FIELD

[0001] This application claims priority to U.S. Provisional Application Serial No. 62/117,386, filed February 17, 2015, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present application relates to an apparatus for generating microdroplets of sample fluid without fluidic control. The apparatus may be useful to include in systems for nucleic acid amplification such as Polymerase Chain Reaction (PCR) and digital Polymerase Chain Reaction (dPCR) When used for dPCR, the system may include other components useful in dPCR such as a needle loading module, a sample loading module, and/or a detection module, e.g., a fluorescence detection module.

BACKGROUND

[0003] PCR, or Polymerase Chain Reaction, is a technology that can rapidly amplify specific genes or DNA sequences in vitro. The technology is capable of rapidly amplifying a specific DNA sequence, yielding multiple million copies of the sequence in a short amount of time.

[0004] To date, PCR technology has undergone three generations of evolution. First- generation PCR utilizes a machine that amplifies a target gene, and employs agarose gel electrophoresis to analyze the reaction product. With second-generation PCR (Real-Time PCR, or qPCR), fluorescence reagents are included in the reaction system. The fluorescence reagents are capable of indicating reaction progress and provide real-time monitoring of the accumulation of reaction products. The Ct value derived from the fluorescence signal curve of a qPCR reaction generally allows quantitative determination of the concentration of the starting target gene. Third-generation PCR technology, often referred to as digital PCR (dPCR, or Dig-PCR), detects and quantifies nucleic acids by directly measuring the number of target molecules without relying on any normalization standard or external standards. In this manner, the absolute number of target molecules can be determined, with a lower limit being a single copy of the molecule. The strategy used by dPCR can be referred to as a "Divide and Conquer" strategy where a sample is first diluted and divided into thousands to tens of thousands of micro reaction chambers, so that each reaction chamber contains only either zero or one copy of the target gene sequence (a small number of reaction chambers may contain multiple copies). By counting the number of reaction chambers with positive amplification results, researchers can determine the absolute number of target gene molecules in the original sample. Because of the binary Yes/No amplification states when analyzing results from each reaction chamber in dPCR, there is no need to determine the crossing point between a set threshold value and the fluorescence signal (as in qPCR). In general, data analysis in dPCR is completely independent of determining a Ct value, thereby significantly reducing the influence by the amplification efficiency in dPCR and improving the tolerance to inhibitors in the PCR reaction. The standard division process of the reaction system in dPCR can greatly reduce the concentration of background sequences that could compete with the target sequence; therefore, dPCR may be especially suitable for detection of rare mutations in a complex background. In comparison to traditional PGR technology, dPCR is considered to have multiple advantages, including low required sample amount, reduced consumption of reagents, absolute quantification of nucleic acid molecules, reduced interference among different copies within the same sample, and superb sensitivity and specificity.

[0005] Two currently available types of dPCR products on the market are microfluidic chip- based dPCR and microdroplet-based dPCR. The principle behind microfluidic chip-based dPCR generally relies on microfluidic devices, which perform highly parallel analysis within the same dPCR reaction. Multilayer soft lithography (MSL) technology is often used to design the integrated microfluidic routes and controls the cost of manufacturing the microfluidic chips. In theory, any molecular reaction that can be carried out in a test tube can be realized on a microfluidic chip. On the same piece of MSL chip, thousands to tens of thousands of individual PCR reactions can be executed, reducing the consumption of reagents and the necessary step for transferring liquids. The microfluidic valves can distribute single copies of sample among a large number of individual microwells, and dPCR can be automatically executed on the MSL chip. In a microfluidic chip, a positive reaction contains at least one target molecule, and for very dilute samples, the number of positive reactions is very close to the number of target molecules. In a sample with a relatively higher

concentration, a positive reaction can contain more than one target molecule, Poisson distribution can be used to calculate the number of target molecules. Representative companies that have been developing microfluidic chip-based dPCR technologies include Fluidigm and Life Technologies.

? [0006] With respect to microdroplet-based dPCR, the process generally includes generating microdroplets with a sample mixture and then controlling the flow of the microdroplets through an inert oily carrier fluid along microchannels. The microdroplets typically do not interfere with each other so that each microdroplet can be considered to be an independent microscale PCR reaction device. The microdroplets are usually far smaller in volume than microweils within a microwell plate (widely used in first and second generation PCR technology, such as 96-well plates), to the extent that microdroplet-based dPCR can be adapted to single-cell analysis. The data analysis method of microdroplet-based dPCR is similar to that used in chip-based dPCR, However, microdroplet-based dPCR is thought to be more versatile than chip-based dPCR given that microdroplets can be formed by various different processes. Researchers can even design a unique experimental procedure for each microdroplet. Representative companies that have been developing microdroplet-based dPCR technology include Bio-Rad and Raindance Technologies.

[0007] Droplet generation technology based on microfiuidics has undergone rapid development in recent years. Here the formation of droplets is generally due to the instability at the boundary between the distributed phase and the continuous phase within a

microchannel. Droplets with miniscule volume can be generated, which can further be subjected to merging, reaction, and division. However, in order to generate suitable droplets on a chip, certain conditions, including specific flow rate, surface tension at the oil-water boundary, as well as conformation and surface modification of the microchannels must be satisfied. The adjustable range of volume of the droplets is therefore limited by the aforementioned factors. Additionally, after the droplets are formed in the microchannels, specific procedures and devices are required to transfer the droplets to a storage apparatus in order to set the conditions of individual droplets, rendering steps such as localization, retrieval, and analysis of the droplets rather inconvenient.

[0008] Other droplet generation technologies rely on capillary tubes or microchannels to inject or eject micro-volumes of liquid, and to infuse the liquid into microweils or spots on a base chip. This technology, in principle, is a simple strategy to generate droplets. However, in actual execution, when a droplet is leaving the capillar}' tube, there exists surface tension between the droplet and the contiguous liquid within the tube, as well as affinity between the droplet and the surface of the tube opening, both of which affect the volume of the droplet. To overcome these factors, measures that include the use of piezoelectric materials, heat- induced expansion, and ultrasound, are employed to increase the momentum of the droplet while leaving the capillar tube in order to overcome the surface tension. Furthermore, the opening of the microchannel, and silylation or provision of a coating treatment to the surface of the microchannel, can be used to reduce the affinity between the droplet and the tube opening. However, such methods have many disadvantages, including reliance on expensive and complicated fluidic driving devices, and difficulty in precisely controlling droplet volume

[0009] Alternatively, the opening of the microchannel or the capillary tube can be directly pointed at a base chip (or a microwell) to spot the sample such that when the sample droplet contacts the base chip, the affinity and boundary tension between the droplet and the base chip overcomes the affinity between the droplet and the tube opening. However, for biological samples having a complicated composition and non-uniform viscosity, there still lacks a widely applicable method to completely overcome the surface tension between the droplet and the opening of the microchannel in order to avoid accumulation of residual sample at the opening of the microchannel. Accumulation of residual sample at the opening of a microchannel can result in deviation in the droplet volume, clogging of microchannel s, and cross-contamination among samples.

