wells 321-326 individually. In another embodiment, the fluid may be driven by motion of a syringe pump, peristaltic pump, or other means. In the embodiment shown, well 323 is configured to hold a volume of sample of about 5-50 μ L; the well 324 is configured to hold a volume of about 50-1500 μ L of a packing medium (e.g., oil), and the well 321 is configured to hold a PCR mixture. In some embodiments, the groove 322 may be configured to receive a coded media (e.g., a fluorescent bead or dye). Each of the wells 321, 322, 323, and 324 contains a port configured to be in fluid connection with an inlet on the microfluidic network of the attached chip for loading the sample, PCR mix, and optional encoding medium, and encapsulating the cells in an encapsulation medium. The wells 325 and 326 may be used as an embodiment in the form of waste wells, wherein the cells are sorted prior to encapsulation. In addition, more than one groove 325 and 326 may be fluidly connected to multiple air channels on a connected PCR chip to allow for thermal expansion and contraction of the sample stream within the microfluidic channels on the chip while preventing contamination of the sample stream from the external environment. The cover 340 includes a silicone gasket to assist it in sealing the box 341. It also included a 0.1 μm polypropylene filter to create a gas-permeable, liquid-tight interface between the cartridge body and the external environment. Other embodiments of the disposable cartridge are further detailed in co-pending patent application serial No. 11/781848 entitled "cell sorting system and method," which is incorporated herein by reference in its entirety.

As described above, the method and apparatus for performing phenotype-genotype correlation can be applied to samples on the scale of hundreds of cells to thousands of cells to tens of thousands to hundreds of thousands of cells. The instrument platform 300 and disposable sample loading cartridge 320 may be used with a variety of different capillary or microfluidic network chips configured to process up to one hundred cell samples, or up to one thousand cell samples, or up to ten thousand cell samples, or up to one hundred thousand cell samples.

Fig. 8 illustrates an embodiment of a microfluidic chip-on-chip 400 and a capillary 420 for use with a disposable cartridge 320. The microfluidic chip 410 is configured to be coupled to the optical window region of the cartridge 320, for example, by a UV adhesive coupling. When the chip is attached to the cartridge 320, input ports 411, 412 and 413 are provided on the microfluidic chip to be coupled with the PCR mixture well 321, the packing medium well 324 and the sample input well 323. The sample input port 413 is in fluid connection with the sample flow channel 409, whereby, in use, sample cells contained in the sample reservoir 323 are transported into the sample flow channel 409. A PCR mixture reservoir 411 is fluidly connected to PCR flow channels 405a, b, which intersect sample flow channel 409 at a T-junction to introduce a PCR mixture into the sample cell stream flowing through sample flow channel 409. In some embodiments, the flow conditions of the PCR mixture are controlled such that the introduction of the PCR mixture also organizes the sample stream into a single cell stream. Additionally, the diameter of the sample flow channel 409 may be configured to organize the sample into a single bundle of cell flows when the sample is introduced at the sample cell input 413. Packing medium inlet 412 is in flow communication with packing flow channel 407, which intersects sample flow channel 409 at an L-junction downstream of the PCR mixture intersection. A coating medium (e.g., oil) is flowed into the cell and PCR mixture stream at the "L" junction between sample flow channel 409 and coating flow channel 407 under suitable flow conditions to pinch off the cell and PCR mixture droplets into a nanoliter micro-container stream (preferably containing individual cells surrounded by oil coating medium). Flow conditions in the encapsulation flow channel 407 are controlled to ensure that 0 or one cell is encapsulated in each micro-container. For example, microfluidic channels matched for flow cytometry typically have a cross-section of 50-100 μm by 150-300 μm, and available Teflon capillaries can be as small as 400 μm in diameter, so the nanoliter micro-container 408 will have a diameter of 30-400 μm. Setting and controlling flow conditions in a microfluidic channel network can be accomplished by direct drive pumps, pneumatic pumps, electrodynamics, capillary action, gravity, or other methods of generating fluid flow.

The micro-containers 408 are then flow loaded into a microfluidic capillary 420 for amplification of genetic material and measurement of gene expression in the micro-containers. As described above, the flow conditions of sample flow channel 409 and packing flow channel 307 can be further controlled to vary the size of the nano-liter micro-containers and/or the spacing between nano-liter micro-containers as described above, which can provide a unique order of micro-containers for encoding and indexing the relative positions of the micro-containers. In the illustrated embodiment, the distance (d) between the micro-containers in the capillary is about 500-1000 μm, and the total length (L) of the micro-flow capillary 420 is 500-1000 mm to provide a capacity of about 1000 micro-containers. In another embodiment, the spacing of the droplets and/or the overall length of the microfluidic capillary may be adjusted to accommodate larger or smaller samples. In use, the capillary tube is shaped into a serpentine arrangement containing a plurality of adjacent parallel sections connected by two end fittings. For example, in some embodiments, the capillary tube is divided into 10-20 parallel sections. Cutting the joint to leave a plurality of adjacent parallel segments each 40-50 mm long. The capillary may be divided into multiple segments prior to the amplification process to minimize the chain reaction of thermal expansion between PCR amplification cycles at the micro-container locations within the capillary, thereby improving the ability to track the individual micro-container locations, thereby allowing the determination of the amplification products to be correlated with the previous phenotypic determination of each individual micro-container.

