WO2018075577A1

WO2018075577A1 - Methods of integrated microfluidic processing for preventing sample loss

Info

Publication number: WO2018075577A1
Application number: PCT/US2017/057083
Authority: WO
Inventors: Xiaoliang Sunney Xie; Chi-Han Chang; Dong XING
Original assignee: President And Fellows Of Harvard College
Priority date: 2016-10-18
Filing date: 2017-10-18
Publication date: 2018-04-26

Abstract

The present disclosure provides methods of pre-loading processing and loading of an aqueous sample solution to a device such as a microfluidic device or chip in order to prevent sample loss due to dead volume retention of the aqueous sample solution or sample sticking to wall of test tubes or reaction containers, methods of integrated processing of an aqueous sample solution on an on-chip well and direct feeding of the processed in-well solution into microfluidic circuits for further processing without any transfer apparatus in order to prevent sample loss, methods of utilizing phase separation of different matters for reactions with any number of steps of reagent addition at any desired volume or mass ratios determined by the chosen reaction and transfer apparatus while preventing loss of rare or valuable samples, methods of fabricating a portable all-in-one device and using it for applications including isothermal reactions, and methods of using a portable and integrated device at a remote site for medical doctors, scientists, police officers, and the like to perform on-site studies.

Description

Methods of Integrated Microfluidic Processing for Preventing Sample Loss

RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Application No. 62/409,440 filed on October 18, 2016 which is hereby incorporated herein by reference in its entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under 5DP1CA186693 from the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

Microfluidic devices have been increasingly used in a wide range of applications. For example, microfluidic devices are used in the biomedical field with the laboratories on a chip because they allow the integration of many medical tests on a single chip. Microfluidic devices are used in cell biology research because micro-channels have the same characteristic size as biological cells or intra-cellular components. Thus, microfluidic chips allow easy manipulations of single cells or even single molecules such as chromosomal DNA. Microfluidic devices are also used in protein crystallization because they allow the generation on a single chip of a large number of crystallization conditions including temperature, pH, and humidity. Further, microfluidic devices are used in many other areas including drug screening, glucose tests, chemical microreactor, electrochemistry, and microprocessor cooling or micro fuel cells. All of these applications involve small amounts of samples. For example, single-cell whole genome amplification and sequencing is used with microfluidic devices in studies where cell-to-cell variation and population heterogeneity play a key role, such as tumor growth, stem ceil reprogramming, embryonic development, etc. Single cell

I whole genome amplification and sequencing is important when the cell samples subject to sequencing are valuable or rare or in small amounts. Important to accurate single-ceil genome sequencing is the initial amplification of the genomic DNA which can be in small amounts. For example, the amount of genomic DNA from a single human cell is around 6 pico-grams, and the reaction volume for amplifying such amount of DNA is typically in the range of micro-, nano-, or pico-iiter scales.

Although micro- to pico-liter-scaie reactions can be well performed in microfluidic devices, users need to transfer aqueous solution into microfluidic chips to initiate a reaction, and it is during this transferring process that dead volume at similar volume scales almost always occurs. (See FIG. 4A and 4B.) Moreover, DNA or RNA molecules can stick to surfaces of reaction tubes or containers or transfer apparatus, further leading to loss of genetic materials for analysis. This is less an issue when the amount of sample is large, such as when studying targeted genomic DNA regions from millions of cells together. However, for applications such as whole-genome amplification (WGA) for a single human cell, the starting sample amount is very small, and a loss of as little as 10% of the genomic DNA solution would result in a loss of more than 300 million base pairs of genomic sequence, severely limiting the coverage of genetic analyses.

Additional microfluidic applications including digital PGR, such as droplet digital PGR (ddPCR), also require dead volume prevention technologies or surface-binding avoidance methods for rare or valuable samples. In particular, for ddPCR applications, those of skill in the art typically need to select an instrument that retains the least amount of dead volume in order to obtain an accurate quantification of, for example, the number of nucleic acid molecules in the sample, this is especially important when users need an absolute quantification rather than a relative or qualitative measure. However, dead volume prevention in commercial ddPCR systems known to those of skill in the art, such as the RainDrop System (RainDance Technologies) and the QX200 systems (Bio-Rad), remains an open question. Even skilled users cannot avoid a dead volume of around 1% to 10%, or even more than 10% due to technical limitations. In the example of ddPCR, a 10% dead volume would contribute to an error rate of 10%, in addition to other potentially existing errors that should not be ignored for precise and accurate measurements.

The importance of preventing dead volume and surfacing binding cannot be overemphasized for sensitive analyses of rare or valuable samples, especially for microfluidic applications in which the amounts of samples or reagents need to be finely monitored. There is certainly a demand for methods of integrated microfluidic processing of small amounts of samples, such as genomic DNA from a single cell or a small group of ceils, in order to prevent sample loss due to sample transfer, handling or loading, which can lead to dead volume and/or surface binding. Such methods are critical for a wide range of applications such as sensitive genetic analyses for single cells.

SUMMARY

The present disclosure provides methods of pre-loading processing and loading of an aqueous sample solution into a device such as a microfluidic device or chip that prevents sample loss due to dead volume retention of the aqueous sample solution or sample sticking to the wails or surfaces of experimental apparatus, and methods of integrated processing of aqueous sample in an on-chip well and direct feeding, without transfer apparatus such as pipet, micropipette or the like, of the processed sample in well into microfluidic circuits beneath the well for further processing in order to prevent sample loss due to dead volume retention of the aqueous sample solution or sample sticking to the walls or surfaces of experimental reaction apparatus or transfer apparatus. According to one aspect, the present disclosure provides a method of loading an aqueous sample solution via a syringe that prevents sample loss due to dead volume retention of the aqueous sample solution in syringe needle and syringe tip including pre-filling the syringe connected to one end of a tubing via the syringe needle with a suitable hydrophobic material, wherein the hydrophobic material is drawn into the syringe from another end of the tubing by pulling back syringe plunger, and wherein the hydrophobic material at least fills in the needle and tip of the syringe where dead volume occurs, placing the other end of the tubing into the aqueous sample solution and continue to pull back the syringe plunger to draw an amount of aqueous sample solution into the tubing, and pushing forward the syringe plunger to load the aqueous sample solution into a device.

In certain embodiments, the present disclosure provides that the hydrophobic material comprises air, gas or hydrophobic liquid that does not mix with the aqueous solution or adversely affect the sample in the aqueous solution. In one embodiment, the present disclosure provides that when the hydrophobic liquid is used as the hydrophobic material, a small amount of air is drawn into the tubing before drawing the aqueous sample solution to separate the aqueous solution from the hydrophobic liquid for easy visualization. In one embodiment, the present disclosure provides that sample loss due to dead volume retention by the syringe needle or the syringe tip is prevented because the aqueous sample solution only fills in the tubing. In one embodiment, the present disclosure provides that the hydrophobic liquid comprises oil. In another embodiment, the present disclosure provides that the oil comprises fluonnated oil. In yet another embodiment, the present disclosure provides that the fluorinated oil comprises 3-ethoxyperfluoro(2-methylhexane). In a further embodiment, the present disclosure provides that the hydrophobic liquid further comprises a surfactant. In one embodiment, the present disclosure provides that the aqueous sample solution is loaded into a microfluidic device for further processing. In one embodiment, the present disclosure provides that a substantially entire amount of the sample is loaded into the device. In certain embodiments, the present disclosure provides that the sample can be biological or non-biological. In some embodiments, the present disclosure provides that the biological sample comprises tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, cells and fluids present within a subject. In other embodiments, the present disclosure provides that the biological sample comprises nucleic acids, genomic DNAs, proteins and the like. In certain embodiments, the present disclosure provides that the aqueous sample solution further comprises biological, chemical and/or buffer reagents. In one embodiment, the present disclosure provides that the genomic DNA is whole genomic DNA obtained from a single cell. In certain embodiments, the present disclosure provides that the genomic DNA is from a prenatal cell, a cancer cell, a circulating tumor cell, a single prenatal cell, a single cancer cell or a single circulating tumor cell.

According to one aspect, the present disclosure provides a method of pre-loading processing of an aqueous sample solution in a test tube or reaction container before transferring and loading the solution into a microfluidic chip, wherein the aqueous solution is surrounded by a hydrophobic liquid in the test tube or reaction container, thereby preventing contact between the sample and the wall of the test tube or reaction container and preventing sample loss due to molecular sticking to the wall of the test tube or reaction container. In certain embodiments, the volume ratio between the added aqueous reagent to the existing aqueous solution in the test tube or container can range from 10,000: 1, 1,000: 1, 100: 1 , 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1 ,000, 1 : 1.00, 1 : 10, or 1 : 1 or other ratios as long as the test tube, container or reaction or transfer apparatus can accommodate the volumes of the reagents.