[0010] Overall, currently available technologies to generate microscale reaction chambers for dPCR, including microfluidic-based dPCR and microdroplet-based dPCR, require

complicated and precise flow control for the microstream or microdroplets, as well as microchannels with complex designs to execute the experiments. Consequently, current dPCR systems tend to be expensive and oftentimes incompatible with the traditional 96-weil plate format. Accordingly, new dPCR systems and methods for microdroplet generation and control are clearly needed,

SUMMARY

[0011] The systems described herein may be useful in generating microdroplets of a sample fluid. In some instances, the systems are configured to perform digital PCR on the sample fluid. Other applications may include single cell analysis, DNA sequencing, drug selection, microbiological analysis, and clinical disease diagnosis. The systems generally comprise a microdroplet generation assembly that differs from Inkjet technology by forming the microdroplets directly in a fluid. A further distinction is that the sample fluid is contained within a needle lumen of the droplet generation assembly and is flushed therefrom into a collection reservoir instead of being loaded into an injection chamber. Depending on the intended use, the system may be configured to include the microdroplet generation assembly alone, or in combination with a sample needle loading/sample loading module, a PCR module, and/or a fluorescence detection module. When employed in digital PCR, the systems described herein may include at least a sample needle loading/sample loading module, a microdroplet generation assembly, and a PCR module. Here a fluorescence detection module may also be included if desired. The various devices, assemblies, subassemblies, modules, etc., will generally be configured to provide a fully automated digital PCR system,

[0012] In general, the microdroplet generation assemblies described herein include an elongate needle (sample needle) having a proximal end, a distal end, and a lumen extending therebetween, a deformable injection chamber in fluid communication with the needle lumen and containing a carrier fluid, where the deformable injection chamber comprises a proximal end, a distal end, and an actuation mechanism configured to deform a wall of the injection chamber; and a reservoir for collecting a microdroplet, where the reservoir comprises a reservoir fluid. The deformation of the injection chamber compresses a carrier fluid contained therein, resulting in a pressure wave of carrier fluid that forces both the carrier fluid and the sample fluid through the needle. As the sample fluid is injected into a reservoir fluid, one or more microdroplets of the sample fluid are formed. The needles empl oyed will usually be disposable so that they can be exchanged for new needles when a different sample fluid is to be loaded.

[0013] The actuation mechanism of the microdroplet generation assembly m ay compri se a piezoelectric ceramic material that deforms upon application of an applied voltage and frequency. The piezoelectric ceramic material may have a driven frequency ranging from about 0.1 to about 2 kHz. The driven peak-to peak voltage of the piezoelectric ceramic material may range from about 10 to about 1,000 Vpp.

[0014] The proximal end of the needle may comprise a conical hub configured for removable attachment to the distal end of the injection chamber. Other suitable hub geometries may be employed. The distal end of the needle typically comprises an orifice in fluid communication with the needle lumen . The orifice may have a diameter of ranging from about 10 microns to about 2.0 mm. In some instances, the needle is a disposable needle. [0015] A sample fluid is loaded and contained within the needle. The sample fluid may be an aqueous fluid. Exemplar}' sample fluids may comprise a plurality of nucleic acids such as DNA and/or R A.

[0016] With respect to the carrier fluid and the reservoir fluid, any suitable fluid may be used. The carrier fluid and the reservoir fluid can be the same or different. The carrier fluid and the reservoir fluid may be an oily fluid. For example, the carrier fluid and/or the reservoir fluid may comprise mineral oil. In other instances, the carrier fluid and/or the reservoir fluid may comprise silicone oil .

[0017] The reservoir of the microdroplet generation assembly may be a single well (reservoir) of a microwell plate. The microwell plate may comprise any number of wells depending on the experimental design. For example, when microdroplets are generated for PCR, the microwell plate may include 96 wells. The microwell plate may comprise a plurality of rows, where each row includes a plurality of reservoirs. Each row may contain microdroplets of the same volume. Alternatively, each row may contain microdroplets of different volume.

[0018] The reservoirs may include one or more (a plurality) of microdroplets of a sample fluid. The one or more microdroplets of the sample fluid may comprise at least one nucleic acid such as DNA and/or RNA. The one or more microdroplets of the sample fluid may have a volume ranging from about 1 nl to about 150 ni. For example, the volume of the

microdroplets may be about I nl, about 5 nl, about 25 nl, or about 125 nl.

[0019] The microdroplet generation assemblies described herein may be useful to include in a PCR system, e.g., a dPCR system. Accordingly, such a system may comprise a

microdroplet generation assembly and a dPCR module, e.g., a temperature-controlled dPCR module. Similar to that mentioned above, a sample fluid is loaded and contained within the needle. The sample fluid may be an aqueous fluid. Exemplar,' sample fluids may comprise a plurality of nucleic acids such as DNA and/or RNA.

[0020] Additionally, the carrier fluid and the reservoir fluid can be the same or different in the PCR system. The carrier fluid and the reservoir fluid may be an oily fluid. For example, the carrier fluid and/or the reservoir fluid may comprise mineral oil. In other instances, the carrier fluid and/or the reservoir fluid may comprise silicone oil. [0021] Furthermore, the reservoir of the system may be a single well (reservoir) of a microwell plate. The microwell plate may comprise any number of wells depending on the experimental design. For example, when microdroplets are generated for PGR, the microwell plate may include 96 wells. The microwell plate may comprise a plurality of rows, where each row includes a plurality of reservoirs. Each row may contain microdroplets of the same volume. Alternatively, each row may contain microdroplets of different volume.

[0022] The PGR system may also include a sample needle loading/sample loading module. If desired, a detection module, e.g., a fluorescence detection module can further be included. The microdroplet generation assembly, the sample needle loading/sample loading module, the PGR module, and the detection module may be configured to operate as an automated digital PGR system,

[0023] Additionally, methods for microdroplet formation are described herein. The methods generally include a step of loading a sample fluid into a needle of a microdroplet generation assembly, where the needle has a proximal end and a distal orifice, and a lumen extending therebetween. The microdroplet generation assembly will generally further comprise a reservoir containing a reservoir fluid and an injection chamber in fluid communication with the needle lumen and containing a carrier fluid, where the injection chamber comprises an actuation mechanism. The actuation mechanism may comprise a piezoelectric ceramic material that deforms as described above. The methods may also include the steps of placing the needle orifice into the reservoir fluid, activating the actuation mechanism to deform a wall of the injection chamber, and forming a microdroplet of the sample fluid by ejecting a volume of the sample fluid from the needle into the reservoir fluid while the needle orifice is disposed in the reservoir fluid. The needle may be replaced, e.g., when a different sample fluid is to be loaded.

[0024] More specifically, activation of the actuation mechanism generates a flow of carrier fluid from the injection chamber through the lumen of the needle to thereby eject the volume of the sample fluid into the reservoir fluid. Here the ejected volume of the sample liquid directly correlates to an amplitude of deformation of the wall of the injection chamber. The actuation mechanism of the microdroplet generation assembly may comprise a piezoelectric ceramic material that deforms upon application of an applied voltage and frequency. The piezoelectric ceramic material may have a driven frequency ranging from about 0.1 to about

"7 2 kHz. The driven peak-ΐο peak voltage of the piezoelectric ceramic material may range from about 10 to about 1 ,000 Vpp.