FIGS. 9A-C show a thermal control assembly for use with a capillary based on the PCR system described above. Once the micro-containers are loaded into the capillary 420 and the capillary is divided into a plurality of sections 421, the sections 421 are mounted into an aluminum block 422 having a plurality of rectangular grooves 423 or capillary holders. The rectangular grooves may be about 1mm deep, or 2mm deep, or 3mm deep, or 4mm deep, or 5mm deep. In one embodiment, as shown in FIG. 9B, the width of the rectangular slot 423 is slightly narrower than the cross-sectional diameter of the capillary, so that there is thermal contact between the bottom and side walls of the slot and the capillary; in some embodiments, the width of the rectangular slot is slightly greater than the diameter of the capillary when the capillary is in close proximity to the bottom and side walls of the aluminum block. In another embodiment, as shown in fig. 9C, the bottom capillary holder 424 is shaped to correspond to the capillary section diameter so that there is more thermal contact between the aluminum block 422 and the capillary section 421. In both embodiments, the width of the rectangular groove side wall is equal to or greater than the diameter of the capillary. In some embodiments, a glass cover 425 of about 1mm thickness may be mounted on top of the aluminum block to prevent conduction to the ambient air and block heat radiation. In addition, deeper grooves can be used without a glass cover. In another embodiment, the sidewalls of the rectangular grooves can be made thinner than the diameter of the capillaries, which advantageously reduces the spacing between the rectangular grooves, thereby increasing the density of capillaries in the heating block. In the described embodiment, a glass cover is used over a rectangular channel of height equal to the capillary diameter to maintain good thermal conductivity between the thinner side walls; or making the height of the rectangular groove at least 1.5 times the diameter of the capillary. For example, in one embodiment, if the depth of the groove is at least 1.5 times the diameter of the capillary, the sidewall has a thickness of about 0.1mm to 0.2 mm. Once the aluminum block 422 is loaded with the plurality of capillary segments 421, the temperature of the aluminum block can be raised from 60 ℃ to 95 ℃ at 1 ℃/S to cycle the capillary segments 421 through the temperature profile for PCR amplification. Any suitable number of cycles of heating element cycling temperature described above may be used to undergo the desired PCR.

FIG. 10 illustrates an embodiment of a microfluidic chip 500 for single cell phenotypic and genotypic analysis and correlation on larger samples. The microfluidic chip 500 is shown to be approximately 75mm by 75mm and has a capacity of up to 10,000 micro-containers. The microfluidic chip is preferably an optically transparent substrate to allow the cell detection light to be projected into the microchannel. The substrate is typically, but not limited to: glass, quartz, plastics (e.g., Polymethylmethacrylate (PMMT), etc.), and other moldable or workable polymers (e.g., polydimethylsiloxane, PDMS, or SU 8).

In use, the microfluidic chip 500 is attached to a disposable sample loading cartridge containing the sample, the PCR mixture, and the encapsulation medium, as described above. When the chip 500 is mounted on the optical window of the disposable cartridge, the chip 500 has a cell inlet 513, a PCR mixture inlet 512, and an oil inlet 511 configured to be connected to a cell well, a PCR mixture well, and an oil well on the disposable cartridge. The cell input port 515 is in fluid connection with the sample flow channel 509 in order to transport, in use, cells contained in the sample well of the disposable cartridge into the sample flow channel 509. A PCR mixture inlet 411 is fluidly connected to PCR flow channels 505a, b, which intersect sample flow channel 409 to introduce a PCR mixture into the cell stream flowing through sample flow channel 409. In some embodiments, the flow conditions of the PCR mixture are controlled, whereby introduction of the PCR mixture pools the sample stream into a single cell stream. In addition, when a sample is introduced into the cell sample input part 513, the diameter of the sample flow cell 509 may be configured to collect the sample into a single cell flow. Oil inlet 512 is in fluid connection with packing flow channel 507, which intersects sample flow channel 409 at an L-shaped junction downstream of the PCR mixture intersection. An oil stream is injected into the cell and PCR mixture stream at the "L" junction between sample flow channel 409 and packing flow channel 407 under suitable flow conditions to clamp the cell and PCR mixture droplets into a nanoliter micro-container stream (preferably containing individual cells surrounded by an oil-coating medium). In some embodiments, to limit the number of encapsulated cells to a preselected subpopulation that can still be inversely correlated with phenotypic assays (screening), a sorting zone is provided to sort the sample prior to encapsulation. For example, sorting may be performed to discard red blood cells and pool only nucleated cells. The cross-sectional diameter of the nano-liter micro-containers is preferably larger than the sample cells and smaller than the cross-sectional diameter of the flow channels. For example, the diameter of the cross section of the micro-container is preferably 30 to 400 μm depending on the size of the flow channel.

As described above, the flow conditions in packing flow channel 507 are controlled to ensure that a majority of the micro-containers preferably contain one cell or 0 cell, e.g., in some embodiments, at least 90% of the micro-containers contain 1 or 0 cell, or at least 80% of the micro-containers contain 1 or 0 cell, or at least 70% of the micro-containers contain 1 or 0 cell, or at least 60% of the micro-containers contain 1 or 0 cell. Additionally, in some embodiments, the flow conditions of the package flow channel 507 and/or the sample flow channel 509 can be further controlled to periodically change the micro-container size and/or the spacing between micro-containers to create a coded signal for indexing and tracking individual micro-containers. Setting and controlling flow conditions in a microfluidic channel network can be accomplished by direct drive pumps, pneumatic pumps, electrodynamics, capillary action, gravity, or other methods of generating fluid flow.

The micro-container stream is then loaded into a serpentine channel 520, which serpentine channel 520 is in fluid connection with a sample flow channel 509 (for amplifying target sequences in sample cells). The serpentine analysis channel 509 is divided into a plurality of parallel segments 521 a-j by adjacent segments 521 a-j connected by a plurality of bends 531 a-i. Preferably the serpentine channel is divided into 50 to 100 parallel sections each 50 to 100mm long depending on the sample size and the required spacing between the micro-containers. Air channels 540 a-j are coupled in flow communication with each end of each parallel section 521 a-j of serpentine channel 520. The plenums 540 a-j are configured to accommodate thermal expansion and contraction of the micro-container flow in each section 521 a-j between temperature cycles during amplification. The serpentine channel 520 is divided into a plurality of parallel sections connected to the air channels to isolate movement of the micro-containers caused by thermal expansion of each micro-section 521 a-j, thereby eliminating the chain reaction of thermal expansion along the entire serpentine channel 520. Thus, movement of the micro-containers within serpentine channel 520 during temperature cycling can be greatly reduced and the ability to track individual micro-containers within a large volume of sample (e.g., contained within serpentine channel 520) is improved. Minimizing micro-container movement during temperature cycling may also minimize the risk of contamination of micro-containers moving back and forth within the space occupied by adjacent micro-containers and reduce the likelihood of adjacent micro-containers coalescing.