In one embodiment, the present disclosure provides a method for pre-loading processing of samples to prevent sample loss using any outer phase and inner phase can be any phase or any combination of phases (e.g. liquid, solid, or gas) involving liquid mixture or powder or bubbles as long as the outer phase can surround but does not mix or react with the reaction phase so that molecular surface sticking or dead volume retention can be prevented.

According to another aspect, the present disclosure provides a method of processing an aqueous sample solution in an on-chip well that prevents sample loss due to sample sticking to wall of the well including adding a volume of hydrophobic liquid to the on-chip well, and adding the aqueous sample solution to the on-chip well, wherein the aqueous sample solution forms a droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from contacting or sticking to the wall of the well, wherein the bottom of the on-chip well has an opening that connects to a microfluidic circuit underneath, and wherein the hydrophobic liquid including the aqueous sample solution droplet is directly fed into the microfluidic circuit right beneath the well via an external force. In one embodiment, the present invention provides a method, a device, and a procedure wherein a microfluidic chip is integrated with a test tube or well or the like in which reactions from nano-iiter to micro-liter or milii-iiter scales can be performed and the solution can be fed directly into the microfluidic circuit beneath the tube without any transferring procedure using pipet or other transfer apparatus that may lead to sample loss as a result of molecular sticking on the transfer apparatus.

According to another aspect, the present disclosure provides a method of preprocessing of an aqueous sample solution before loading into a microfluidic chip that prevents sample loss due to sample sticking to wall of a test tube or a reaction container comprising adding a volume of hydrophobic liquid to the test tube or reaction container, and adding the aqueous sample solution into the test tube or reaction container already containing the hydrophobic liquid, wherein the aqueous sample solution forms a droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the test tube or reaction container. In certain embodiments, the present disclosure provides that multiple aqueous sample solutions can be added. In one embodiment, the present disclosure provides that all added aqueous sample solution will merge together within seconds of addition to form a single droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the test tube or reaction container. In another embodiment, the present disclosure provides that biochemical reactions can happen in the aqueous sample solution. In one embodiment, the present disclosure provides that the hydrophobic liquid further comprises a surfactant. In certain embodiments, the present disclosure provides that the sample can be biological or non-biological. In some embodiments, the present disclosure provides that the biological sample comprises tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, cells and fluids present within a subject. In other embodiments, the present disclosure provides that the biological sample comprises nucleic acids, genomic DNAs, proteins and the like. In one embodiment, the present disclosure provides that the aqueous sample solution further comprises biological, chemical and/or buffer reagents.

According to another aspect, the present disclosure provides an integrated method of processing an aqueous sample solution on an integrated microfluidic device that prevents sample loss due to solution transfer or sample molecule sticking to wall of any transfer apparatus or test tube or reaction container comprising: adding a volume of hydrophobic liquid to the on-chip well, and adding the aqueous sample solution to the on-chip well for reaction in the well, wherein the aqueous sample solution forms a droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the well, wherein the bottom of the on-chip well has an opening that connects to a microfluidic circuit underneath, and wherein the hydrophobic liquid including the aqueous sample solution droplet is directly fed into the microfluidic circuit right beneath the well via an external force for further microfluidic processing of the sample without any transferring processes by pipet or other transfer apparatus.

In certain embodiments, the present disclosure provides that multiple aqueous sample solutions can be added. In one embodiment, the present disclosure provides that all added aqueous sample solution will merge together within seconds of addition to form a single droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the test tube or reaction container. In another embodiment, the present disclosure provides that biochemical reactions can happen in the aqueous sample solution. In certain embodiments, the present disclosure provides that the external force comprises suction or pumping. In one embodiment, the present disclosure provides that the hydrophobic liquid further comprises a surfactant. In certain embodiments, the present disclosure provides that the sample can be biological or non-biological. In some embodiments, the present disclosure provides that the biological sample comprises tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, cells and fluids present within a subject. In other embodiments, the present disclosure provides that the biological sample comprises nucleic acids, genomic DNAs, proteins and the like. In one embodiment, the present disclosure provides that the aqueous sample solution further comprises biological, chemical and/or buffer reagents.

In certain embodiments, the present disclosure provides a method of preventing sample loss due to surface contact by making use of the phase separation phenomena between different phases of matter for processing rare or valuable samples. In one embodiment, the reactants can be added to the test tube or reaction container in as many steps as desired and, for each step, the volume or mass ratio between the added aqueous reactant to the existing reactant in the test tube or container can range from 10,000: 1, 1,000: 1, 100: 1, 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the test tube, container or reaction or transfer apparatus can accommodate the volumes of the reagents while preventing contact of the reactants with surface of the test tube or reaction container in order to prevent sample loss.

In certain embodiments, various phases can be used for phase separation for processing rare or valuable samples. In one embodiment an inner phase such as an aqueous sample solution can be separated from an outer phase such as a hydrophobic liquid according to the method described herein. In some embodiments, the present disclosure provides a method for preventing sample loss by selecting the outer phase and inner phase so that the reaction in the inner phase can be isolated and prevented from contacting the wall of the well. In some embodiments, the outer phase or inner phase can be any type of phase or any combination of phases (e.g. liquid, solid, or gas) involving liquid mixture or powder or bubbles as long as the outer phase can surround but does not mix or react with the inner reaction phase so that molecular surface sticking or dead volume retention can be prevented.

According to one aspect, the present disclosure provides a method for making a portable (or hand-held) and integrated all-in-one device that can be carried or shipped to a remote site for medical doctors, nurses, scientists, police officers, trained workers and the like to perform on-site medical diagnosis, forensic identification, archaeological or other scientific studies, or additional purposes involving processing rare or valuable samples. In one aspect, the device can be used for isothermal processing of nucleic acid samples using an integrated all-in-one device without using any thermocycling instrument while preventing loss of valuable or rare samples. Further features and advantages of certain embodiments of the present disclosure will become more fully apparent in the following description of the embodiments and drawings thereof and from the claims,

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 A is a schematic of an exemplar}' embodiment of microfluidic circuits for use in preparing sample droplets. The shape and structure of the aqueous solution inlet were empirically found to allow aqueous solution to flow into the narrow channel while preventing the liquid from being trapped in any part of the region. The squares and other shapes in the aqueous inlet region were found to effectively filter out unwanted particles at a minimal cost of surface area increase. Although adding more squares or other shapes might lead to better filtering, it will increase surface area and increase the chance of DNA sticking to the surface. (Note that the oil inlet shown in the next figure contains many squares, but that is fine because the DNA solution is not in contact with those surfaces, and so no issue of DNA sticking is present there.) For the design shown in this figure, adding an additional row of shapes has not provided more effective filtering but only increased the surface area; while decreasing one row of shapes has led to unwanted particles passing into downstream channels and clogging of the circuit. The current design reflects a balance between effective filtering and sample loss prevention. FIG. IB is a zoom-in view of the orange box in FIG. 1A. FIG. 1C is a zoom-in view of the blue box in FIG. B, and shows the droplet generation junction: When the aqueous solution ("Aq") flowing to the right encounters a "focusing flow" of oil that comes from both sizes of the horizontal channel, the Aq will spontaneously become surrounded by oil given that the surface of the channel is very hydrophobic and so contact between Aq and the channel surface is unfavorable. This is how and why droplets can form spontaneously when Aq and oil are pumped into the device based on the knowledge known to those of skill in the art. The droplets generated from a horizontal channel of dimensions 25 x 25 μηι (width x height) typically have a volume of approximately 7 pico-liters, although the size can be tuned to be smaller if the flow rate ratio of Aq to oil is smaller and vice versa.