[0025] A sample fluid is loaded and contained within the needle. The sample fluid may be an aqueous fluid. Exemplary sample fluids may comprise a plurality of nucleic acids such as DNA and/or RNA.

[0026] With respect to the carrier fluid and the reservoir fluid, any suitable fluid may be used. The carrier fluid and the reservoir fluid can be the same or different. The carrier fluid and the reservoir fluid may be an oily fluid. For example, the carrier fluid and/or the reservoir fluid may comprise mineral oil . In other instances, the carrier fluid and/or the reservoir fluid may comprise silicone oil.

[0027] The reservoir of the microdroplet generation assembly may be a single well (reservoir) of a microwell plate. The microwell plate may comprise any number of wells depending on the experimental design. For example, when microdropiets are generated for PGR, the microwell plate may include 96 wells. The microwell plate may comprise a plurality of rows, where each row includes a plurality of reservoirs. Each row may contain microdropiets of the same volume. Alternatively, each row may contain microdropiets of different volume.

[0028] The reservoirs may include one or more (a plurality) of microdropiets of a sample fluid. The one or more microdropiets of the sample fluid may comprise at least one nucleic acid such as DNA and/or RNA. The one or more microdropiets of the sample fluid may have a volume ranging from about I nl to about 150 nl. For example, the volume of the

microdropiets may be about 1 nl, about 5 nl, about 25 nl, or about 125 nl.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 A is a flow chart that illustrates an exemplary digital PGR process performed by the systems described herein.

[0030] FIG. I B shows a perspective view of an exemplary digital PGR system .

[0031] FIG. 1C shows another perspective view of the exemplary digital PGR system of FIG. IB. [0032] FIGS. 2A-2E depict an exemplary niicrodroplet generation assembly. FIG. 2 A shows a perspective view of the exemplar}' niicrodroplet generation assembly. FIGS. 2B and 2C show unassembled views of the niicrodroplet generation assembly of FIG. 2 A. FIG. 2D shows a cross-sectional view taken through a portion of the niicrodroplet generation assembly of FIG. 2 A. FIG. 2E is a side, cross-sectional view of the niicrodroplet generation assembly of FIG 2 A.

[0033] FIG. 3 A depicts perspective views of an exemplar}' sample needle loading/sample loading module.

[0034] FIG 3B depicts another perspective view of the exemplar}' sample needle

loading/sample loading module of FIG. 3 A, the module including the sample loading needles.

[0035] FIG. 3C illustrates an exemplary process of sample needle loading and sample fluid loading.

[0036] FIG. 4 depicts an exemplary temperature-controlled PGR module. [0037] FIG. 5 depicts an exemplary fluorescence detection module.

[0038] FIGS. 6A and 6B illustrate an exemplary method of how the sample loading module and microdroplet generation assembly automatically work together to deliver microdroplets into a 96-well plate.

[0039] FIG. 7 illustrates another exemplary method of sample uptake and distribution.

[0040] FIG. 8A is a schematic diagram that shows an exemplary microdroplet generation assembly of a dPCR system and an exemplary method of forming the microdroplets using the assembly.

[0041] FIG. 8B is a schematic diagram that shows an exemplary temperature-controlled PGR reaction using the dPCR microdroplet generation assembly of FIG. 8 A.

[0042] FIG. 8C is a schematic representation that shows fluorescence detection results using the dPCR microdroplet generation assembly of FIG 8 A. DETAILED DESCRIPTION

[0043] Described herein are systems in which microdroplet formation may be useful. For example, PGR systems, including digital PGR systems, are described that generate microdroplets of sample fluid without relying on fluidic control. The systems generally comprise a microdroplet generation assembly that may form microdroplets directly in a fluid. Furthermore, the sample fluid contained within a needle lumen of the droplet generation assembly is flushed therefrom into a collection reservoir instead of being loaded into an injection chamber. In addition to PGR, the systems described herein may be used for single cell analysis, DNA sequencing, drug selection, microbiological analysis, and clinical disease diagnosis. The systems may be used for other applications and indications where

microdroplet formation or analysis of microdroplets may be useful.

PGR SYSTEMS

[0044] The PGR systems described herein generally include four components or modules, although depending on the indication, additional (or fewer) components can be employed. The four components will usually be a microdroplet generation assembly, a sample needle loading/sample loading module, a PGR module, and a fluorescence detection module. The modules may be used in the process of sample preparation, microdroplet preparation, PGR, fluorescence detection, and data analysis, which is diagrammed in the flowchart provided in FIG. 1 Λ

[0045] For example, when digital PGR is to be performed, the dPCR system may include the four components as configured in FIGS. IB and 1C. In FIG. IB, a dPCR system (10) is shown that comprises a housing (12). Disposed within the housing (12) is a sample needle loading/sample loading module ( 14), a microdroplet generation assembly (16), and a PGR module (18), Referring to another view of the dPCR system (10), as shown in FIG. IC, the housing (12) further includes a fluorescence detection module (11). It is understood that components of the system can be arranged within the housing in other suitable ways. A more detailed description of the dPCR system components is provided below. Although a system for dPCR has been disclosed, it is understood that the microdroplet generation assembly described herein and further below can be used in systems having other applications. MICRODROPLET GENERATION ASSEMBLY

[0046] The systems described herein generally include a microdroplet generation assembly for forming microdroplets of a sample fluid. The microdroplet generation assembly may comprise a needle having a proximal end, a distal end, and a lumen extending therebetween; a deformabie injection chamber in fluid communication with the needle lumen and containing a carrier fluid, where the deformabie injection chamber comprises a proximal end, a distal end, and an actuation mechanism configured to deform a wall of the injection chamber; and a reservoir for collecting a microdroplet, where the reservoir comprises a reservoir fluid. The relationship between the properties of the sample fluid and the carrier fluid, and the sample fluid and the reservoir fluid, influences microdroplet formation. In general, the sample fluid and the reservoir fluid are immiscible to prevent formed microdroplets from coalescing, and the sample fluid and the carrier fluid are immiscible so that they remain separated within the injection chamber. The sample fluid typically comprises an aqueous fluid, and the carrier fluid and the reservoir fluid typically comprise an oily fluid. Unlike the technology used in Inkjet printing where a sample fluid is loaded into and contained within an injection chamber, the microdroplet generation assembly described here includes a sample fluid that is loaded into and contained within a needle of the assembly. Furthermore, instead of forming microdroplets on a surface, the needle of the microdroplet generation assembly is placed into a reservoir fluid and the microdroplets formed while a portion of the needle, e.g., the needle orifice, is immersed in the reservoir fluid.

[0047] The needle of the microdroplet generation assembly will generally comprise a proximal end, a distal end, and a lumen extending therebetween, as stated above. Needle length may range from about 10 to about 15 mm. The proximal end may include a hub configured for removable attachment to the distal end of an injection chamber. For example, the hub may include threads on its internal surface (or a portion thereof) for pairing with external threads on the distal end of the injection chamber so that rotation of the hub in one direction about the external threads attaches the hub to the injection chamber. Here rotation in the opposite direction would remove or detach the hub from the injection chamber.