In use, gas channels 541 a-j are sealed with high pressure seals during loading of serpentine channel 520 to maintain a positive pressure on serpentine channel 520 and ensure that the micro-containers are loaded in the serpentine channel. In some embodiments, first, the high pressure seal may comprise a mechanical membrane and one or more rubber gaskets to seal each vent 540 a-j during sample loading. In addition, the airway may be covered with a photolithographic material that can be etched after loading or a trap that can be actuated by thermal expansion pressure (overpressure). Once loaded into the serpentine channel 520, the first high pressure seal can be released or removed. A second seal is then used to isolate the air channels 540 a-j from the external environment to prevent contamination of the micro-containers. The second seal is preferably mechanically compliant to accommodate thermal expansion and contraction of the micro-containers in each section 521 a-j during thermal cycling. For example, in some embodiments, the second seal may comprise a thin or flexible membrane attached to the airways 540 a-j. In addition, the air channels 540 a-j may be in fluid communication with more than one common air channel on the disposable cartridge, which may be sealed from the outside environment with a flexible membrane or a lid with a filter.

FIG. 11 illustrates another embodiment of a microfluidic chip 600 for single cell phenotypic and genotypic analysis and correlation of samples at a larger scale, such as samples containing up to 100,000 micro-containers. The chip 600 is divided into 4 independent loading and analysis zones 610a, b, c, d, each having a serpentine channel 620 a-d, the serpentine channels 620 a-d being configured to accommodate up to 25,000 micro-containers with a spacing between the micro-containers of about 100-200 μm. It is conceivable to divide the chip into more or less loading zones depending on the required chip capacity. On the disposable cartridge, each of the loading regions 610 a-d is configured to have cell inlets 613 a-d, PCR mixture inlets 612 a-d, and oil inlets 611 a-d connected to a cell well, a PCR mixture well, and an oil well. Each loading region 610 a-d on the chip 600 preferably has the same size as the 10,000 PCR capacity chip described above, so that the relative positions of the cell input, PCR input, and oil input are similar. In use, the PCR chip 600 may be attached to a power station that temporarily attaches each loading zone 610 a-d to a disposable cartridge for loading the chip 600 with sample and PCR mixture, priming the oil to pack the sample and PCR mixture in a nano-liter micro-container, and loading the micro-container into a serpentine analysis zone in the same manner as described above for 10,000 PCR microfluidic chips 500 having one loading and analysis zone. In addition, the cartridge and the chip may be integrated into one integrated device.

For example, in some embodiments, the disposable cartridge may have a common well for both the packaging medium and the PCR mixture, which may be alternately connected to each chip, e.g., using the power stage described above, or multiplexed to each chip or loading area. In addition, the disposable cartridge has a plurality of separate oil wells and PCR mixture wells, each of which is connected to one loading region. Also, in some embodiments, the cartridge may have one sample well that is multiplexed to each cell input using more than one valve and flow channel. In addition, in some embodiments, the cartridge has a plurality of sample wells, each of which is independently connected to one cell input and analysis zone. The cartridge is also configured to allow light and loading zones for performing phenotypic analysis.

As described above, each loading and analysis region 610 a-d in the reference microfluidic chip 500, 100,000PCR chip 600 contains a serpentine channel 620 a-d fluidly connected to the serpentine channel 620 a-d in fluid communication with the sample flow channels 609 a-d (for target sequence amplification of sample cells in each micro-container). Serpentine analysis channel 620 is divided into a plurality of parallel segments connected by a plurality of bends. Each serpentine channel 620 a-d is preferably divided into 50-100 parallel segments, each about 50-100 mm long, depending on the sample size and the spacing between the desired micro-containers. As described above, vent 640 is fluidly connected to each end of each parallel segment of serpentine channels 620 a-d to seal serpentine channels 620 a-d during the loading process and allow for thermal expansion and contraction of the serpentine channels while isolating the serpentine channels from the external environment during the amplification process.

In another embodiment, a sample preloaded into a microwell array chip can be phenotyped and correlated. FIG. 12 shows the steps for genotyping data analysis and correlating phenotype-genotype data derived from samples pre-loaded into a microwell array. In step 700, the cells in the sample input 70 are independently analyzed for selected phenotypic characteristics. In some embodiments, prior to isolating the cells into the nanopores, the phenotypic analysis is performed in a microfluidic environment (e.g., by flow cytometry as described above). Alternatively, after isolation of cells in individual nanopores, phenotypic assays can be performed using scanning cytometry. As described above, in some embodiments, a multiplex-type assay can be performed using, for example, a multiplex fluorescence detector or a combination of a light scattering analyzer and fluorescence.

In step 702, cells may be sorted based on a user selected parameter (e.g., a measured phenotypic characteristic). The target cells can then be directed to a target flow channel for delivery to the nanopore array. Non-target cells can be directed to a waste flow channel connected to a waste reservoir.

In step 704, target cells are sequentially placed in the nanopores of the one or more arrays. Preferably, each nanopore may contain one cell. The nanopores are preloaded with reagents required for gene expression assays and fluorescent detector molecules or probes that, when excited, emit a level of fluorescence proportional to the amount of amplified product. In some embodiments, the number of available wells preferably exceeds the number of cells tested to ensure limited dilution. The holes are isolated from each other using a hydrophobic fluid cap (e.g., oil). Phenotypic information for the cells in each nanopore can be indexed and recorded based on the precise location of each cell in the array.

In step 706, the individual cells are genotyped. Cells may be lysed using heat, laser, ultrasound, or chemical lysis, or any other suitable technique known in the art. The array is thermally coupled to a block heater and the nanopores are subjected to more than one temperature required to amplify a gene product by isothermal amplification or Polymerase Chain Reaction (PCR) cycles. The array is subjected to the desired number of amplification cycles and the amplified gene products are determined.