FIG. 2 is a schematic showing a typical flow-focusing microfluidic circuits for making sample droplets. (Macosko et al., Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets, Cell, 161 (5) 1202-14, 2015) hereby incorporated by reference in its entirety. The principles of droplet generation are the same as those described for FIG. 1 C, except that here, two types of aqueous solution are mixed together before the aqueous mixture encounters a focusing flow of oil and form droplets. Two aqueous inlets are present here because the authors had to inject another aqueous solution containing other components for mixing, and the channel widths are larger here since the authors had to make larger droplets for their application. For the example given in this document, only the difference between aqueous inlet 1 in this figure and the aqueous inlet in FIG. 1A will be discussed, although one skilled in the art can readily appreciate designing more inlets or channels of different sizes for customized needs. Here, in the region of aqueous inlet 1 , liquid sometimes get trapped at the regions marked by the green circles, leading to dead volume that should be prevented when the input sample amount is small. Accordingly, the shape of the aqueous inlet illustrated in FIG. 1 A is designed to avoid liquid trapping in order to prevent loss of rare or valuable sample. FIGS. 3A-E show an exemplar)-' embodiment of a droplet microfluidic system for processing genomic DNA samples for amplification and sequencing. FIG. 3A shows that both hydrophobic liquid ("oil") and genomic DNA (gDNA) solution are pumped into the microfluidic chip that will make droplets of chosen sizes. FIG. 3B shows that a "dead volume-prevention circle" of tubing is used to prevent valuable gDNA solution from being trapped in the syringe needle or syringe tip as dead volume. FIG. 3C shows that this exemplary microfluidic circuit allows making millions of pico-liter droplets in minutes. FIG. 3D shows that droplet formation is monitored under the microscope. FIG. 3E shows that all droplets are collected from the microfluidic chip and therm oeycled. Finally, all droplets are merged together, and the aqueous gDNA solution is collected for sequencing or further analyses.

FIGS, 4A-4B show dead volume in a syringe needle that need to be prevented. FIG. 4A shows residual liquid (red) left in the needle that cannot be pumped into microfluidic devices; this is an example of where dead volume occurs. The dead volume in a syringe needle is typically more than 10 μΐ, which can lead to loss of more than 10% of the total sample reaction volume. FIG. 4B is a schematic drawing that puts the syringe needle and tubing of FIG. 4A in connection with the microfluidic chip in an experiment.

FIGS. 5A-5B show that by containing all the genetic materials in aqueous solution ("Aq") within the connection tube, filling the upstream volume (especially the syringe needle) with the 3-ethoxyperfluoro(2-methylhexane) ("HFE oil"), aqueous dead volume is avoided. FIG. 5A is a picture showing a syringe and needle filled with HFE oil and a connection tube containing upstream FIFE oil and downstream Aq to be injected into the microfluidic chip. FIG. 5B is a schematic drawing of FIG, 5A. FIG. 6 is a picture showing that a hydrophobic liquid can surround Aq and does not mix with it, preventing the genetic materials or other molecules in Aq from sticking to the wall of the tube. Adding both the Aq and 1 11 1 ^' oil in tube J to the FIFE oil and Aq in tube 2 and centnfugation will result in a single Aq droplet as depicted in tube 3 : The red and blue aqueous solutions initially separated by HFE oil will mix together spontaneously into a single (purple) droplet surrounded by HFE oil after centnfugation. The purple droplet in tube 3 is still isolated in HFE oil and not in contact with the wall of the tube.

FIGS. 7A-7C show that biochemical reactions of desired volumes and steps are performed in an aqueous compartment surrounded by a hydrophobic liquid within an on-chip well. FIG. 7A is a schematic drawing that shows that because the aqueous reaction mixture is surrounded by HFE oil, genetic materials will not be lost due to binding or sticking to the well's inner surface. Upon completion of reactions in the well, the solution is directly sucked or pumped into the microfluidic circuit for further processing, such as encapsulation into droplets for temperature cycling. This integrated all-in-one device and method prevent dead volume and surface sticking all together. FIG. 7B is a picture showing an integrated chip with a reaction on-chip well; microfluidic circuits are right beneath the wells. FIG. 7C is a picture showing that a chip and on-chip well can be in a "multi-well plate" format to scale up the throughput.

DETAILED DESCRIPTION

The present disclosure provides methods that prevent sample loss during transfer or handling of a sample solution or sample liquid. The sample solution according to the present disclosure is present in small amounts and sample loss will greatly affect the outcome of sample analyses. In some embodiments, an aqueous sample solution is transferred from a syringe to a device such as a microfluidic device or chip. The aqueous sample solution is trapped in the syringe needle or syringe tip as dead volume which leads to loss of valuable sample. In some embodiments, a hydrophobic phase such as a hydrophobic liquid or gas is used to pre-fil! the syringe that is connected to a connection tube so that the dead volume is occupied by the hydrophobic phase which would otherwise be occupied by the aqueous sample solution. The aqueous sample solution is later drawn into the connection tube and then pushed out to fill in the microfluidic device by the hydrophobic phase. Sample loss due to dead volume retention is thus prevented since the dead volume is filled by the hydrophobic phase. In other embodiments, an aqueous sample solution needs to react in a reaction chamber such as a well or tube before being loaded into a device such as a microfluidic device or chip. The sample in the aqueous solution can stick to the inner wall of the reaction tube or well and leads to sample loss. In some embodiments, an integrated setup of on-chip well is used to avoid sample loss. In some embodiments, an on-chip well is first loaded with a hydrophobic liquid. Aqueous samples and reagents are then added into the well in any- desired number of steps. All added aqueous samples and reagents will spontaneously merge together within seconds or upon centrifugation into a single droplet surrounded by the hydrophobic liquid; sample loss due to sticking to the wall of the tube or well is thus prevented. The bottom of the on-chip well has small opening or open port that directly connects to the microfluidic channel s/circuits, allowing direct loading of the hydrophobic liquid and the sample droplet into the microfluidic channel s/circuits without any intermediate processes involving pipets or other transfer apparatus. The disclosure provides a method of keeping the aqueous sample solution in the connection tube to avoid dead volume retention in syringe needle or syringe tip and the method can be used in combination with the on-chip well method as herein described for sample handling and transfer to minimize sample loss.

Microfluidic devices or chips are known to a skilled in the art. These devices/chips have been used in a wide range of applications in biology, chemistry and biomedical fields that deal with the flow of liquids or gases inside micrometer-size channels. According to some embodiments, the present disclosure contemplates a microfluidic device or chip that includes a set of micro-channels etched or molded into a material such as glass, silicon, plastics or polymer such as PDMS, for PolyDimethylSiloxane. The micro-channels (circuits) forming the microfluidic chip are connected together in order to achieve the desired features such as mixing, pumping, sorting, or controlling bio-chemical environment of samples. According to some embodiments, the network of micro-channels trapped into the microfluidic chip is connected to the outside by inputs (inlets) and outputs (outlets) affixed to or pierced through the chip, as an interface between the macro- and micro-world, it is through these holes of inputs (inlets) and outputs (outlets) that the liquids (such as aqueous solutions containing samples) are injected and removed from the microfluidic chip. According to some embodiments, liquids are injected and removed from the microfluidic device/chip via tubing, syringe adapters or even simple holes in the chip with external active systems including pressure controller, push-syringe, peri static pump or other pumping systems such as osmotic pumps, or passive ways (e.g. hydrostatic pressure).

Methods of fabricating a microfluidic chip are known in the art. According to some embodiments, the fabrication process of a microfluidic chip is based on photolithographic methods, derived from the well -developed semiconductor industry. Because of the development of specific processes such as deposition and electrodeposition, etching, bonding, injection molding, embossing and soft lithography (especially with PDMS), the disclosure also contemplates the use of diverse materials for microfluidics chips such as polymers (e.g. PDMS), plastics (e.g. molded plastics), ceramics (e.g. glass), semi-conductors (e.g. silicon), metal, cellulose (e.g. paper), biomaterials, or other materials from which flow circuit can be generated. Soft-lithography does not require hundreds of square meters of clean room space. A little bench space under a lab fume hood is sufficient to place essential rapid PDMS prototyping instruments to quickly assess microfluidic concepts and obtain publishable results.

The present disclosure provides a portable (or hand-held) and integrated all-in-one microfluidic device that can be carried or shipped to a remote site for medical doctors, nurses, scientists, police officers, trained workers and the like to perform on-site medical diagnosis, forensic identification, archaeological or other scientific studies, or additional purposes involving processing rare or valuable samples. In certain embodiments, the ail-in-one microfluidic device includes single or multiple wells or reaction tubes/containers glued or fixed to the microfluidic device where the bottom of the well or tube has an opening or open port that allows direct passage of the liquid from the well or tube to the circuits or channels of the microfluidic device or chip such as an open-port chip or through-hole chip for further processing. In some embodiments, on-chip well in single or multi-well format is used. The on-chip well can be glued or fixed to the surface of the chip or be integrated in the chip such that no leakage of solution is allowed. In some embodiments, an external force or pressure is applied to the all-in-one microfluidic device or chip that is fixed to the well or tube to draw or pump the liquid into the micro-channels or micro-circuits. In one aspect, the integrated all-in- one device can be used for isothermal processing of nucleic acid samples without using any thermocycling instalment while preventing loss of valuable or rare samples.