Alternatively, the hub and distal end of the injection chamber could be removably attached by a friction fit between the two elements. The hub portion of the needle may be conicaily shaped. This configuration may be beneficial because it may minimize the amount of residual sample liquid held by the hub after microdroplet ejection. The distal end of the needle comprises an orifice in fluid communication with the needle lumen. The orifice may have a diameter ranging from about 10 microns to about 2.0 mm. In some variations, the orifice diameter may range from about 10 microns to about 1.0 mm. In further variations, the orifice diameter may range from about 10 microns to about 50 microns. In one instance, the orifice diameter is about 50 microns. The inner diameter of the needle may range from about 5 to about 250 microns, from about 40 to about 200 microns, from about 25 to about 150 microns, or from about 10 to about 100 microns. In one variation, the needle may have an inner diameter of about 50 microns. The outer diameter of the needle may range from about 10 to about 500 microns. Some variations of the needle are configured to be disposable,

[0048] The needle may be made from any suitable material . Exemplary materials include without limitation, TEFLON® thermoplastic polymer (polytetrafluoroethylene or PTFE), silicone, and stainless steel. In one variation, the needle is made of stainless steel. The needles may also be surface treated with a material imparting lower surface tension at the needle orifi ce. For example, the outer wall of the needles can be coated with a lubricious material, e.g., by silanization, using 1 H, 1H, 2H, 2H-perflurooctyltrichlorosilane. It is understood that other suitable materials can be used to treat the surface of the needle to reduce the surface tension at the needle orifice.

[0049] In contrast to Inkjet technology, a sample fluid is loaded and contained within the needle, not within an injection chamber. The sample fluid may include a plurality of nucleic acid sequences. Some variations of the sample fluid include DNA as the target nucleic acid . In other variations, the sample fluid includes RNA as the target nucleic acid. Exemplary target nucleic acids include, but are not limited to, genomic DNA, fragments of genomic DNA (e.g., sheared or restriction-digested genomic DNA), mitochondria DNA, cDNA, total RNA, mRNA, non-coding RNA (e.g. tRNA, rRNA and long non-coding RNA/incRNA), small RNA (e.g., microRNA, siRNA and piRNA), next-generation sequencing library, other forms of DNA or RNA, biochemically modified (e.g., methylated or damaged) DNA or RNA, chemically treated DNA or RNA (e.g., methylated DNA), or any combination thereof.

Target nucleic acids may be purified samples with only a single sequence, a single nucleic acid species, or multiple sequences, or multiple nucleic acid species. Alternatively, complex biological samples containing target nucleic acids, which further comprise non-nucleic acid molecules, such as proteins, lipids, carbohydrates and small molecules, can be directly used in the sample fluid without nucleic acid extraction or purification steps. Examples of such complex biological samples may include total ceil lysates, crude extracts from formalin-fixed, paraffin-embedded (FFPE) samples, and microbial or environmental nucleic acids samples (e.g. extracts of soil). Current chip-based dPCR and microdroplet-based dPCR on the market, which rely on microfiuidic control to generate reaction microwells, typically have difficulty handling complex biological samples with high viscosity. However, the microdroplet generation assembly described herein can be used to sample fluids with various physical properties (e.g., high viscosity) by adjusting control parameters, such as carrier fluid ejection flow rate and/or vibration amplitude or frequency. In other instances, the sample fluid comprises a reagent, e.g., enzymes (such as DNA polymerase and optionally, RNA reverse transcriptase), nucleotide mixtures (such as dNTP, NTP), buffer, RNase inhibitor, primers (such as target specifi c primers, random primers, and/or oligo(dT)), or one or more

fluorescent probes. Exemplary fluorescence probes suitable for dPCR reactions include nonspecific DNA Binding Dye (such as SYBR ^'8, green I), or sequence-specific fluorescently labeled probes that are positioned between the PGR primers (such as TaqMan* molecular beacons, or SCORPION ^® probe).

[0050] The injection chamber of the microdroplet generation assembly may have a

cylindrical geometry, with a diameter of about 35 mm and a height of about 10 mm, but an suitable shape can be employed. The injection chamber generally contains a carrier fluid that is used to flush a sample fluid out of the needle and into a reseivoir fluid. Exemplary carrier fluids typically have a density that is lower than the density of the sample fluid so that the microdroplet will fall to the bottom of the reservoir due to gravity. For example, the sample fluid may have a density of about 1.0 g/cm ³ (similar to water), and the carrier fluid may have a density of about 0.8 g/cm ^J . The carrier fluids typically also have one or more properties that prevent the microdroplets from breaking or merging with each other during the PGR process. For example, the carrier fluid can be one that is immiscible with the sample fluid. Here the carrier fluid can be an oily fluid and the sample fluid can be an aqueous fluid. In some variations, the carrier fluid comprises mineral oil. In other variations, the carrier fluid comprises silicone oil. In one variation, the carrier fluid and the reservoir fluid are the same. For example, the carrier fluid and the reservoir fluid may both comprise mineral oil.

[0051] In order for the carrier fluid to force a sample fluid out of the needle, a pressure wave of the carrier fluid is generated. The actuation mechanism of the microdroplet generation assembly will generally be configured to deform a wall of the injection chamber to generate this wave of fluid. In some variations, the actuation mechanism comprises a chip made from a piezoelectric material, e.g., a piezoelectric ceramic material. Exemplary piezoelectric ceramic materials include without limitation, barium titanate, bismuth ferrite, bismuth titanate, lead zirconate titanate, potassium niobate, lithium niobate, lithium tantalite, sodium bismuth titanate, sodium niobate, sodium potassium niobate, sodium tungstate, zinc oxide, and combinations thereof.

[0052] The piezoelectric chip is capable of deformation (e.g., expanding and contracting) in response to a driven frequency ranging from about 0.1 to about 2 kHz and a driven peak-to peak voltage ranging from about 10 to about 1,000 Vpp, from about 10 Vpp to about 500 Vpp, from about 10 to about 100 Vpp, from about 10 to about 50 Vpp, or from about 10 to about 25 Vpp. For example, the driven frequency may be about 10 Vpp, about 20 Vpp, about 30 Vpp, about 40 Vpp, about 50 Vpp, about 60 Vpp, about 70 Vpp, about 80 Vpp, about 90 Vpp, about 100 Vpp, about 150 Vpp, about 200 Vpp, about 250 Vpp, about 300 Vpp, about 350 Vpp, about 400 Vpp, about 450 Vpp, about 500 Vpp, about 550 Vpp, about 600 Vpp, about 650 Vpp, about 700 Vpp, about 750 Vpp, about 800 Vpp, about 850 Vpp, about 900 Vpp, about 950 Vpp, or about 1,000 Vpp. The driven frequency generally controls the speed of droplet formation so that formation is efficient (not too slow) but not so fast that the droplets will collide with one another. In use, deformation of the piezoelectric chip deforms a wall of the injection chamber, which in turn compresses the carrier fluid within the injection chamber to thereby create a wave of carrier fluid. The wave of carrier fluid travels through the needle to eject a sample fluid therefrom in the form of a microdroplet.