In step 708, the determination of the genotype is decoded. Fluorescence detection molecules or probes are excited with a light source (e.g., a stationary or scanning laser, UV lamp, light emitting diode) and an image of the array is collected with an optical detector (e.g., CCD imaging, photomultiplier tube, photodiode, or photodiode array) to determine the intensity of the fluorescence signal in each nanopore and detect the amount of amplified product. In some embodiments, an optical detector (e.g., a CCD camera) is used to capture an image of the entire array. In addition, the nanopores are interrogated sequentially by scanning an excitation light source or scanning a detector (e.g., an optical fiber connected to a photomultiplier tube). The images are sent to a processor for image processing and correlated with previously recorded phenotypic determinations for each nanopore, providing associated phenotypic and genotypic data 74 for each cell.

As shown in fig. 13A-B, in some embodiments, the phenotypic assay of cells may be performed sequentially in a microfluidic environment, and then the cells loaded in a microwell array for amplification and genotyping. Here, in step 700, a phenotypic analysis is performed by introducing cells 808 into the microfluidic flow channel 709. As described above, the flow channel is configured to reduce the cross-sectional diameter of flow channel 709, for example, to render the cells into a single cell stream for phenotypic analysis. The cells 808 are sequentially interrogated at the analysis zone 705 to determine more than one phenotypic characteristic of the cells. In some embodiments, the cells are sorted into target cells and non-target cells based on the determined phenotypic characteristic or any other parameter selected by the user. The presence of target cells having the desired characteristics can be detected in the fluid stream by fluorescence, forward scatter, or any suitable imaging or detection format. Target cells are then directed to the target flow channel 711 for transport to the individual nanopores 801 a-f on the nanopore array chip 800 using dielectrophoresis, pneumatic switches, or ovofluidic or optical switches as described in co-pending patent application serial No. 11/781848 entitled "cell sorting system and method," which is incorporated by reference herein in its entirety. Non-target cells are directed to a waste channel 710 that connects to a waste reservoir.

In step 704, the target channels 711 are connected to the microfluidic channels 803 of the nanopore array chip 800 to transport the target cells to the individual nanopores 801 a-f. Target cells 808 are placed in each of the nanopores 801 a-f in sequence. The desired PCR reagents and fluorescent detector or label that emits a level of fluorescence proportional to the amount of amplification product are preloaded into each nanopore location 801 a-f. The location of each cell in the nanopore array creates an index that can be subsequently used to correlate the phenotypic assay of each cell with the genotypic assay based on the location in the array. Each nanopore 801 a-f preferably has a volume of a few nanoliters to accommodate individual cells and reagents required to amplify a target DNA sequence. For example, in one embodiment, the holes are 100 μm by 100 μm square and have a depth of 70 μm. The nanopores 801 a-f can be microfabricated using a variety of materials, including but not limited to: glass, quartz, plastics (e.g., Polymethylmethacrylate (PMMT), etc.), and other moldable or workable polymers (e.g., polydimethylsiloxane, PDMS, or SU 8). The depth of the micro flow channel 803 connecting the nano holes 801 a-f is usually, but not limited to, in the range of 10 μm to 100 μm. The width of the microfluidic channel is usually, but not limited to, 1-5 times the depth. Once the cells are loaded into individual nanopores 801 a-f, oil or any other suitable encapsulation medium is flowed through microfluidic channel 803 to isolate individual cells with PCR reagents in each well.

Next, in step 706, the isolated cells are lysed and cycled through the temperature profile required to complete the polymerase chain reaction. For example, as described herein, the array 800 is cycled through a first temperature of about 96 ℃, a second temperature of about 60 ℃, and a third temperature of about 72 ℃. In some embodiments, the nanopore array temperature is adjusted to produce the appropriate temperature for PCR by using one or more elements known in the art (e.g., a heating block, an integrated heating wire, a Peltier heater) or by circulating hot/cold fluid or hot/cold air. The desired number of PCR cycles is performed and the amplification product is determined. The fluorescent probes or labels in each nanopore are excited by a light source (e.g., fixed and scanning laser, UV lamp, light emitting diode) and an image of the nanopore is collected with an optical detector 815 (e.g., CCD imaging, photomultiplier tube, photodiode, or photodiode array) to determine the fluorescent signal intensity. In another embodiment, UV light can be used to determine the absorbance of the nucleic acid product. The determination of genotypes is then indexed by position on the array. Thus, the phenotype and genotype of individual cells can be correlated based on location on the nanopore array.

In another embodiment shown in FIG. 14, a cell stream 808 in a flow path 709 is sorted into a plurality of target arrays 901 a-i. In the case of multiple target arrays, a network of sorting switches is used to sort the cell stream multiple times according to multiple assay phenotypic characteristics. For example, as shown below, cells are sorted by three lines at each sorting fork using 4 sorting switches 725 a-d. The sorting network shown is capable of dividing the cells into 9 different arrays 901 a-i based on the measured phenotype prior to sorting. As shown in fig. 14B, although individual cells are not correlated back to their phenotype, a cell-by-cell statistical distribution of gene expression for each array can be determined and statistics can be correlated to the user-selected 9 phenotypic clusters.

As shown in fig. 15A-B, in some embodiments, after the cells are stored and isolated in individual nanopores 801 a-f, the phenotype of each cell is determined by scanning cytometry. Here, the cells are labeled for phenotypic analysis, for example using antibodies conjugated with fluorescent molecules (such as fluorescein isothiocyanate, also known as FITC), nuclear stains, intracellular dyes, or cell-directed synthetic fluorescent proteins (e.g., green fluorescent protein, also known as GFP). The cells are then placed in nanopores 801 a-f containing the PCR reagents required to amplify the target sequence and fluorescent probes or labels to detect the amount of amplified target sequence, and isolated. The nanopores 801 a-f may be square and flat-bottomed, or preferably tapered to a small flat bottom, so as to allow the cells to automatically collect at the bottom of the well, which reduces the number of optical scanners required to determine the phenotype of the cells. Individual cells are placed in each of the nanopores 801 a-f. The fluorescent labels on the cells in the nanopore array 800 are excited using a light source 840 (e.g., fixed and scanning laser, UV lamp, light emitting diode) and each nanopore in the array 800 is sequentially interrogated with a fluorescence detector 812 to determine one or more phenotypic characteristics of the cells. The location and phenotypic measurement of each cell in the array is recorded to facilitate correlation of subsequent genotypic measurements of individual cells with phenotypic measurements. The isolated cells were lysed and the cells were cycled through a PCR temperature profile multiple times to amplify the genetic material, as described above. The fluorescent label or probe in each nanopore is excited using a laser 840 and the fluorescence signal intensity is measured with a fluorescence detector 812 to determine the amount of amplified genetic material after each PCR cycle. The results of the genotyping at each position in the array are recorded and sent to a processor to correlate them with the previously recorded phenotyping of each nanopore position, thereby correlating the phenotyping of each cell with the genotyping. In practice, encoding and decoding can be performed by adding a coordinate system to the aperture array.

