WO2020016905A1

WO2020016905A1 - Quantification of concentration of a component in a sample fluid

Info

Publication number: WO2020016905A1
Application number: PCT/IN2019/050527
Authority: WO
Inventors: Priyanka VALLOLY; Jatin PANWAR; Rahul Roy
Original assignee: Indian Institute of Science IISC
Current assignee: Indian Institute of Science IISC
Priority date: 2018-07-15
Filing date: 2019-07-15
Publication date: 2020-01-23
Anticipated expiration: 2021-01-15

Abstract

Apparatuses and methods for quantification of a component in a sample fluid are provided. Apparatuses comprises a microfluidic device to provide compartments of sample fluid by interspersing with carrier phase; a suction unit coupled to an outlet of a microfluidic device to drive the sample fluid, the carrier phase, and the compartments; a reservoir to collect the compartments; and an imaging unit to quantify the concentration of the component in the sample fluid based on distribution of compartments and dilution factor.

Description

QUANTIFICATION OF CONCENTRATION OF A COMPONENT IN A SAMPLE FLUID

TECHNICAL FIELD

[0001] The present subject matter relates to microfluidic devices, and in particular, to microfluidic devices for high dynamic range quantification of molecules and cells.

BACKGROUND

[0002] For the purpose of amplification and quantification of nucleic acids, microfluidic platforms use isothermal amplification methods like Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequence Based Amplification (NASBA), Loop-Mediated Isothermal Amplification (LAMP), Helicase Dependent Amplification (HDA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), and Multiple Displacement Amplification (MDA). Quantification of amplified nucleic acids has also been incorporated on microfluidic devices. Typically, sample preparation for the amplification and quantification is conducted prior to introduction of the sample to the microfluidic platforms.

BRIEF DESCRIPTION OF DRAWINGS

[0003] The detailed description is described with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The same numbers are used throughout the drawings to reference like features and components.

[0004] Fig. 1(a) illustrates a bifurcation junction of an example microfluidic device, in accordance with an implementation of the present subject matter.

[0005] Fig. 1(b) illustrates flow rates of inlets to the bifurcation junction, in accordance with an implementation of the present subject matter. [0006] Fig. 1(c) depicts step function provided to the valves for“n” discrete positions between the fully open and fully closed configuration, in accordance with an implementation of the present subject matter.

[0007] Fig. 1(d) illustrates a side view of a microfluidic device, in accordance with an implementation of the present subject matter.

[0008] Fig. 1(e) depicts a first connection of the microfluidic device, in accordance with an implementation of the present subject matter.

[0009] Fig. 2 illustrates another example method of forming compartments with gradient concentration, in accordance with an implementation of the present subject matter.

[00010] Fig. 3 depicts yet another microfluidic device for on-chip amplification and quantification of nucleic acids, in accordance with an implementation of the present subject matter.

[00011] Fig. 4(a) depicts an apparatus, in accordance with an implementation of the present subject matter.

[00012] Fig. 4(b) depicts a method for quantification, in accordance with an implementation of the present subject matter.

[00013] Fig. 4(c) depicts an example method for serial dilution, in accordance with an implementation of the present subject matter.

[00014] Fig. 5 depicts formation of compartments (a) in the absence of valves (b) in the presence of valves, in accordance with an implementation of the present subject matter.

[00015] Fig. 6 illustrates a schematic of series of compartments which may or may not comprise nucleic acids, in accordance with an implementation of the present subject matter.

[00016] Fig. 7(a) illustrates graphs indicating rate kinetics of amplification in compartments, in accordance with an implementation of the present subject matter. [00017] Fig. 7(b) depicts a correlation between copy number in bulk and copy number per compartment, in accordance with an implementation of the present subject matter.

[00018] Fig. 8 depicts an example graph for quantification of ON and OFF compartments, in accordance with an implementation of the present subject matter. DETAILED DESCRIPTION

[00019] The present subject matter provides a modular microfluidic device for use in formation of compartments with a concentration gradient. Further, the microfluidic device can also be used for high throughput quantification of nucleic acids, proteins, single cells, and molecules. While the present subject matter has been explained with reference to nucleic acids amplification and quantification, other molecules and cells can also be quantified in a sample.

[00020] Studies have been conducted to incorporate amplification and detection of nucleic acids in microfluidic platforms, hereinafter, also referred to as lab-on-chip platforms. However, for this, samples are generally preprocessed separately from the microfluidic platforms and the preprocessing requires skilled professionals. Preprocessing, among other steps, includes serial dilution of the sample which is time consuming and tedious. Further, serial dilution is generally done manually and, therefore, is associated with a significant standard error, especially, while pipetting volumes as low as a few microliters.

[00021] On-chip serial dilution microfluidics platforms, where the serial dilution is performed on the microfluidics platforms, generates a concentration gradient in a continuous phase. However, such microfluidics platforms do not provide compartmentalization and, therefore, cannot be used to form localized and discrete concentrations. Further, typically, conventional microfluidic devices can measure concentrations within known ranges.