[0053] When piezoelectric actuation mechanisms are used, the surface tension and viscosity of the sample fluid may be adjusted to a value beneficial for microdroplet formation. For example, the viscosity can be adjusted to be between about 0.5 to about 40 CP, and the surface tension can be adjusted to be between about 20 to about 70 dynes/cm. Surface tension and viscosity can be modified by changing the concentration of solute and solvent, or by adding viscosity and/or surface tension modifying agents such as glycol, glycerin, alcohols, and/or surfactants.

[0054] The volume of the formed microdroplet may depend on the amount (amplitude) of deformation of the actuation mechanism, e.g., the piezoelectri c chip. The amplitude of deformation is typically a few microns. The volume of the microdroplets may range from about 0.02 nl to about 150 nl, or from about 0.25 nl to about 150 nl. For example, the volume of the microdroplets may be about 1 nl, about 5 nl, about 10 ni, about 15 nl, about 20 nl, about 25 nl, about 50 nl, about 100 nl, about 125 nl, or about 150 nl. In some variations, the microdroplet includes at least one nucleic acid sequence. In other variations, the

microdroplet includes at least one DNA sequence.

[0055] To reiterate, the volume of the microdroplets m ay be determined by the voltage applied to the piezoelectric chip. Higher voltage typically leads to larger deformation of the piezoelectric chip, thereby resulting in a larger volume change of the chamber, and a larger volume of droplets ejected out by the chamber. The speed of the ejected droplets may be determined by the frequency of the electrical voltage applied to the piezoelectric chip.

Appropriately set vibrational frequency can on one hand reduce the time used to generate the microdroplets as well as avoid fusion and overlapping of the microdroplets due to overly fast droplet generation.

[0056] A reservoir of the microdroplet generation assembly collects the one or more formed microdroplets of a sample fluid, and may be a single well of a microwell plate. However, any suitable number of wells may be employed. When used in digital PCR, the microwell plate may comprise 24 wells, 96 wells, or 384 wells, etc, and may be made from a material suitable for heat insulation. The reservoir will generally include a reservoir fluid. It may be beneficial to use a reservoir fluid that has a density lower than that of the density of the sample fluid so that the microdroplet of sample fluid will fall to the bottom of the reservoir due to gravity, as well as a reservoir fluid having properties that prevent the microdroplets from breaking or merging during the PCR process. In some variations, the reservoir fluid comprises mineral oil. In one variation, the reservoir fluid and the carrier fluid are the same. For example, the reservoir fluid and the carrier fluid may both comprise mineral oil. The number of microdroplets that can be generated per reservoir may generally depend on the volume of the microdroplet and the surface area of the reservoir. For example, if the volume of one microdroplet is about 1 nl, there could be about 1,000 microdroplets in one well of a 96-well plate,

[0057] The microdroplet generation assemblies described herein generally form the microdroplets while the needle orifice is submerged in a reservoir fluid. The needle orifice may be positioned to be between about 2 mm and about 5 mm from the bottom of the reservoir. For example, the needle orifice may be positioned about 2 mm, about 3 mm, about 4 mm, or about 5 mm from the bottom of the reservoir. In one variation, the needle orifice is positioned at least about 2 mm from the bottom of the reservoir to prevent the microdroplet from breaking upon hitting the reservoir bottom,

[0058] The microdroplet generation assemblies may form microdroplets using various other mechanisms that do not require complex microfluidic control. For example, microdroplets may be formed using vibrational, ultrasonic, or electrostatic mechanisms.

[0059] In yet further variations, the microdroplet generation assembly includes a cartridge comprising a plurality of injection chambers, e.g., four to eight injection chambers. As further described below, needles can be removably attached to the plurality of injection chambers and a sample fluid loaded therein by a sample needle loading/sample loading module. Using this design, different tests or experiments (e.g., on different sample fluids) can be performed by only replacing the needle and without the risk of contamination between samples and contamination of the reusable (non-disposable) parts of the system, thus significantly reducing the cost of the process,

[0060] As previously stated, the microdroplet generation assemblies described herein generally include an elongate needle (sample needle) having a proximal end, a distal end, and a lumen extending therebetween; a deformabie injection chamber in fluid communication with the needle lumen and containing a carrier fluid, where the deformabie injection chamber comprises a proximal end, a distal end, and an actuation mechanism configured to deform a wall of the injection chamber, and a reservoir for collecting a microdroplet, where the reservoir comprises a reservoir fluid. Referring to FIGS. 2A-2E, parts of an exemplary microdroplet generation assembly are shown, FIG. 2A depicts a substantial portion of the microdroplet generation assembly, where the assembly (20) comprises a cartridge (22) including a plurality of injection chambers (not shown), piezoelectric chips (24), and needles (26). The cartridge (22) is coupled to a robotic arm (28) that moves the cartridge (22) within the system so that needles (26) can be attached and detached from associated injection chambers (not shown) and sample fluid loaded into the needles (26).

[0061] In FIG. 2B, an unassembled view of the microdroplet generation assembly (20) is provided to show further details of the various elements of the assembly (20). Here input connectors (30) are coupled to injection chambers (not shown) disposed within cartridge (22) for attachment of a carrier fluid delivery device. The piezoelectric ceramic chips (32) are fastened to the cartridge (22) by a pressure ring (42). An output connector (36) removably attaches a distal end of an injection chamber within cartridge (22) to a needle (26),

[0062] The cartridge may be formed by a plurality of bodies. Referring to FIG. 2C, additional staictural detail of the bodies are provided. In the figure, an individual body (40) comprises a piezoelectric ceramic chip (32), a sealing ring (34), a pressure ring (42), an output connector (36), and a needle (26). As shown in the cross-sectional views of FIG. 2D, piezoelectric chip (32) is disposed between the sealing ring (34) and the pressure ring (42) in a fully assembled body, and piezoelectric chip (32) forms one wall of the injection chamber (44). FIG. 2E shows a side, cross-sectional view of the body (40), the piezoelectric ceramic chip (32), and the injection chamber (44).

[0063] As further described below, the microdroplet generation assembly and sample needle loading/sample loading module generally work together to take up sample fluid and create microdroplets of the sample fluid to add to a microwell plate,

SAMPLE NEEDLE LOADING/SAMPLE LOADING MODULE,

[0064] In addition to a microdroplet generation assembly, the PCR systems described herein, including dPCR systems, may also include a sample needle loading/sample loading module, which functions to attach and detach needles (sample needles) to an output connector, e.g., as shown in FIG. 2B, and to load a sample (sample fluid) into the needles. After the sample is taken up, microdroplets of predetermined size may be generated and distributed into a microwell plate, e.g., a 96-well plate. The sample needle loading/sample loading module may include any suitable number of needles and sample-containing centrifuge tubes.