In some embodiments, as shown in fig. 15A and fig. 18, it is desirable to recover genetic material from a selected portion of cells amplified and assayed in a nanopore array based on phenotypic and genotypic assays of the cells. Before or after the cells are placed in the nanopore array, the cells may be interrogated for more than one phenotypic assay as shown in fig. 13 and 15, or if the cells are collected based solely on genotype information, they may be placed directly in the nanopore array 800. The cells are isolated, lysed, and subjected to one or more temperature thermocycling arrays 800 to amplify the gene expression signature, as described above. The amplified genetic material is measured with an optical detector 815 and correlated with phenotypic information for each nanopore location. Genetic material is then harvested from the nanopore of interest based on the associated phenotype-genotype information using an automated micropipette 820 to extract material from the individually addressable well of interest.

The alternative embodiment of the method of using nanopores shown in FIGS. 13 and 15 is for genotyping only. Cells can be isolated, lysed, thermocycled, and genotypic information determined for the cells as previously described in fig. 13 and 15 to obtain parallel single cell genotypic data on tens to hundreds of thousands of cells. Because of the small volume (usually nanoliters), the cost per reaction per cell is comparable to the cost per reaction for the same number of cells in bulk.

FIGS. 18A-18B show micrographs of cell separations performed in various flow formats. Specifically, micrographs 18A and 18B show plastic formed devices, while micrographs 18C and 18D show PDMS formed devices. In fig. 18A, a sample mixture contained in a solution separated by a spacer is formed at a sample mixture input portion 1002, a spacer input portion 1004 (e.g., containing a spacer oil (e.g., fluorocarbon oil)), a solution input portion 1006, and finally an output portion 1008 of an intersection 1000. FIG. 18B shows a micrograph measured with a 400 μm ruler. Fig. 18C shows a similar structure with the same conventional elements as fig. 18, and has been numbered in a consistent manner.

FIG. 19 shows oscillometric cell forward scatter and droplet forward scatter as indicated by the markers. The y-axis represents intensity and the x-axis represents time. As can be seen from the Forward Scatter (FSC) of the drops, the drops have a fairly regular periodicity and indicate the boundary between one drop and the next. In contrast, the number of peaks in cell Forward Scatter (FSC) indicates the number of cells in each drop.

Fig. 20 shows a micrograph of the 6 capillaries marked 1011 to 1016 in the picture from top to bottom. The coding of the material is influenced by the drop length variation. For example, comparing the drop length in the capillary 1013 to the length in the adjacent capillary 1012 shows a significant change in drop length.

FIG. 21 is a further micrograph of an encoding mode affected by varying the droplet length. Capillaries 1021, 1022, and 1023 are encoded from top to bottom in the micrograph. Both length and position may form a varying order, which is expressed in the "Morse code" pattern.

FIGS. 22A and 22B show the results of actual detection of gene expression. Figure 22A shows parallel 20 capillaries, each with multiple reaction volume filled. Fig. 22A shows the results of phenotype CD 34. Fig. 22B shows the results after qPCR. Light reaction droplets represent positive gene expression. Some of the drops are marked with arrows added to the micrograph. Typically, there are one or two cells in each drop. About 5% of the cells were CD34 positive KG1A and about 95% of the cells were CD34 negative Jurkat cells. The graph labeled qPCR intensity shows the quantitative PCR intensity as a function of cycle number. On the left side of the x-axis there are 0 cycles and on the right end with about 46 cycles. The CT histogram reflects the amount of CD34 expression from cell to cell.

Although the above invention describes methods and apparatus for correlating nucleic acid amplification to phenotypic data for flow cytometry, it is readily envisioned that other molecular assays (such as DNA methylation, protein abundance, cytokine assays, or other enzyme or protein assays) may be performed in the micro-containers to correlate them to phenotypic measurements for each cell. Furthermore, while the focus of the present invention is on the determination and correlation of individual cells within a micro-container, it will be appreciated that the devices and methods described above can be used to correlate phenotype-genotype data determined on multiple cells.

While the above invention has been described in detail by way of illustration and example with specific objects, it will be apparent to those skilled in the art that it is readily understood. Certain changes and modifications may be made in accordance with the teachings of the present invention without departing from the spirit and scope of the present invention.

The present invention also includes the following aspects,

1. an integrated structure for microfluidic single-cell analysis and correlation, comprising:

● a cartridge, the cartridge comprising:

■ the optical window is provided with a light guide,

■ a plurality of slots comprising at least:

a sample tank,

Fluid troughs, and

a reagent tank, and

● chip, the chip comprising at least:

■ a cell input channel in fluid connection with the sample well,

■ a first fluid input channel in fluid communication with the fluid sink and the cell input channel,

■ a second fluid input channel in fluid communication with the reagent reservoir and the cell input channel,

■ a serpentine channel comprising a first end in fluid communication with the cell input channel downstream of the first and second fluid input channels and a plurality of parallel partitions having first and second ends and in fluid communication with each other,

■ a plurality of air channels located at a first end and a second end of the plurality of partitions,

the chip is positioned adjacent to the optical window.