The present disclosure further provides a method of making/fabricating the portable (or hand-held) and integrated all-in-one microfluidic device wherein polydimethyisiloxane, or other non-solid materials that can solidify upon certain treatments such as baking, and the reaction containers such as test tubes are assembled into an all-in-one device for precise and accurate processing of rare or valuable samples. As used herein, samples can include any biological or non-biological material that are compatible with a microfluidic device for processing. In some embodiments, biological samples dissolved in an aqueous solution are used. In certain embodiments, biological samples of nucleic acids such as DNA, genomic DNA, RNA or protein are used. In some embodiments, reaction reagents, chemical reagents and buffer reagents are included or added in the aqueous sample solution for a specific application.

In some embodiments, a connection tube or tubing is used to transfer or load the aqueous sample solution into a microfluidic device. The tubing is connected at one end to an inlet of a microfluidic device and at the other end to a syringe needle or syringe tip. The sample solution is drawn into the tubing and syringe by pulling back a syringe plunger. The sample solution is loaded into the microfluidic device by pushing forward a syringe plunger. A skilled in the art can readily appreciate that equivalents of a push-syringe, such as a pressure controller, an injector or a pump or the like can also be used.

In some embodiments, hydrophobic material is used to fill in the dead volume to prevent sample loss. In some embodiments, hydrophobic material includes hydrophobic liquids, gases or air that does not mix with or adversely affect the samples or reactions in the aqueous solution. In some embodiments, hydrophobic liquid is used to surround the aqueous sample solution in reaction well or tube to prevent samples from sticking to the inner wall of the well or tube. In some embodiments, the well or tube is fixed to the microfluidic device where the bottom of the well or tube has an opening or open port that allows direct passage of the liquid from the well or tube to the circuits or channels of the microfluidic device or chip such as an open-port chip or through-hole chip for further processing. In some embodiments, on-chip well is used. The on-chip well can be in single or multi-well format. The on-chip well can be glued or fixed to the surface of the chip or be integrated in the glass such that no leakage of solution is allowed, in some embodiments, an external force or pressure is applied to the microfluidic device or chip that is fixed to the well or tube to draw or pump the liquid into the micro-channels or micro-circuits.

In some embodiments, after sample is loaded, the sample is encapsulated into a droplet with a mixture of reaction reagents using a flow-focusing microfluidic device such as the devices described in Macosko et al., Highly Parallel Genome-wide Expression Profiling of Individual Ceils Using Nanoliter Droplets, Cell, 161 (5): p. 1202-14, 20 5 and Klein et al., Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells, Cell, 2015, 161(5): p. 1 187-1201 each of which is hereby incorporated by reference in its entirety, such that each droplet contains a sample microparticle, a cell or a molecule. A typical flow circuit is illustrated in FIG. 2 which includes in fluid communication via microchannels two aqueous phase inlets and a hydrophobic liquid inlet (referred to as an oil inlet), a combination zone for combining the two aqueous solutions, and a combination zone for combining the aqueous phase with the oil phase which is in further fluid communication by a microchannel to an emulsion droplet outlet region. The aqueous mix is combined with the reagents and the combination is then formed into microdroplets with one sample microparticle, a cell or a molecule per microdroplet.

A suitable hydrophobic phase is one that allows aqueous droplets to be generated when an aqueous medium is introduced into the hydrophobic phase. Suitable oil phases are known to those of skill in the art into which an aqueous phase input spontaneously results in aqueous droplets or isolated volumes or compartments surrounded by the oil phase. An exemplary hydrophobic phase includes a hydrophobic liquid, such as an oil, such as a fluorinated oil, such as 3-ethoxyperfluoro(2-methylhexane), and a surfactant. An exemplary hydrophobic phase including a suitable oil and a surfactant is commercially available as QX200^1M Droplet Generation Oil for Evagreen (Bio-Rad), a hydrophobic surfactant- containing liquid that does not mix with aqueous solution or adversely affect biochemical reactions in aqueous solution. Surfactants are well known to those of skill in the art, and includes 008-FluoroSurfactant (RAN Biotechnologies), Pico-Surf^lM 1 (Dolomite Microfluidics), Proprietary Oil Surfactants (RainDance Technologies), fluorosurfactants discussed in Mazutis, L., et ai. Single-ceil analysis and sorting using droplet-based microfluidics, Nature Protocols, 2013, 8, p. 870-891, and other surfactants described in Baret, J.-C, Lab on a Chip, 2012, 12, p. 422-433 each of which is hereby incorporated by reference in its entirety.

When the oil phase and the aqueous phase are combined in the combination region or the emulsion droplet outlet region, the aqueous phase will spontaneously form droplets surrounded by the oil phase. According to one aspect, a flush volume of a hydrophobic fluid, such as an oil which may not contain a surfactant as none is needed for a flush volume, upstream of the aqueous phase either within the microfluidic design or within a syringe or injector used to input the aqueous sample phase into the microfluidic design is used to displace any aqueous phase that may otherwise occupy a dead volume to minimize loss of original aqueous phase introduced into the microfluidic chip design. Useful microfluidic chip designs can be created using AutoCAD software (Autodesk Inc.) and can be printed by CAD Art Services Inc. into a photomask for microfluidic fabrication. Molds or masters can be created using conventional techniques as described in (Mazutis et al., Single-cell analysis and sorting using droplet-based microfluidics, Nature Protocols, 8 (5) 870-891 , 2013) hereby incorporated by reference in its entirety. Microfluidic chips can be made from the master by curing uncured PDMS (Dow Corning Sylgard 184) poured onto the master and heated to curing to create a surface with trenches or circuits. Inlet and outlet holes are created and the cured surface with the circuits is placed against a glass slide and secured to create the microchannels and the microfluidic chip. Before use, the interior of the niicrofluidic chip can be treated with a compound for improving the hydrophobicity of the interior of the microfluidic chip and washed to remove potential contamination. On-chip wells or tubes as herein described can be fixed to the microfluidic chip to connect the inside of the well to the microchannels.

After microfluidic chip processing, the sample droplets can be processed with temperature cycles or even sorted as described in (Mazutis et al., Single-cell analysis and sorting using droplet-based microfluidics, Nature Protocols, 8 (5) 870-891, 2013), hereby incorporated by reference in its entirety, and finally lysed or demul sifted by adding perfluorooctanol (TCI Chemicals) to the droplets and after shaking by hand or vortexing and centrifugation, all aqueous solution initially separated in microdroplets will merge into one large droplet from which aqueous solution containing the sample can be collected.

In some embodiments, samples in the aqueous solution are treated or reacted with suitable reagents before, during or after being processed in a microfluidic device or chip according to a desired application using methods known to those of skill in the art. In an exemplary embodiment, single-cell DNA fragments for whole genome amplification and sequencing can be amplified within microdroplets using methods known to those of skill in the art. Reagents and hardware for conducting amplification reactions are commercially available. Microdroplets may be formed as an emulsion of an oil phase and an aqueous phase. An emulsion may include aqueous droplets or isolated aqueous volumes within a continuous oil phase. Emulsion whole genome amplification methods are described using small volume aqueous droplets in oil to isolate each fragment for uniform amplification of a single cell's genome as described in Fu, Y, et al., Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification, Proceedings of the National Academy of Sciences of the United States of America, 2015, 1 2(38): p. 1 1923-8; Sidore, A. M., et al., Enhanced sequencing coverage with digital droplet multiple displacement amplification, Nucleic Acids Research, 2015, Dec. 23; Nishikawa; Y, et al., Monodisperse picoliter droplets for low-bias and contamination -free reactions in single-cell whole genome amplification, PLoS One, 201 5, September 21 ; Rhee, M., et al., Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples, PLoS One, May 4, 2016, each of which is hereby incorporated by reference in its entirety. By distributing each fragment into its own droplet or isolated aqueous reaction volume, each droplet is allowed to reach saturation of DNA amplification. The amplicons within each droplet are then merged by demulsifi cation resulting in an even amplification of all of the fragments of the whole genome of the single cell ,

DNA to be amplified may be obtained from a single cell or a small population of cells. Methods described herein allow DNA to be amplified from any species or organism in a reaction mixture, such as a single reaction mixture carried out in a single reaction vessel. In one aspect, methods described herein include sequence independent amplification of DNA from any source including but not limited to human, animal, plant, yeast, viral, eukaryotic and prokaryotic DNA, and synthetic DNA.