[0065] In some variations, the sample needle loading/sample loading module comprises an equal number of needles and sample-containing centrifuge tubes. For example, as shown in FIG. 3A, the sample needle loading/sample loading module may be configured to hold 24 sample-containing centrifuge tubes and 24 sample loading needles. Here the sample needle loading/sample loading module (50) is structured to hold three rows of centrifuge tubes (52) and three rows of sample loading needles (54). The rows (52, 54) with centrifuge tubes (51) and sample needles (53) contained therein are depicted in FIG, 3B. Each row contains 8 centrifuge tubes or 8 needles. At the rear of the module are needle unloading slots (56), which can unload one row of sample loading needles at a time in conjunction with the sample loading device. The module may further include a needle recycling bin (58) (FIG. 3A) to temporarily store used sample loading needles,

[0066] In use, as illustrated in FIG. 3C, the sample needle loading/sample loading module (50) may perform the following steps: 1) attach a row of 8 sample loading needles to output connectors (60) by pressing them in the downward direction onto the proximal end of the sample loading needles (53); 2) translate a robotic arm (62) to move the sample loading needles (53) and position them within centrifuge tubes (51) to load a sample fluid (not shown) from the centrifuge tubes (51) into the needles (53); and 3) detach used needles (64) from their respective output connectors by placing them into a needle unloading slot (56), as previously described, whereby the needles drop into the needle recycling bin.

[0067] Generally, the sample needle loading/sample loading module, in conjunction with the microdroplet generation assembly, can achieve fully automated sample loading. In some instances, 24 samples can be distributed in a 96-well plate at a time. Each sample loading needle typically only takes sample fluid from a single centrifuge tube; the sample in each centrifuge tube may then be added into a series of four consecutive wells on the 96-weli plate in the form of microdroplets of the same volume or up to four different volumes. Referring to FIG. 7, a needle (66) is used to take up sample in the centrifuge tube (68), which is then distributed into each of the four wells indicated by dashed lines in the 96-well plate. Each well may receive microdroplets of the same or different volumes. The same scheme generally applies to the other needles, centrifuge tubes, and wells accordingly. For example, as shown in FIGS. 6A-6B, 8 needles (61) can be used to take up sample from centrifuge tubes (63) for distribution into each of 8 wells in a 96-well plate (65).

PGR MODULE

[0068] The systems described herein generally include a PGR module. The systems may be configured to perform dPCR. In one variation, the PGR module is a temperature-controlled PGR module. In another variation, the PGR module is a temperature-controlled dPCR module. An exemplary temperature-controlled dPCR module is shown in FIG . 4. In the figure, temperature-controlled dPCR module (70) includes an electric heater (72), a fan (74), a cooling wind channel (76), and a thermoelectric cooling (TEC) chip (78). In this instance, air heated by the electric heater (72) is evenly blown onto the bottom of a multiwell plate (e.g., 96-weli plate) (80) to heat the samples therein. When rapid cooling is needed, the TEC chip (78) is activated to cool air within the cooling wind channel (76). Fan (74) then blows the cool air onto electric heater (72) and the 96-well plate (80), in order to rapidly cool the reaction system. The temperature obtained by a temperature sensor (not shown), which is located near the 96-well plate, controls the heating temperature, thereby realizing the necessary and precise temperature environment for the reaction.

DETECTION MODULE

[0069] The PGR systems described herein may include a detection module for detection of a signal from the microdroplets. In one variation, the detection module is a fluorescence detection module. For example, after the sample has undergone temperature-controlled PGR, a main carrier stage transfers the sample to the fluorescence detection module to examine the reaction results. For example, as shown in FIG. 5, when the main carrier stage with the 96- well plate reaches its set position in the fluorescence detection module (82), an electrical motor (84) drives a ball screw (86) to translate a microscope lens (88) along a linear guide (90) to the position of each well of the 96-well plate successively. At each detection position, the microscope lens (80) focuses the light beam from a light source (92) to the microdroplets lying flatly on the bottom of the well, wherein the fluorescent probe in the microdroplet is excited to produce a fluorescence signal. Here the microscope lens (88) collects the signal and sends it back to the fluorescence detection camera (94) to collect data. A schematic diagram showing fluorescence detection results of reactions using the dPCR system as described herein is provided in FIG. 8C.

[0070] The aforementioned light source can be a laser, a mercury arc discharge lamp, a metal halide lamp, a xenon lamp, a LED (Light Emitting Diode), or any suitable light source that has prominent emission lines at the excitation wavelength(s) of the fluorophore(s) in the fluorescence probe(s) used in the dPCR assay. The light source may further comprise a bandpass interference filter to select a specific band of wavelengths that are used to illuminate the fluorophore.

[0071] Some variations of the fluorescence detection module allow execution of a multicolor dPCR, in which multiple fluorescent probes are used, for example, to allow

simultaneous quantification of multiple target nucleic acid sequences using a uniquely colored sequence-specific fluorescent probe for each target nucleic acid. Multi-color dPCR requires the fluorescence detection module to have a light source emitting at multiple wavelengths (e.g., red, green and/or blue light) and the ability to rapidly switch between different wavelengths of light to excite multiple f!uorophores within the same microdroplet. Typically, to achieve the rapid switch between different wavelengths, multiple filters, beam splitters, rotating filter wheels, tunable optical filters, and/or monochromators are employed in combination with the light source.

[0072] To reduce signal-to-background ratio, the lens may further comprise an emission filter to block excitation light while passing the fluorescence signal from the fluorophore(s). The fluorescence detection camera can be a CCD (charge-coupled device) camera or a CMOS image sensor, optionally with multicolor sensing capability, to provide high resolution and sensitivity to fluorescent signals,

[0073] The PCR modules generally comprise a microdroplet formation module and a PGR module. The microdroplet formation module may include a microdroplet generation assembly as described herein but is not so limited. It is understood that the microdroplet formation module may employ other devices, components, assemblies, etc. that form microdroplets using different technologies. For example, a microdroplet formation module that solely uses vibration to form the microdroplets can be employed. As previously described, the microdroplet generation assembly may include an injection chamber having a proximal end and a distal end, and a carrier fluid disposed within the injection chamber. The carrier fluid may comprise mineral oil. In one variation, the injection chamber comprises a wall partially formed by a deformable, piezoelectric ceramic material. A needle is generally coupled to the distal end of the injection chamber, and generally includes a proximal hub and a distal orifice. The proximal hub may have any suitable geometry, and in some instances is conical ly shaped. The needle is generally configured to have a sample fluid, e.g., an aqueous sample fluid, loaded into and contained therein. The PCR system may further comprise a reservoir fluid, where the system is configured to submerge the distal orifice of the needle in the reservoir fluid. The reservoir fluid may comprise mineral oil.

[0074] The PCR module may comprise any suitable module capable of amplifying a target nucleic acid. For example, the PCR module may be a temperature-controlled PCR module, a dPCR module, or a temperature-controlled dPCR module.