[00022] Digital Polymerase Chain Reaction (dPCR) improves upon the current PCR practices by dividing a reaction mixture comprising nucleic acid into multiple, smaller reactions. The sample in the reaction mixture is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. Micro well plates, capillaries, oil emulsion, and arrays of miniaturized chambers with nucleic acid binding surfaces can be used to partition the samples.

[00023] A PCR solution which consists of template DNA (or RNA), fluorescence- quencher probes, primers, and a PCR master mix, which contains DNA polymerase, dNTPs, MgCl2, and reaction buffers at optimal concentrations. The PCR solution is divided into smaller reactions and are then made to run PCR individually. After multiple PCR amplification cycles, the samples are checked for fluorescence with a binary readout of“0” or“1”. The fraction of fluorescing compartments is recorded. The partitioning of the sample allows one to quantify the number of different molecules by assuming that the molecule population follows the Poisson distribution, thus accounting for the possibility of multiple target molecules inhabiting a single compartment.

[00024] However, conventionally used techniques for compartment formation cannot be easily scaled up. Further, there can be redundancy when multiple compartments include the same target nucleic acids. Electronics enabled serial dilution microfluidics platforms are known. However, these microfluidics platforms use electronic assistance for motion of the compartment which, consequently, compromises modularity and also limits its application as a high-throughput platform. Further, these methods can, generally, not be used in complex biomolecular analyses, such as molecular quantification without considerable modifications made to flow patterns, residence time, and temperature controls.

[00025] The present subject matter provides an apparatus comprising a microfluidic device which provides serial dilution, geometry mediated passive flow control for compartmentalization, and on-chip amplification and analysis of nucleic acids. The serial dilution, hereinafter also referred to as compartment formation with concentration gradient, is programmable and helps in generating compartments that contain a distribution of sample concentration ranging from zero copy of molecules per compartment to the actual molecular concentration in the sample. The microfluidic device can be incorporated with temperature control for quantification of molecules at real-time, i.e., during amplification or at end-point, i.e., after completion of amplification. The apparatus can also be used as a concentration dispenser into paper microfluidic strips or precast compartments.

[00026] The apparatus may be used for viral load quantification for prognosis, proximity nucleic acid ligation assays for expression level quantification of different proteins, effect of enzyme concentration on reactions, high throughput quantification and isolation of rare cells, drug response monitoring, and the like. The apparatus provides accurate and automated measurement of concentration of cells and biomolecules like nucleic acids or proteins in a given sample with minimum reagent volume.

[00027] The microfluidic device of the apparatus comprises a sample inlet port for inlet of the sample fluid. The sample fluid comprising the component to be estimated and a marker ingredient. The microfluidic device further comprises a carrier inlet port for inlet of a carrier fluid and an outlet port for outlet of compartments of the sample fluid interspersed with the carrier fluid. A first connection may be formed at an intersection of a carrier phase microchannel and a supplier microchannel. The carrier phase microchannel is to carry the carrier fluid from the carrier inlet port to the first connection and the supplier microchannel is to supply the sample fluid to the first connection. The sample fluid is partitioned into the compartments interspersed with the carrier fluid at the first connection. The compartments have varying concentrations of the component and corresponding concentrations of the marker ingredient. A downstream microchannel extends from the first connection to the outlet port to allow outflow of the compartments.

[00028] A suction unit can be coupled to the outlet port of the microfluidic device to provide a suction force to draw the sample fluid and the carrier fluid from respective inlet ports through the first connection to form the compartments and to draw the compartments out from the outlet port. The suction unit is controllable to apply the suction force based on a function of the capillary number of the downstream microchannel and resistance of the first, second, and downstream microchannels to form the compartments of substantially equal size.

[00029] The apparatus further comprises a reservoir to collect the compartments of the sample fluid and an imaging unit to quantify the concentration of the component in the sample fluid. The imaging unit can include a stage to position the reservoir; an image capturing device to capture an image of the compartments in the reservoir; and a processor to process the image to determine the concentration of the component. The apparatus can also have additional components, such as laser, illumination sources, and the like, which are not discussed herein for the sake of brevity.

[00030] The processor can determine, from the image, the quantity/ concentration of the component based on digital analysis.

[00031] Digital analysis works by diluting molecules like nucleic acids to an average of < 0.3 molecule per compartment. When the signal is amplified using enzymatic reactions (or other assays), the compartments either turn‘on’ or remain‘off for the signal based on whether a molecule was present or not, respectively in the compartment. This gives a high-contrast digital signature at the end of the reaction and does not require continuous monitoring. Since the distribution of the molecules into the compartments will follow a Poisson distribution, a ratio of ‘on’ and ‘off compartments are defined by the average concentration per compartment. By comparing this ratio over several compartments, the concentration of the molecule in the sample can be determined.

[00032] In case of serial dilution quantification, an array of compartments is generated per sample; the array includes compartments that are diluted over a range of concentrations that will be estimated based on dilution factor using a tracer dye. The high concentration droplets will always be‘on’ but diluted droplets will display no amplification whose position is dependent on the original concentration of the sample. Concentration is recovered using the on/off ratio, i.e., distribution of compartments with component, combined with the tracer dye concentration, i.e., dilution factor. The Poisson distribution may be applied to each dilution condition and deconvolution of the‘on-off ratio over the entire range of concentrations may be used to improve the accuracy of the estimate.