In some embodiments, the methods for preventing sample loss as described herein are used with a method of single cell whole genome amplification and sequencing which includes fragmenting double stranded genomic DNA from a single cell and genomic DNA fragments are amplified. The amplicons are sequenced using, for example, high-throughput sequencing methods known to those of skill in the art.

In an exemplary embodiment, methods are provided for the amplification and sequencing of substantially the entire genome without loss of representation of specific sites (herein defined as "whole genome amplification"). In a specific embodiment, whole genome amplification comprises amplification of substantially all fragments or all fragments of a genomic library. In a further specific embodiment, "substantially entire" or "substantially all" refers to about 80%, about 85%, about 90%, about 95%>, about 97%, about 99%, or about 99.9%> of all sequences in a genome.

In some embodiments, the DNA sample is genomic DNA, micro dissected chromosome DNA, yeast artificial chromosome (YAC) DNA, plasmid DNA, cosmid DNA, phage DNA, PI derived artificial chromosome (PAC) DNA, or bacterial artificial chromosome (BAG) DNA, mitochondrial DNA, chloroplast DNA, forensic sample DNA, or other DNA from natural or artificial sources to be tested. In another preferred embodiment, the DNA sample is mammalian DNA, plant DNA, yeast DNA, viral DNA, or prokaryotic DNA. In yet another preferred embodiment, the DNA sample is obtained from a human, bovine, porcine, ovine, equine, rodent, avian, fish, shrimp, plant, yeast, virus, bacteria or ancient organisms. Preferably the DNA sample is genomic DNA.

According to certain aspects when amplifying small amounts of DNA such as DNA from a single cell, a DNA column purification step is not carried out so as to maximize the small amount (~6 pg) of genomic DNA that can be obtained from within a single ceil prior to amplification. The DNA can be amplified directly from a cell lysate or other impure condition. Accordingly, the DNA sample may be impure, unpurified, or not isolated. Accordingly, aspects of the present method allow one to maximize the amount of genomic DNA for amplification and reduce loss due to purification. According to an additional aspect, methods described herein may utilize amplification methods other than PCR.

In certain exemplary embodiments, cells are identified and then a single cell or a plurality of cells is isolated. Cells within the scope of the present disclosure include any type of cell where understanding the DNA content is considered by those of skill in the art to be useful. A ceil according to the present disclosure includes a cancer ceil of any type, hepatocyte, oocyte, embryo, stem cell, iPS cell, ES cell, neuron, erythrocyte, melanocyte, astrocyte, germ cell, oligodendrocyte, kidney cell and the like. According to one aspect, the methods of the present invention are practiced with the cellular DNA from a single ceil. A plurality of cells includes from about 2 to about 1,000,000 ceils, about 2 to about 10 cells, about 2 to about 100 cells, about 2 to about 1,000 cells, about 2 to about 10,000 cells, about 2 to about 100,000 cells, about 2 to about 10 cells or about 2 to about 5 ceils.

Nucleic acids processed by methods described herein may be DNA and they may be obtained from any useful source, such as, for example, a human sample. In specific embodiments, a double stranded DNA molecule is further defined as comprising a genome, such as, for example, one obtained from a sample from a human. The sample may be any sample from a human, such as blood, serum, plasma, cerebrospinal fluid, cheek scrapings, nipple aspirate, biopsy, semen (which may be referred to as ejaculate), urine, feces, hair follicle, saliva, sweat, immunoprecipitated or physically isolated chromatin, and so forth. In specific embodiments, the sample comprises a single cell. In specific embodiments, the sample includes only a single cell.

In particular embodiments, the amplified nucleic acid molecule or group of molecules from the sample provides diagnostic or prognostic information. For example, the prepared nucleic acid molecules from the sample may provide genomic copy number and/or sequence information, allelic variation information, cancer diagnosis, prenatal diagnosis, paternity information, disease diagnosis, detection, monitoring, and/or treatment information, sequence information, and so forth. As used herein, a "single cell" refers to one ceil. Single cells useful in the methods described herein can be obtained from a tissue of interest, or from a biopsy, blood sample, or cell culture. Additionally, cells from specific organs, tissues, tumors, neoplasms, or the like can be obtained and used in the methods described herein. Furthermore, in general, cells from any population can be used in the methods, such as a population of prokaryotic or eukaryotic single celled organisms including bacteria or yeast. A single cell suspension can be obtained using standard methods known in the art including, for example, enzymatically using trypsin or papain to digest proteins connecting cells in tissue samples or releasing adherent cells in culture, or mechanically separating cells in a sample. Single cells can be placed in any suitable reaction vessel in which single cells can be treated individually. For example a 96- well plate, such that each single cell is placed in a single well .

Methods for manipulating single cells are known in the art and include fluorescence activated cell sorting (FACS), flow cytometry (Herzenberg., PNAS USA 76: 1453-55 1979), micromanipulation and the use of semi-automated cell pickers (e.g. the Quixeli^1M ceil transfer system from Stoeiting Co.). Individual cells can, for example, be individually selected based on features detectable by microscopic observation, such as location, morphology, or reporter gene expression. Additionally, a combination of gradient centritugation and flow cytometry can also be used to increase isolation or sorting efficiency.

Once a desired cell has been identified, the cell is lysed to release cellular contents including DNA, using methods known to those of skill in the art. The cellular contents are contained within a vessel or a collection volume. Alternatively, the ceil lysate can be separated into two or more volumes such as into two or more containers, tubes or regions using methods known to those of skill in the art with a portion of the cell lysate contained in each volume container, tube or region. Genomic DNA contained in each container, tube or region may then be amplified by methods described herein or methods known to those of skill in the art.

The amplified DNA can be sequenced by any suitable method. In particular, the amplified DNA can be sequenced using a high-throughput screening method, such as Applied Biosy stems' SOLiD sequencing technology, Thermo Fisher's Ion sequencing platforms, or Illumina's Genome Analyzer. In one aspect of the invention, the amplified DNA can be shotgun sequenced. The number of reads can be at least 10,000, at least 1 million, at least 10 million, at least 100 million, or at least 1000 million. In another aspect, the number of reads can be from 10,000 to 100,000, or alternatively from 100,000 to 1 million, or alternatively from 1 million to 10 million, or alternatively from 10 million to 100 million, or alternatively from 100 million to 1000 million. A "read" is a length of continuous nucleic acid sequence obtained by a sequencing reaction.

The amplification and sequencing methods are useful in the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the genomic DNA in order to determine whether an individual is at risk of developing a disorder and/or disease. Such assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the onset of the disorder and/or disease. Accordingly, in certain exemplary embodiments, methods of diagnosing and/or prognosing one or more diseases and/or disorders using one or more of expression profiling methods described herein are provided. As used herein, the term "biological sample" is intended to include, but is not limited to, tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, cells and fluids present within a subject.

In certain exemplary embodiments, electronic apparatus readable media comprising one or more genomic DNA sequences described herein is provided. As used herein, "electronic apparatus readable media" refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon one or more expression profiles described herein.

As used herein, the term "electronic apparatus" is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatuses suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like, and local and distributed processing systems.

As used herein, "recorded" refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising one or more expression profiles described herein. A variety of software programs and formats can be used to store the genomic DNA information of the present invention on the electronic apparatus readable medium. For example, the nucleic acid sequence can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of data processor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon one or more expression profiles described herein.

It is to be understood that the embodiments of the present invention which have been described are merely illustrative of some of the applications of the principles of the present invention. Numerous modifications may be made by those skilled in the art based upon the teachings presented herein without departing from the true spirit and scope of the invention. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of the present invention. These examples are not to be construed as limiting the scope of the invention as these and other equivalent embodiments will be apparent in view of the present disclosure, figures and accompanying claims.

EXAMPLES Example I

Microfluidic Design and Processing 1. Flow circuits design: The flow circuit for genomic DNA amplification was drawn on a computer using the AutoCAD software (Autodesk Inc.), and was printed by CAD Art Services Inc. into a photomask for microfluidic fabrication. The flow circuit for genomic DNA amplification (FIG. 1 A) was modified from a conventional flow-focusing droplet generation design (FIG. 2) (Macosko et al., Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoiiter Droplets, Cell 161 (5) 1202-14, 2015) hereby incorporated by reference in its entirety. The surface area along the aqueous flow path was minimized to prevent potential sticking of DNA fragments on the surfaces.