[0075] The PCR systems described herein generally further comprise a sample needle loading/sample loading module. The sample needle loading/sample loading module may include a plurality of unloading slots and/or a recycling bin. In addition to the aforementioned modules, the PCR systems generally also include a detection module for detecting a signal from a microdroplet. In some variations, the detection module is a fluorescence detection module,

[0076] The PCR system typically uses a robotic arm that has been configured to move between the various modules of the system, e.g., the microdroplet formation module, the PCR module, the sample needle loading/sample loading module, and the detection module.

Movement of the robotic arm is generally automated and controlled by user input at a control interface. It is understood that fewer modules, additional modules, and/or different modules can be employed in the PCR system or systems for different applications.

METHODS

[0077] Methods for microdroplet formation are described herein. The methods generally include a step of loading a sample fluid into a needle of a microdroplet generation assembly, where the needle has a proximal end and a distal orifice, and a lumen extending therebetween. The microdroplet generation assembly will generally further comprise a reservoir containing a reservoir fluid and an injection chamber in fluid communication with the needle lumen and containing a carrier fluid, where the injection chamber comprises an actuation mechanism. The actuation mechanism may comprise a piezoelectric ceramic material that deforms as described above. The methods may also include the steps of placing the needle orifice into the reservoir fluid, activating the actuation mechanism to deform a wall of the injection chamber, and forming a microdroplet of the sample fluid by ejecting a volume of the sample fluid from the needle into the reservoir fluid. The needle may be replaced, e.g., when a different sample fluid is to be loaded.

[0078] The microdroplet generation assemblies employed in the methods may be any one previously described herein. For example, referring to FIG. 8A, the microdroplet generation assembly may include a sample needle (96) comprising a proximal end (98), a distal end (100), and a lumen (102) extending therebetween. An orifice (104) is provided at distal end (100), which is in fluid communication with the lumen (102). The proximal end (98) may have a conical shape, which may be useful in minimizing the amount of residual sample left in the needle after the microdroplets have been ejected, as previously described. The cone shaped proximal end (98) generally holds a small amount of sample fluid (106) (sample), and is configured to be removably attached to an injection control chamber (108). The injection control chamber (108) comprises a piezoelectric ceramic chip (1 10) on one side of the outer surface, which can change the volume of the chamber (108) by vibration of the piezoelectric chip (1 10) in the direction of the arrows, and which is controlled by an electrical circuit. Microdroplets can also be formed by vibration of the needle.

[0079] More specifically, by changing the vibrational amplitude of the piezoelectric chip of the injection control chamber, the volume and number of generated droplets can be precisely controlled. Microdroplets of different sizes can be stored in different reservoirs. By comprehensively analyzing the number of positive PCR reactions for microdroplets of different sizes, a smaller total number of microdroplets can be used to achieve the same dynamic range and resolution as those required for analysis of microdroplets of uniform size. In a digital PCR with a single microdroplet volume, the upper limit of quantification is determined by the volume of each well in a microwell plate. The number of wells may also affect the upper limit of quantification. The lower detection limit may be determined by the total volume. Therefore, if a system needs to achieve a relatively large dynamic range, tens of thousands, even millions of wells have to be designed. Such a requirement poses a significant challenge to the design and throughput of the system, and increases the complexity and reagent consumption of the experiment. Digital PCR with multiple volumes can overcome the abovementioned issues.

[0080] For example, the design of wells with multiple volumes decouples the direct relationship between the total volume and the smallest volume of the wells. Wells with the smallest volume enable the detection of highly concentrated samples. On the other hand, wells with large volumes improve the precision of the measurement. Using the "Most Probably Number" theory for the analysis, multi-volume dPCR can achieve the same dynamic range and resolution using only a few hundred wells as those achievable with tens of thousands of wells in a single-volume dPCR. Accordingly, using the dPCR systems described herein, a strategy of dividing the same sample into microdroplets of at least four different volumes (such as 1 nL, 5 nL, 25 nL, and 125 nL) can be employed. Such a strategy can effectively overcome the common disadvantage of a small dynamic range, as experienced with currently available dPCR systems.

[0081] The method may include the step of ejecting a sample fluid in the form of

microdroplets, where the sample fluid is loaded into and contained within a sample needle. Alternatively, the method may include the steps of: 1) loading a sample fluid into the sample needle from a sample loading container, where the sample fluid taken up is stored in the cone shaped proximal end of the needle; and 2) ejecting the sample fluid out of the needle orifice in the form of microdroplets. In some variations, the method includes the steps of: 1) loading a sample fluid into the sample needle from a sample loading container, where the sample fluid taken up is stored in the cone shaped proximal end of the needle; 2) loading a reservoir fluid into the reservoir; and 3) ejecting the sample fluid out of the needle orifice in the form of microdroplets. In other variations, the method may include the steps of: 1) loading a sample fluid into the sample needle from a sample loading container, 2) loading a reservoir fluid into the reservoir; and 3) submerging a portion of the needle, e.g., the needle orifice in the reservoir fluid, and 4) ejecting the sample fluid out of the needle orifice in the form of microdroplets while the needle orifice is submerged in the reservoir fluid. In further variations, the method includes the steps of: 1) attaching the sample needle to the distal end of the injection control chamber via an output connector; 2) loading a carrier fluid

(immiscible with the sample fluid to be tested) into the injection control chamber via an input connector; 3) loading a sample fluid to be tested into the sample needle from a sample loading container (e.g., a centrifuge tube), where the sample fluid taken up is stored in the cone shaped proximal end of the needle; 4) loading a reservoir fluid into the reservoir (e.g., reservoir ( 1 12) in FIG. 8A); 5) submerging the orifice of the needle beneath the surface of the reservoir fluid inside the reservoir; 6) controlling the piezoelectric chip on the outside surface of the injection control chamber to allow the chip to deform and vibrate at high frequency in order to squeeze a carrier fluid inside the chamber and create a wave of carrier fluid; and 7) using the wave of carrier fluid to eject the sample fluid in the cone shaped proximal end and lumen of the needle out of the needle orifice in the form of microdroplets. In other variations, the method includes the steps of: Adjusting the surface tension of the sample fluid can help to generate microdroplets having a uniform size. Due to gravity, the generated microdroplets sink to the bottom of the reservoir and automatically lay flat as a droplet array.

[0082] Microdroplets produced by any one of the above processes can be used for dPCR. For example, uniform-sized droplets can be subjected to a temperature-controlled system, where arrays of droplets in the reservoir undergo nucleic acid amplifi cation (PGR reaction). Given that the number of microdroplets is typically larger than the number of nucleic acid molecules, the difference between droplets with amplification and without amplification can be taken advantage of by using fluorescence detection methods to determine the number of droplets having amplified a specific target sequence, thereby achieving quantitative analysis of the nucleic acid molecules. Such automatic, non-fluidic control of droplet generation, when integrated with dPCR technology, is beneficial to realizing quantitative examination of low-concentration nucleic acids in a micro well. Furthermore, the process is simple and highly efficient, and can be implemented at a low cost. Moreover, there is no need for any chip or like structures to rapidly and efficiently realize the dPCR process, avoiding complicated chip manufacture and design of integrated microfluidic routes,

[0083] In some variations, a method for performing digital PGR includes sample preparation, droplet generation, PCR reaction, fluorescence detection, and data analysis, as illustrated in FIG. 1. Digital PCR may be carried out by any one of the systems described herein. In one variation, dPCR can be carried out by a system comprising a microdropiet generation assembly and a PCR module as described herein, e.g., a temperature-controlled PCR module. In another variation, dPCR can be carried out by a system comprising a microdropiet generation assembly, a PCR module, and a sample needle loading/sample loading module. In yet further variations, dPCR can be carried out by a system comprising a microdropiet generation assembly, a PCR module, a sample needle loading/sample loading module, and a fluorescence detection module. The microdropiet generation assembly, PCR module, sample needle loading/sample loading module, and fluorescence detection module may be configured to operate as an automated dPCR system.