2. The integrated structure of item 1, further comprising a lid, the lid comprising at least:

● a pneumatic channel having an inlet and connected to at least one of the sample cell and the fluid cell, an

● a filter disposed between the air pressure channel inlet and at least one of the sample well and the fluid well.

3. The integrated structure of item 2, further comprising:

● manifold connecting pneumatic pressure from a source to the pneumatic pressure channels without intervening tubes.

4. The integrated structure of item 1, wherein the cartridge further comprises a second fluid slot and the chip further comprises a coding region, wherein the second fluid slot is in fluid connection with the first fluid input channel and a cell input channel downstream of the second fluid input channel.

5. The integrated structure of item 1, wherein the cartridge further comprises a waste reservoir and the chip further comprises a sorting region, wherein the cell input channel is in fluidic connection with the waste reservoir.

6. The integrated structure of item 3, wherein said sorting region comprises a lateral force switch.

7. The integrated structure of item 6, wherein the lateral force switch is generated using optical force, dielectrophoresis, or fluidic pulsing.

8. The integrated structure of item 1, wherein the serpentine channel is 2.5-5 m long.

9. The integrated structure of item 1, wherein said serpentine channel comprises 50-100 parallel partitions.

10. The integrated structure of item 1, wherein the plurality of airways has a first seal configured to seal the serpentine channel during loading and a second seal configured to isolate the airways from an external environment.

11. The integrated structure of item 10, wherein the second seal is mechanically compliant to thermal expansion and thermal contraction.

12. The integrated structure of item 10, wherein the first enclosures each comprise a mechanical mechanism.

13. The integrated structure of item 10, wherein the first seals each comprise a photosensitive material.

14. The integrated structure of item 10, wherein the first seals each comprise a hydrophobic material.

15. The integrated structure of item 10, wherein the second seals each comprise a thin film configured to isolate the serpentine channel from an external environment.

16. The integrated structure of item 1, wherein the cartridge further comprises a vent channel having a filter, and the plurality of vent channels are in fluid connection with the vent channel.

17. The integrated structure of item 1, further comprising a thermal component in operative connection with the serpentine channel to regulate the temperature of the fluid in the serpentine channel.

18. The integrated structure of item 1, wherein the thermal assembly comprises a heating element and a thermal control element to repeatedly cycle the temperature of the fluid in the serpentine channel.

19. The integrated structure of item 18, wherein the heating element comprises a heating block.

20. The integrated structure of item 19, wherein the heating element comprises hot air.

21. The integrated structure of item 1, further comprising a plurality of chips, wherein the cartridge is configured to connect the plurality of slots and the plurality of chips.

22. The integrated structure of item 21, wherein the cartridge further comprises one or more valves to fluidly connect the plurality of wells to the plurality of chips.

23. The integrated structure of item 1, further comprising a plurality of chips, wherein the cartridge further comprises a plurality of sample wells each configured to be in fluid connection with a cell input on one of the plurality of chips.

24. The integrated structure of item 23, wherein the cartridge further comprises a plurality of fluidic slots each configured to be in fluid connection with a first fluidic input channel on one of the plurality of chips and a plurality of reagent slots each configured to be in fluid connection with a second fluidic input channel on one of the plurality of chips.

25. An integrated structure for microfluidic single-cell analysis and correlation, comprising:

● a cartridge, the cartridge comprising:

■ the optical window is provided with a light guide,

■ a plurality of slots comprising at least:

a sample tank,

Fluid troughs, and

a reagent tank is filled with the reagent,

■ cover, the cover comprising at least:

at least one pneumatic channel having an inlet and being connected to at least one of the sample well and the fluid well, and

a filter interposed between the air pressure channel inlet and at least one of the sample slot and the fluid slot, and

a manifold connecting a gas pressure from a source to the gas pressure channel without an intervening tube,

● chip, the chip comprising at least:

■ a cell input channel adapted to receive one or more cells in a fluid medium and in fluid connection with the sample well,

■ a first fluid input channel in fluid communication with the fluid sink and the cell input channel,

■ a second fluid input channel in fluid communication with the reagent reservoir and the cell input channel,

■ output channel in fluid connection with the cell input channel downstream of the first and second fluid input channels,

the chip is at least partially disposed adjacent to the optical window; and

● capillary tube, it connects with said output channel circulation.

26. The integrated structure of item 25, wherein the capillary is 500-1000 mm long.

27. The integrated structure of item 25, wherein the first fluid input channel is in fluid connection with a cell input channel downstream of the second fluid input channel.

28. The integrated structure of item 25, wherein the cartridge further comprises a waste reservoir and the chip further comprises a sorting region, wherein the cell input channel is in fluidic connection with the waste reservoir.

29. A microfluidic chip for single cell analysis and correlation, comprising:

● a loading area comprising:

■ a cell input channel configured to be in fluid communication with a sample source,

■ a first fluid input channel having first and second ends, the first end configured to be in fluid connection with a source of reagent, the second end configured to be in fluid connection with a cell input channel, and

■ a second fluid input channel having first and second ends, the first end configured to be fluidly connected to a fluid source and the second end fluidly connected to a cell input channel downstream of the first fluid input channel;

● an analytical zone comprising:

■ a serpentine channel comprising a first end in fluid communication with the cell input channel downstream of the first and second fluid input channels and a plurality of parallel partitions having first and second ends and in fluid communication with each other.

■ a plurality of air channels located at the first and second ends of the plurality of partitions.

30. The microfluidic chip of item 29, wherein the plurality of gas channels further comprises a first seal configured to seal the serpentine channel and a second seal configured to isolate the gas channels from an external environment.

31. The microfluidic chip of item 30, wherein the first encapsulants each comprise a mechanical mechanism.

32. The microfluidic chip of item 30, wherein the first encapsulants each comprise a photosensitive material.

33. The microfluidic chip of item 30, wherein the first encapsulants each comprise a hydrophobic material.

34. The microfluidic chip of item 30, wherein the second encapsulants each comprise a membrane configured to isolate the serpentine channel from an external environment.