2, Shape of the aqueous inlet region:

The flow circuit design uses a "microphone-trumpet" shape at the aqueous inlet region, so that the DNA solution injected at the left end of the shape can be focused towards the narrow channel on the right. (FIG. 1A) This design prevents liquid from being trapped in the inlet region as in certain droplet generation circuits in which some liquid needs to undergo sharp turns before reaching the narrow parts downstream. (FIG. 2)

3. Filter in the aqueous path:

A filter unit composed of closely spaced squares or other shapes is needed in the DNA solution (aqueous) inlet as well as the oil inlet to prevent dusts or other objects from flowing downstream into the circuit. Objects flowing into the circuits happen from time to time (as dusts are floating around in air), and can lead to very different droplet sizes that impact amplification evenness (since a bigger droplet may contain multiple DNA molecules). Filtering shapes are needed to ensure stable droplet formation.

However, there cannot be too many squares or shapes in the aqueous inlet circuit; otherwise there would be too much surface area and too much probability for DNA molecules to stick to the surfaces. One row of shapes, such as squares, was found to be insufficient for blocking unwanted objects. Two to three rows of shapes were found to effectively serve the filtering purpose with minimal surface area in the circuit, and are thus used in the design for the flow circuit, (Note that the filter unit in the oil inlet contains many more shapes, this is fine because DNA solution is not in contact with those surfaces, and so no issue of DNA sticking is present there.)

4. Microfluidic fabrication:

Microfluidic chips were made from molds (masters) fabricated using conventional procedures described in reference (Mazutis et al., Single-cell analysis and sorting using droplet-based microfluidics, Nature Protocols, 8 (5) 870-891, 2013) hereby incorporated by reference in its entirety. Briefly, SU8 3025 (MicroChem) was spun at 3000rpm on a silicon wafer that was pre-baked at 200°C for 5 min. and pre-cieaned with Oxygen plasma at 35W with lOsccm for 90s right before use. After the wafer with SU8 was baked at 95 °C for 1 1 min., it was exposed to 180 mJ/cm² of UV light under the photomask, and was baked at 65 °C for 1 min. and then 95°C for 3 min. The wafer with SU8 was then soaked in propylene glycol monomethyl ether acetate ("PGMEA") (MicroChem) for 6 min. (Under a photomask, some SU8 is exposed to UV and some is not. Exposed SU8 will polymerize due to the UV light, and unexposed SU8 is subsequently washed away by PGMEA, leaving a solid pattern that is defined by the photomask, which is initiall designed on a computer.) This completes the fabrication of a master.

To make microfluidic chips from a master, uncured polydimethylsiloxane (PDMS) (Dow Corning Sylgard 184) was poured onto the master and baked at 65 °C overnight for curing. The cured (solidified) PDMS was cut from the wafer, and holes were punched on the PDMS using a lmm biopsy puncher (Miltex). (There are 3 holes for each device since a device contains 2 inlets and 1 outlet.) The PDMS surface with circuits and a glass slide were treated with Oxygen plasma at 25 W with lOsccm for 10s, and two treated surfaces were placed against each other for bonding. Once the PDMS bonds to the glass after the surface activation by plasma, the fabrication of a microfluidic chip is completed,

5. Surface treatment and preparation for genomic DNA amplification and sequencing:

Each microfluidic chip was treated with Aquapel (Aquapel) to make the channel surfaces hydrophobic, as described in reference (Macosko et al., Highly Parallel Genome- wide Expression Profiling of Individual Cells Using Nanoliter Droplets, Cell, 161 (5) 1202- 14, 2015) hereby incorporated by reference in its entirety. Before starting each genomic DNA amplification experiment, the device was washed with nuclease-free water to remove potential contamination, and then washed with droplet generation oil such as the Bio-Rad Droplet Generation Oil for Evagreen. The droplet generation oil is in a syringe connected to the oil inlet of the chip via polyethylene tubing (Scientific Commodities # BB31695-PE/2), as depicted in FIG. 3 A. The outlet of the chip is connected to a 1.5ml DNA LoBind tube via polyethylene tubing for droplet collection. (FIG. 3A)

6. Dead-volume prevention:

To load genomic DNA (gDNA) solution into the chip without dead volume, a 1ml syringe connected to a 140cm-long polyethylene tubing via syringe needle was pre-filled with 3-ethoxyperiluoro(2 -methyl hexane) ("FIFE oil"). The gDNA solution containing fragmented DNA was then sucked into the tubing without touching the syringe needle or syringe, where dead volume occurs. (FIG. 3B) To distinguish HFE oil from gDNA solution inside the polyethylene tube (which are both transparent), a small amount of air was sucked into the polyethylene tube before sucking in the gDNA solution to separate both types of liquids. Note that the air-liquid interfaces can be easily visualized, but the HFE oil-gDNA solution interface can hardly be seen; therefore the small segment of air separating HFE oil and gDNA solution helps visualize where the gDNA solution resides in the polyethylene tubing so that a user can prevent the gDNA solution from reaching the syringe needle or the syringe. This method ensures that all gDNA solution be pumped into the chip without remaining in the syringe needle or the syringe; the solution is pushed fully into the chip by the HFE oil that does not mix with it.

7. Droplet generation, thermocycling, and product collection:

By pumping the gDNA solution and the droplet generation oil into the chip using the configuration shown in Fig. 3 A-C, droplets will form spontaneously in the flow circuit as shown in FIG. 3D. The droplets were then aliquoted into PCR tubes for thermocycling for amplification. Afterwards, 50μ,1 of periluorooctanol (TCI Chemicals) was added to each PCR tube; after shaking by hand and centrifugation, all droplets would break, and the aqueous solution containing amplified DNA is finally pooled together for downstream analyses such as sequencing using sequencing workflow and platform known to those of skill in the art, such as the sequencing workflow and platform of Illumina HiSeq 2500. (FIG. 3E)

Example II

This example describes embodiments according to the disclosure that uses three approaches that all make use of phase separation between hydrophobic and aqueous liquids to prevent loss of samples such as genetic materials.

Approach 1 :

Preventing genetic materials from sticking to container surfaces by phase separation As an exemplar' embodiment, 3-ethoxyperfluoro(2-methylhexane) (HFE oil), a hydrophobic liquid that does not mix with aqueous solution ("Aq") nor affect biochemical reactions in Aq was used. When HFE oil and Aq are added into the same container, they will separate spontaneously within seconds, and sequentially added aqueous reagents will all merge together into one single contained aqueous droplet surrounded by HFE oil. (See FIG. 6) The HFE oil can surround aqueous solution and does not mix with it, preventing genetic material s in the aqueous solution from sticking to the inner wall of the container such as a tube or a well.

After a tube or a well is loaded with HFE oil, aqueous solution containing genetic materials and reagents can be added in as many steps as possible. All added aqueous solution will merge together to an aqueous droplet within seconds of reagent addition or, in certain cases, centrifugation after reagent addition, and biochemical reactions can happen within the droplet after merging, with the aqueous solution still separated from the wall of the container. The aqueous solution within the droplet can then be transferred into a microfluidic chip or for further processing.

This way, the aqueous solution does not have any contact with the wall of the container or test tube, and aqueous reagents can be added in as many steps as desired in such a way that, in each step of reagent addition, the volume ratio between the added aqueous reagent to the existing aqueous solution in the test tube or container can range from 10,000: 1, 1,000: 1, 100: 1, 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the test tube, container or reaction or transfer apparatus can accommodate the volumes of the reagents.