[0084] The carrier fluid and the reservoir fluid of the dPCR system may be the same or different. In some variations, the carrier fluid and the reservoir fluid may both comprise an oily fluid such as mineral oil. The sample fluid contained within the needle(s) of the system may then comprise an aqueous fluid. In some instances, the sample fluid is aqueous, and comprises a plurality of nucleic acid sequences. The nucleic acid sequences may comprise DNA or RNA. For example, the sample preparation method may include loading 24 prepared samples (DNA amplification reaction mix or RNA reverse transcription reaction mix) into the centrifuge tubes of the fi rst three rows in a sample loading needle/sample loading module, where each row has 8 centrifuge tubes; placing 24 sample loading needles into the last three rows of the module; placing a 96-well plate on the main carrier stage of the system (a standard 96-well plate has wells arranged in an 8 columns by 12 rows array); and placing the sample loading needle/sample loading module into the system. These steps can be automatically executed by the system. [0085] With respect to droplet generation, the method may include moving the robotic arm of the system to position the microdroplet generation assembly over the sample loading needle/sample loading module to obtain sample loading needles of the first row. A carrier fluid can then be added to each well (reservoir) of a row on a microwell plate. After loading the carrier fluid, the robotic arm returns to the top of the sample needle loading/sample loading module and loads a sample fluid from the first row of centrifuge tubes, and then returns to the top of the main carrier stage to add the sample fluid in the form of

rnicrodroplets (as previously described herein) to each well of the row on the microwell plate. The microwell plate may comprise any suitable number of wells and rows. When the microwell plate comprises a plurality of rows, each row of wells may contain rnicrodroplets of the same volume. In other instances, each row of wells may contain rnicrodroplets of different volume.

[0086] In some variations, the method may include moving the robotic arm of the system to position the microdroplet generation assembly over the sample loading needle/sample loading module to obtain sample loading needles of the first row. A carrier fluid can then be added into each well of a 96-well plate. After loading the carrier fluid, the robotic arm returns to the top of the sample needle loading/sample loading module and loads a sample fluid from the first row of centrifuge tubes, and then returns to the top of the main carrier stage to add the sample fluid in the form of rnicrodroplets (as previously described herein) for each of the first four rows in the 96-well plate. The volume of the rnicrodroplets added to the first row may be controlled to be about 1 nL, the volume of the rnicrodroplets added to the second row may be controlled to be about 5 nL, the volume of the rnicrodroplets added to the third row may be controlled to be about 25 nL, and the volume of the rnicrodroplets added to the fourth row may be controlled to be about 125 nL. The generated droplets generally lay flat on the bottom of the microweils. After loading the sample, the robotic arm returns to the top of the sample needle loading/sample loading module to discard the 8 sample loading needles using the unloading slot on the module. The process may then be repeated to add sample fluid to microweils in rows 5-12 of the 96-well plate,

[0087] After loading the rnicrodroplets of the sample fluid into the 96-well plate, the main carrier stage may transfer the plate to a temperature-controlled PCR reaction module to perform PCR reaction (PCR reaction step). In some variations, the PCR module is used to execute a digital PCR reaction on nucleic acid molecules contained in the rnicrodroplets. Parameters of the dPCR experiment, such as temperature and duration of each thermocycling step, and total number of cycles, may depend on the subtype of the dPCR assay and the nature of the sample (e.g., whether DNA or RNA molecules are measured, lengths of the molecules, and compl exity of the sampl e). Because traditional PGR consumables can be adapted for dPCR assays using the system described herein, protocols developed for traditional PGR techniques can be readily applied to such dPCR assays. The most commonly used PGR techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, fourth edition (Green and Sambrook, 2012); PGR: The Polymerase Chain Reaction, (Mullis et al ,, eds,, 1994), Real-time PGR for mRNA quantitation (Wong and Medrano, 2005).

[0088] For example, in a typical copy number variation assay that aims to determine the number of copies of a given target locus in a genome with respect to an invariant reference locus, a typical DNA-based PGR protocol can be executed on microdroplets generated from a sample fluid comprising a genomic DNA sample, appropriate primers for amplifying the target locus or the reference locus, ddNTPs, DNA polymerase, a fluorescent probe and buffers. In another variation, thermocycling steps appropriate for one-step or two-step RT PCRs can be carried out by the PG module for a typical gene expression assay that aims to determine the absolute copy number of an mRNA transcript. For example, in a one-step RT PGR protocol, the sample fluid may comprise a sample of total RNA from cells, reverse transcriptase, appropriate primers (e.g. oiigo(dT) and/or random primers, and sequence- specific primers for the target mRNA), DNA polymerase, nucleotide mix, a fluorescent probe, RNAse inhibitor, and buffer.

[0089] During an exemplary dPCR reaction, as illustrated in FIG. 8B, each microwell in a multiwell plate (e.g., a 96-weli plate) contains a number of microdroplets comprising the same sample fluid. The microdroplets may have a volume ranging from about 1 nl to about 150 ni. For example, the microdroplets may have a volume of about 1 nl, about 5 nl, about 25 nl, about 50 nl, about 75 nl, about 100 nl, about 125 nl, or about 150 nl . Each

microdroplet contains zero (empty circles in FIG. 8B left), one copy, or a small number of copies of a target nucleic acid molecule to be amplified (circles with a dot in FIG. 8B left). As the PGR module heats or cools each microwell during thermocycling steps, target nucleic acid molecules within a microdroplet are amplified and retained within the microdroplet. The PGR module ensures minimal disturbance to the microdroplets during the thermocycling steps so that the microdroplets do not rupture or coalesce. At the end of the dPCR procedure, microdroplets that originally contain target nucleic acid molecules accumulate a large number of copies of amplified product molecules (black circles in FIG. 8B right), but microdroplets that originally contain zero target nucleic acid molecules do not accumulate specific amplification product (empty circles in FIG. 8B right). Under certain conditions, a small number of non-specifically amplified nucleic acid molecules can be detected in the microdroplets, but such non-specific amplification can be avoided by optimization of the dPCR protocol or primers. Consequently, a binary YES/NO readout can be obtained for dPCR on a large number of microdroplets in each microwell of a 96-well plate.

[0090] Next, the main carrier stage may transfer the plate to the fluorescence microscopy module for observation (fluorescence detection step). As a final step, the observation results may be sent to the connected computer for analysis (data analysis step).