35. The microfluidic chip of item 29, wherein the serpentine channel is 2.5-5 m long.

36. The microfluidic chip of item 29, wherein the serpentine channel comprises 50-100 parallel partitions.

37. The microfluidic chip of item 29, further comprising a plurality of regions, each of the regions comprising a loading region and an analysis region.

38. The microfluidic chip of item 37, further comprising 4 zones, wherein the serpentine channel in each analysis zone is 2.5-5 m long and is divided into 50-100 parallel segments.

39. A method of correlating phenotypic and genotypic information cell by cell, comprising:

● providing a first solution containing a plurality of cells and at least one reagent for amplifying a target DNA sequence and a second solution immiscible with the first solution;

● analyzing the phenotype of each cell individually in the first solution;

● combining the first solution and the second solution, thereby forming a stream comprising a plurality of nano-liter micro-containers, wherein a majority of the nano-liter micro-containers are wrapped around single cells or are free of cells;

● encoding the nanoliter micro-container stream with a reference signal;

● subjecting the nano-liter micro-containers to thermal conditions suitable for amplification of the target DNA;

● determining gene expression in each microreactor; and

● the reference signal is decoded to correlate the gene expression values in each microreactor with the phenotype of the cells in the microreactors.

40. The method of clause 39, wherein amplifying the target DNA comprises performing isothermal amplification.

41. The method of clause 39, wherein the thermal conditions comprise repeated thermal cycling.

42. The method of clause 41, wherein amplifying the target DNA comprises performing qPCR, real-time PCR, or end-point PCR.

43. The method of item 39, wherein said reference signal is contained in a microreactor.

44. The method of clause 43, wherein the reference signal is created by generating a pseudorandom pattern of microbeads or dyes contained in the nanoliter micro-container stream.

45. The method of item 39, wherein said reference signal is created by generating a pseudo-random pattern of intervals between nano-liter micro-containers.

46. The method of item 39, wherein said reference signal is created by pseudo-randomly varying the size of a nano-liter micro-container.

47. The method of item 39, further comprising imaging the stream of nano-liters micro-containers after encoding the stream of nano-liters micro-containers with the reference signal to create the index.

48. The method of item 39, wherein analyzing the phenotype comprises measuring a fluorescent signal.

49. The method of clause 39, wherein analyzing the phenotype comprises performing a plurality of phenotypic assays.

50. The method of clause 39, further comprising sorting the cells into target cells and non-target cells based on the phenotypic assay, wherein the non-target cells are shunted to a waste tank.

51. The method of clause 39, further comprising lysing the cells in the nanoliter micro-container.

52. The method of clause 51, wherein the cell lysis is performed by osmotic shock, heat, laser lysis, or ultrasound.

53. The method of clause 39, wherein said gene expression is determined by real-time PCR or quantitative PCR.

54. The method of clause 39, wherein determining gene expression comprises determining uptake of genetic material.

55. The method of clause 39, wherein determining gene expression comprises determining uptake of a probe or dye that binds to the DNA product or DNA amplification byproduct.

56. The method of clause 39, further comprising sorting the stream of nanocells based on the determination of gene expression.

57. The method of item 39, further comprising introducing the first solution and the second solution into a microfluidic network.

58. The method of item 57, wherein the microfluidic network comprises:

● a loading zone comprising at least first and second flow paths for combining said first and second solutions, thereby forming a stream comprising a plurality of nano-liter micro-containers;

● a coding region; and

● an analytical zone comprising:

■ serpentine flow channel having a plurality of parallel partitions having first and second ends and being in fluid communication with each other, and

■ a plurality of air channels located at first and second ends of the plurality of partitions; and

● decoding area.

59. The method of clause 39, wherein a flow of nanocells is selected into an array of nanofluidic microwells, whereby each well contains a single microreactor, and wherein the step of subjecting the nanocells to repeated temperature cycling subjects the microwell array to repeated temperature cycling.

60. The method of clause 39, wherein the plurality of nanocapsules comprises at least 1,000 nanocapsules.

61. The method of clause 60, wherein the plurality of nanocontainers comprises at least 10,000 nanocontainers.

62. The method of clause 61, wherein the plurality of nanocapsules comprises at least 100,000 nanocapsules.

63. An integrated system for providing correlated phenotypic and genotypic analysis of individual cells in a microfluidic network, comprising:

● a sample source comprising an aqueous solution containing a plurality of cells;

● a packaging source comprising a hydrophobic fluid;

● a microfluidic network, comprising:

■ a loading area comprising:

a sample flow path connected to a sample source in a flow-through manner,

a lateral flow channel in fluid communication with the fluid slot and the sample flow channel, and

a controller configured to direct flow conditions in the lateral flow channel, whereby the sample source is divided into a plurality of nano-liter micro-containers, and

■ an analytical zone comprising:

a serpentine channel comprising a first end in fluid connection with the reagent input channel and the cell input channel downstream of the lateral flow path, and a plurality of parallel partitions having first and second ends and in fluid connection with each other,

a plurality of airways at first and second ends of the plurality of partitions;

● a first optical detector in operative connection with the sample flow channel, the detector being configured to determine a signal from a nano-liter micro-container in the sample flow channel;

● a thermal assembly comprising a heating element and a thermal control element in operative connection with the analysis zone;

● an excitation light source configured to illuminate the serpentine channel at a particular wavelength;

● a second optical detector configured to detect and assay amplification products from the nano-liter micro-containers in the serpentine channel; and

● a processor connected to the optical detector for recording and correlating the signals detected by the first and second optical detectors.

64. The integrated system of item 63, wherein said microfluidic network further comprises a coded region configured to apply a reference signal to a plurality of nano-liter micro-containers.

65. The integrated system of item 64, further comprising a decoding sensor configured to decode a reference signal encoded on a nano-liter micro-container.

66. The integrated system of item 63, wherein said thermal control assembly is configured to repeatedly cycle the temperature of the analysis zone at a temperature suitable for performing PCR.

67. The integrated system of item 63, wherein the loading zone further comprises a sorting zone configured to determine a physical characteristic of a plurality of cells in the sample source and sort the sample source based on the physical characteristic.