In certain embodiments, the reaction compartment can be a hydrophobic liquid or a mixture of hydrophobic liquid in which reactions can occur, surrounded by aqueous liquid, by another type of non-reacting and non-mixing hydrophobic liquid, or but other phases of liquid or materials that do not mix or react with the reacting liquid or materials, hi another embodiment, the reaction compartment can contain a combination of liquid and solid, liquid and gas, gas and solid or any combination of different phases of matter, wherein solid can be in the form of powder and gas can be in the form of bubbles, all surrounded by yet another phase of liquid or materials that do not mix or react with any matter in the reaction compartment. In a more general embodiment, the reaction compartment can contain any phase or any combination of phases of matter and the surrounding can contain any phase or any combination of phases of matter, as long as the reacting matter can be isolated by the surrounding matter without mixing or reacting with it, so that the reaction matter does not have any contact with the wall of the test tube or reaction container, thereby prevent sample loss due to sample sticking to the wall of the test tube or reaction container. Examples of non- mixing and non-reacting phases of liquid and materials are described in reference (Zarzar et a!., Dynamically Reconfigurable Complex Emulsions via Tunable Interfacial Tensions, Nature, 158, 520-524, 2015) hereby incorporated by reference in its entirety. In certain aspects, the volume or weight ratio between the added reacting matter and existing matter in the test tube or reaction container can range from 10,000: 1, 1,000: 1, 100: 1, 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the test tube or reaction container or transfer apparatus can accommodate the volumes of the reagents while preventing sample sticking to the wall of the test tube or reaction container and thus preventing sample loss.

Approach 2:

Preventing sample loss due to aqueous dead volume using a separated phase After an aqueous sample containing genetic material is prepared in a well or tube, it is to be transferred via a syringe into a microfluidic device for further processing such as for single cell genomic DNA amplification and sequencing. The FIFE oil is used to pre-fill the dead volume of the syringe needle and syringe tip that would otherwise be filled with Aq that contains genetic materials. After drawing the aqueous sample into the connection tube, HFE oil can fully displace Aq into microfluidic chips. (See FIGS. 5A-B.) By containing all the genetic materials in aqueous solution within the connection tube, filling the upstream volume (especially the syringe needle) with HFE oil, sample loss due to aqueous dead volume retention is avoided. The picture and schematic diagram in FIGS. 5A and 5B show a syringe and needle filled with FIFE oil and a connection tube containing upstream FIFE, oil and downstream Aq to be injected into the microfluidic chip.

Approach 3 :

Minimizing sample loss due to dead volume retention and surface binding using integrated microfluidic and phase separation methods

To further prevent sample reagent loss during sample transfer and handling for more sensitive applications, an integrated setup was designed that eliminated the requirement of using syringe to transfer the sample from the container to the microfluidic chip. (So the transfer step between Approach 1 and Approach 2 described above can be eliminated if using Approach 3 as described herein.) In this setup, all biochemical reactions of samples in aqueous solution are performed directly in an on-chip well. Then the solution in the on-chip well is directly fed into microfluidic circuits right beneath the well for droplet generation via an opening at the bottom of the well. (See FIGS. 7A-C.)

According to this approach, an on-chip well is first loaded with HFE oil . Aqueous samples and reagents (Aq) (e.g. cell lysis buffer, polymerase, dNTPs, etc.) are then added into the well in any desired number of steps with desired volumes. All added aqueous samples and reagents will spontaneously merge together within seconds into a single droplet without introducing any electric field or additional chemicals because no surfactant is added to the HFE oil to prevent merging. Since all added Aq merges into one droplet and since the droplet floats in HFE oil (which has larger density than Aq), genetic materials in Aq does not stick to the inner surface of the well.

After the reactions in well are completed, the HFE oil and Aq is directly sucked or pumped into the microfluidic circuit via an opening at the bottom of the on-chip well for on- chip processing, such as encapsulation into droplets for temperature cycling. This alleviates the need for syringe or syringe needles that can introduce dead volume or additional surfaces for DNA or RNA molecules to stick to. Biochemical reactions of desired volumes and steps are performed in a well. Because the aqueous reaction mixture is surrounded by FIFE oil, genetic materials will not be lost due to sticking to the well surface. This integrated all-in-one device method prevents sample loss due to dead volume and surface sticking together.

FIG. 7B shows a realization of an integrated microfluidic chip with an on-chip reaction well; microfluidic circuits are right beneath the well. FIG. 7C shows a realization that a microfluidic chip can also be in a "multi-well plate" format, with microfluidic circuits right beneath the wells. The opening at the bottom of the well can be connected to the micro- ci cuits/channels via an inlet or the like so that the aqueous solution can be directed passed into the micro-circuits/channels without any pipet or transfer apparatus.

This way, the aqueous solution in the on-chip well does not have any contact with the wall of the well, and aqueous reagents can be added in as many steps as desired in such a way that, in each step of reagent addition, the volume ratio between the added aqueous reagent to the existing aqueous solution in well can range from 10,000: 1, 1,000: 1, 100: 1 , 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the well or transfer apparatus can accommodate the volumes of the reagents; and all the aqueous solution can be directly fed into the microfluidic circuit beneath the well without any transferring process.

In certain embodiments, the reaction compartment can be a hydrophobic liquid or a mixture of hydrophobic liquid in which reactions can occur, surrounded by aqueous liquid, by another type of non-reacting and non-mixing hydrophobic liquid, or but other phases of liquid or materials that do not mix or react with the reacting liquid or materials. In another embodiment, the reaction compartment can contain a combination of liquid and solid, liquid and gas, gas and solid or any combination of different phases of matter, wherein solid can be in the form of powder and gas can be in the form of bubbles, all surrounded by yet another phase of liquid or materials that do not mix or react with any matter in the reaction compartment. In a more general embodiment, the reaction compartment can contain any phase or any combination of phases of matter and the surrounding can contain any phase or any combination of phases of matter, as long as the reacting matter can be isolated by the surrounding matter without mixing or reacting with it, so that the reaction matter does not have any contact with the well, thereby prevent sample loss due to sample sticking to the wall of the well. Examples of non-mixing and non-reacting phases of liquid and materials are described in reference (Zarzar et al., Dynamically Reconfigurable Complex Emulsions via Tunable Interfacial Tensions, Nature, 158, 520-524, 2015) hereby incorporated by reference in its entirety. In certain aspects, the volume or weight ratio between the added reacting matter and existing matter in the well can range from 10,000: 1, 1 ,000: 1, 100: 1 , 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the well or transfer apparatus can accommodate the volumes of the reagents while preventing sample sticking to the wall of the well and thus preventing sample loss. According to one aspect, this integrated device, or "ail-in-one" device, can he used to process a single cell or other samples by extracting the nucleic acid (e.g. DNA or RNA), compartmentalize the nucleic acid molecules into compartments (e.g. droplets), and perform isothermal amplification without any thermocycler or PGR instrument. Any medical doctor, nurse, scientist, police officer, trained workers and the like can bring a hand-held all-in-one device together with portable syringe pumps into a remote area for on-site medical diagnosis, for forensic identification, for archaeological or other scientific studies, or for additional purposes involving processing rare or valuable samples.

In one aspect, the integrated all-in-one device can be fabricated using PDMS, or other fluidic material that can solidify due to certain treatments such as baking, and reaction containers such as test tubes (e.g. Axygen® PGR tube). In a non-limiting example, the device can be made by first curing a thin layer of PDMS covering the mierofluidic master so that the layer contains circuit feature, pouring uncured PDMS on the first layer, immersing in the uncured PDMS a test tube whose bottom has an opening (made by drilling or cutting), securing the tube to a suitable location (such as right above the aqueous inlet in the circuit of the first layer) using metal wire or another piece of pre-cut and pre-cured PDMS that can prevent the tube from moving around, curing the second layer of PDMS by baking at 65 °C for at least 2 hours, punching holes at the inlets and outlets, and finally attach the PDMS with integrated test tube (or "well") to glass slide to complete the fabrication of an integrated all- in-one device. FIGS. 7B and 7C show some realization of this example.

Results:

Making use of these techniques, the whole human genome from a single cell has been repeatedly amplified with high genomic coverage and low amplification bias using ~30X sequencing depth, as shown in table 1 below: Table 1.

As shown in the table, at least 18% (100%-82%) of the genomic regions is lost and cannot be analyzed if using the currently available commercial kits for single-cell whole- genome amplification. Using the method described herein, on the other hand, only around 2% of the genomic regions is lost. In addition, the yield of genome amplification is typically 1-2 μg, marking a dramatic amplification efficiency from only around 6pg of single-cell DNA. Sample loss has been prevented using the integrated microfluidic processing described herein.