68. An integrated system of items 67, wherein the sorting region comprises a switch.

69. The integrated system of item 67, wherein the sorting region is fluidly connected to a waste tank.

70. The integrated system of item 63, further comprising a reagent source comprising a solution of one or more reagents required for amplification of the target DNA sequence, and wherein the loading zone further comprises a reagent flow channel in fluid connection with the reagent source and the sample flow channel for introducing the reagent source into the sample flow channel.

71. The integrated system of item 63, wherein said analysis zone further comprises a sorting switch configured to sort nanoliter micro-containers based on the determination of amplification products in the microreactors.

72. The integrated system of item 63, wherein said processor comprises an error correction algorithm.

73. A method of correlating phenotypic and genotypic information in a microfluidic environment on a cell-by-cell basis, comprising:

● providing a sample solution comprising a plurality of cells in an aqueous environment and a coating solution that is immiscible with the sample solution;

● introducing the sample solution and the encapsulation solution into a microfluidic network;

● determining the phenotype of each cell individually in the sample solution;

● combining the sample solution and the encapsulation solution in the microfluidic network, thereby partitioning the sample source into a plurality of nanoliter micro-containers;

● encoding the nanoliter micro-container stream with a reference signal;

● loading a plurality of nano-liter micro-containers in a serpentine channel comprising a plurality of parallel partitions having first and second ends and being in fluid communication with each other and a plurality of aeration channels at the first and second ends of the plurality of partitions;

● subjecting the serpentine channel to repeated temperature cycles to amplify target DNA sequences in the plurality of nanoliter micro-containers;

● determining the amplification products in the plurality of nanoliter micro-containers; and

● to correlate the measurement of the amplification product from each microreactor with a phenotypic measurement of the cells in each microreactor.

74. The method of item 73, wherein the merging step comprises:

● introducing the sample solution into a sample flow path;

● introducing the encapsulation solution into a lateral flow channel in fluid connection with the sample flow channel, and

● directing flow conditions in a lateral flow channel whereby the sample solution in the sample flow channel downstream of the lateral flow channel is divided into a plurality of nano-liter micro-containers.

75. The method of item 73, wherein the microfluidic network further comprises a waste tank, the method further comprising the steps of:

● classifying cells in the sample solution into target cells and non-target cells based on the phenotypic assay, an

● transferring the non-target cells to a waste tank.

76. The method of clause 73, wherein the temperature cycling comprises subjecting the serpentine channel to hot gas.

77. The method of item 73, wherein the plurality of airways includes a mechanical seal configured to seal the serpentine channel during loading.

78. The method of item 77, wherein the plurality of airways is configured as: when the serpentine channel is subjected to repeated temperature cycling, thermal expansion along the nanoliter micro-container flow in the serpentine channel is minimized.

79. The method of item 78, wherein the plurality of gas passages comprise seals configured to isolate the serpentine channel from an external environment during temperature cycling.

80. The method of clause 79, wherein amplifying the target DNA sequence comprises performing quantitative PCR.

81. The method of clause 80, wherein amplifying the target DNA sequence comprises performing a plurality of quantitative PCR cycles.

82. The method of clause 73, wherein more than 90% of the plurality of nanoliter micro containers contain 1 or 0 cells.

83. The method of clause 73, wherein more than 80% of the plurality of nanoliter micro containers contain 1 or 0 cells.

84. The method of clause 73, wherein more than 70% of the plurality of nanoliter micro containers contain 1 or 0 cells.

Claims (11)

1. Integrated system for performing molecular analyses of individual cells, characterized in that it comprises:

a sample source comprising at least one aqueous solution containing a plurality of cells;

a reagent mixture that supports nucleic acid amplification and quantitative analysis of cells;

an array comprising a plurality of nanofluidic microwells that are sized to substantially contain a mixture of 1 single cell from a sample source and a reagent; the nanofluidic microwell to support nucleic acid amplification and quantitative analysis of cells and configured to provide optical access to the contents of the nanofluidic microwell;

a precipitator for subsequently precipitating cells from the sample source in a plurality of nanofluidic microwells of an array;

a packing medium for separating the cell and reagent mixture in the plurality of microwells, said packing medium separating said plurality of microwells from each other by performing a hydrophobic fluid oil seal while allowing a physical inlet to each microwell and functioning to reduce volatilization of the reagent mixture from the microwells;

at least one optical detector operatively connected to the array, the detector being configured to measure a signal from the plurality of microwells and generate output information indicative thereof;

a thermal module comprising a heating element and a thermal control element operably coupled to the array, the thermal module configured to perform thermal cycling of the array at a temperature suitable to effect nucleic acid amplification at the microwells;

a target collector device operable to individually extract nucleic acid material amplified from the plurality of microwells; and

a processor configured to receive the output information from the optical detector.

2. The integrated system for performing molecular analysis of individual cells of claim 1, wherein the optical detector is a cytometer.

3. The integrated system for performing molecular analysis of individual cells of claim 2, wherein the cytometer is a scanning cytometer.

4. The integrated system for performing molecular analysis of individual cells of claim 1, wherein the processor generates phenotypic information associated with the cells on the array.

5. The integrated system for performing molecular analysis of individual cells of claim 1, wherein the processor generates quantitative information of the material on the array.

6. The integrated system for performing molecular analysis of individual cells of claim 1, wherein the nanofluidic microwell is square with a flat bottom.

7. The integrated system for performing molecular analysis of individual cells of claim 1, wherein the nanofluidic microwells are tapered.

8. The integrated system for performing molecular analysis of individual cells of claim 7, wherein nanofluidic microwell is further comprised of flat bottoms.

9. The integrated system for performing molecular analysis of individual cells of claim 1, wherein target collector comprises a micropipette.

10. The integrated system for performing molecular analysis of individual cells of claim 9, wherein the micropipette is an automated micropipette.

11. The integrated system for performing molecular analysis of individual cells of claim 1, wherein the processor establishes a correlation of a phenotype and quantitative information from each chamber of the plurality of microwells.