Example 111 Kits

The materials and reagents required for the disclosed integrated method may be assembled together in a kit. The kits of the present disclosure generally will include at least the samples such as cells, nucleotides, and reaction reagents and buffers such as DNA polymerase, dNTPs, etc. necessary to carry out the claimed method along with primer sets as needed. In a preferred embodiment, the kit will also contain directions for amplifying DNA from DNA samples. Exemplary kits are those suitable for use in amplifying whole genomic DNA. Ei each case, the kits will preferably have distinct containers for each individual reagent, enzyme or reactant. Each agent will generally be suitably aliquoted in their respective containers. The container means of the kits will generally include at least one vial or test tube. Flasks, bottles, and other container means into which the reagents are placed and aliquoted are also possible. The individual containers of the kit will preferably be maintained in close confinement for commercial sale. Suitable larger containers may include injection or blow-molded plastic containers into which the desired vials are retained. Instructions are preferably provided with the kit.

Claims

What is claimed is:

1. A method of loading an aqueous sample solution via a syringe that prevents sample loss due to dead volume retention of the aqueous sample solution in syringe needle and syringe tip comprising: pre-filling the syringe which is connected to one end of a tubing via the syringe needle with a suitable hydrophobic material, wherein the hydrophobic material is drawn into the syringe from another end of the tubing by pulling back syringe plunger, and wherein the hydrophobic material at least fills in the needle and tip of the syringe where dead volume occurs, placing the other end of the tubing into the aqueous sample solution and continue to pull back the syringe plunger to draw an amount of aqueous sample solution into the tubing, and pushing forward the syringe plunger to load the aqueous sample solution into a device.

2. The method of claim 1, wherein the hydrophobic material comprises air, gas or hydrophobic liquid that does not mix with the aqueous solution or adversely affect the sample in the aqueous solution.

3. The method of claim 1, wherein when hydrophobic liquid is used as the hydrophobic material, a small amount of air is drawn into the tubing before drawing the aqueous sample solution to separate the aqueous solution from the hydrophobic liquid for easy visualization.

4. The method of claim 1, wherein sample loss due to dead volume retention by the syringe needle or the syringe tip is prevented because the aqueous sample solution only fills in the tubing.

5. The method of claim 2, wherein the hydrophobic liquid comprises oil.

6. The method of claim 5, wherein the oil comprises fluorinated oil.

7. The method of claim 6, wherein the fluorinated oil comprises 3-ethoxyperfluoro(2- methylhexane).

8. The method of claim 2, wherein the hydrophobic liquid further comprises a surfactant.

9. The method of claim 1, wherein the aqueous sample solution is loaded into a microfluidic device for further processing.

10. The method of claim 1 , wherein a substantially entire amount of the sample is loaded into the device.

11. The method of claim 1 , wherein the sample can be biological or non-biological.

12. The method of claim 11, wherein the biological sample comprises tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, ceils and fluids present within a subject.

13. The method of claim 11, wherein the biological sample comprises nucleic acids, genomic DNAs, proteins and the like.

14. The method of claim 1, wherein the aqueous sample solution further comprises biological, chemical and/or buffer reagents.

15. The method of claim 13, wherein the genomic DNA is whole genomic DNA obtained from a single ceil.

16. The method of claim 13, wherein the genomic DNA is from a prenatal cell.

17. The method of claim 13, wherein the genomic DNA is from a cancer cell.

18. The method of claim 13, wherein the genomic DNA is from a circulating tumor cell.

19. The method of claim 13, wherein the genomic DNA is from a single prenatal cell.

20. The method of claim 13, wherein the genomic DNA is from a single cancer ceil.

21. The method of claim 13, wherein the genomic DNA is from a single circulating tumor ceil.

22. A method of pre-processing of an aqueous sample solution before loading into a microfluidic chip that prevents sample loss due to sample sticking to wall of a test tube or a reaction container comprising: adding a volume of hydrophobic liquid to the test tube or reaction container, and adding the aqueous sample solution into the test tube or reaction container already containing the hydrophobic liquid, wherein the aqueous sample solution forms a droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the test tube or reaction container.

23. The method of claim 22, wherein multiple aqueous sample solutions can be added in as many steps as permitted by the volumes of the reaction or transfer apparatus.

24. The method of claim 23, wherein all added aqueous sample solution will merge together within seconds of addition to form a single droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the test tube or reaction container.

25. The method of claim 22, wherein biochemical reactions can happen in the aqueous sample solution.

26. The method of claim 22, wherein the hydrophobic liquid further comprises a surfactant.

27. The method of claim 22, wherein the sample can be biological or non -biological.

28. The method of claim 27, wherein the biological sample comprises tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, ceils and fluids present within a subject.

29. The method of claim 27, wherein the biological sample comprises nucleic acids, genomic DNAs, proteins and the like.

30. The method of claim 22, wherein the aqueous sample solution further comprises bioiogical, chemical and/or buffer reagents.

31 . The method of claim 22, wherein for each step of reagent addition the volume ratio between the added aqueous reagent to the existing aqueous solution in the test tube or container can range from 10,000: 1, 1,000: 1, 100: 1, 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the test tube, container or reaction or transfer apparatus can accommodate the volumes of the reagents.

32. The method of claim 22, wherein the aqueous sample solution is the inner phase, wherein the hydrophobic liquid is the outer phase, wherein the inner or outer phase can be any type of phase or any combination of phases (e.g. liquid, solid, or gas) involving liquid mixture or powder or bubbles as long as the outer phase can surround but does not mix or react with the inner reaction phase so that the reaction can be isolated to prevent sample loss due to molecular surface sticking or dead volume retention.

33. An integrated method of processing an aqueous sample solution on an integrated microfluidic device that prevents sample loss due to solution transfer or sample molecule sticking to wall of any transfer apparatus or test tube or reaction container comprising; adding a volume of hydrophobic liquid to the on-chip well, and adding the aqueous sample solution to the on-chip well for reaction in the well, wherein the aqueous sample solution forms a droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wall of the well, wherein the bottom of the on-chip well has an opening that connects to a microfluidic circuit underneath, and wherein the hydrophobic liquid including the aqueous sample solution droplet is directly fed into the microfluidic circuit right beneath the well via an external force for further microfluidic processing of the sample without any transferring processes by pipet or other transfer apparatus,

34. The method of claim 33, wherein multiple aqueous sample solutions can be added.

35. The method of claim 33, wherein all added aqueous sample solution will merge together within seconds of addition to form a single droplet that is surrounded by the hydrophobic liquid thereby preventing the sample in the aqueous solution from sticking to the wal 1 of the well .

36. The method of claim 33, wherein biochemical reactions can happen in the aqueous sample solution.

37. The method of claim 33, wherein the external force comprises suction or pumping.

38. The method of claim 33, wherein the hydrophobic liquid further comprises a surfactant.

39. The method of claim 33, wherein the sample can be biological or non-biological.

40. The method of claim 39, wherein the biological sample comprises tissues, cells, biological fluids and isolates thereof, cultured or isolated from a subject, as well as tissues, ceils and fluids present within a subject.

41. The method of claim 39, wherein the biological sample comprises nucleic acids, genomic DNAs, proteins and the like.

42. The method of claim 33, wherein the aqueous sample solution further comprises biological, chemical and/or buffer reagents.

43. The method of claim 33, wherein for each step of reagent addition the volume ratio between the added aqueous reagent to the existing aqueous solution in the well can range from 10,000: 1, 1,000: 1, 100: 1, 10: 1, or 1 : 1 to 1 : 10,000, 1 : 1,000, 1 : 100, 1 : 10, or 1 : 1 or other ratios as long as the well or transfer apparatus can accommodate the volumes of the reagents.

44. The method of claim 33, wherein the integrated microfluidic device is a portable integrated ("all-in-one") device that can be used for isothermal processing of nucleic acid samples.

45. The method of claim 44, wherein the portable (or hand-held) and integrated ail-in-one device can be taken to a remote site by medical doctors, nurses, scientists, police officers, trained workers and the like to perform on-site medical diagnosis, forensic identification, archaeological or other scientific studies, or additional purposes involving processing rare or valuable samples.

46. The method of claim 33, wherein the aqueous sample solution is the inner phase, wherein the hydrophobic liquid is the outer phase, wherein the inner or outer phase can be any type of phase or any combination of phases (e.g. liquid, solid, or gas) involving liquid mixture or powder or bubbles as long as the outer phase can surround but does not mix or react with the inner reaction phase so that the reaction can be isolated to prevent sample loss due to molecular surface sticking or dead volume retention.

47. The method of claim 44, wherein the portable integrated ("ail-in-one") device can be fabricated by assembling polydimethvlsiloxane, or other non-solid materials that can solidify upon certain treatments such as baking, and the reaction containers such as test tubes into an all-in-one device for precise and accurate processing of rare or valuable samples.