[00033] The microfluidic device can be used for both on-chip as well as off-chip amplification and quantification while the existing technologies provide one of either on-chip amplification or off-chip amplification. The compartments provided in the microfluidic device can be chemically barcoded and collected for off-chip amplification (isothermal and non-isothermal including PCR and the like) for further analysis on existing systems like flow cytometer, quantitative polymerase chain reaction (qPCR), and the like. The barcodes carry the signature for dilution factor and thus correlate the compartments to corresponding sample concentration. The barcoded compartments can also be used for concentration based sorting or single cell screening and analysis.

[00034] The above and other features, aspects, and advantages of the subject matter will be better explained with regard to the following description and accompanying figures. It should be noted that the description and figures merely illustrate the principles of the present subject matter along with examples described herein and, should not be construed as a limitation to the present subject matter. It is thus understood that various arrangements may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and examples thereof, are intended to encompass equivalents thereof. Further, for the sake of simplicity, and without limitation, the same numbers are used throughout the drawings to reference like features and components.

[00035] Fig. 1(a) illustrates a bifurcation junction 101 of an example microfluidic device, in accordance with an implementation of the present subject matter. The bifurcation junction 101 can be a substantially y-shaped junction. The bifurcation junction 101 can include two microchannels, namely, a sample microchannel l03a and a diluent microchannel l03b. The sample microchannel l03a can be for flow of a first liquid, for example, a sample fluid comprising the component to be quantified and a marker ingredient. The component may be nucleic acids, cells, proteins, particles, and the like or any organic or inorganic discrete particle that is to be quantified. The marker ingredient may be dyes, microbeads, and the like. The diluent microchannel l03b can be for flow of a second liquid, for example, a diluent. The diluent may be any fluid that is miscible with the sample fluid. For example, if water is miscible with the sample fluid, the diluent fluid may be an aqueous based fluid. In another example, the sample microchannel 103 a may be for flow of a first solution which has to be serially diluted and the diluent microchannel l03b may be for flow of a diluent to serially dilute the first solution. The sample microchannel l03a can extend from a sample inlet port and the diluent microchannel can extend from a diluent inlet port to the bifurcation junction 101.

[00036] Each of the sample microchannel 103 a and the diluent microchannel l03b can comprise a sample control valve l05a and a diluent control valve l05b. The sample control valve l05a and the diluent control valve l05b control a supply of the sample fluid and the diluent fluid to the bifurcation junction 101. The sample control valve l05a and the diluent control valve l05b are controllable to vary the flow rates of the sample fluid and the diluent fluid to provide a constant flow rate of diluted sample fluid of varying dilution levels.

[00037] A supplier microchannel l03c can be formed downstream of the bifurcation junction 101 to receive the sample fluid and the diluent fluid to mix them. The dimensions of the supplier microchannel l03c can be varied to obtain compartments of different sizes. For example, the width of the supplier microchannel l03c may be in the range of 150 - 200 pm to obtain compartments of adequate volume and stability. The supplier microchannel l03c can form a first connection 107 with a carrier phase microchannel 109. The first connection 107 is formed at an intersection of the carrier phase microchannel 109 and the supplier microchannel.

[00038] The carrier phase microchannel 109 is for passing of a carrier fluid which encapsulates the mixture of the sample fluid and diluent which is received from the supplier microchannel l03c to form compartments. The carrier phase microchannel 109 can extend between a carrier inlet port and the first connection 107. In one example, the first connection 107 is a T-junction. The first connection 107 extends into a downstream microchannel 111. The downstream microchannel 111 can extend between the first connection 107 and an outlet port to allow outflow of the compartments from the first connection 107. To form the compartments having a concentration gradient, the sample control valve l05a and the diluent control valve l05b can be controlled as shown in Fig. 1(b).

[00039] With the sample control valve l05a and the diluent control valve l05b in operation, flow rate of the first liquid and the second liquid can be controlled prior to their merging at bifurcation junction. Each valve has“n” discrete operating positions or steps between“fully open” to“neutral” to“fully closed”. The valves operate in synchronization keeping flow rate after the y-junction constant (Qd) which in turn, makes the valve operation independent of compartment size and frequency. If first valve l05a moderates the flow rate of the carrier phase stream by a factor of x, then the flow rate of the second liquid is moderated by a factor of 1 - x by the second valve l05b where 0 < x < l; x = l : fully open; x = 0: fully closed; x = 0.5: neutral. At any instance during operation, the flow rate of both streams is Qdx and Qd(i-x), respectively, which regulates the volumetric contribution of each sub-stream to the equivalent dispersed phase stream flow rate (Qd) during pinch-off resulting in compartments of desired dilution factors. Fig. 1(c) depicts step function provided to the valves for“n” discrete positions between the fully open and fully closed configuration.

[00040] Fig. 1(d) illustrates a side view of a microfluidic device 100, in accordance with an implementation of the present subject matter. In an example, the microfluidic device 100 comprises three layers. It is to be understood that any number of layers maybe provided. In an example, all three layers are fabricated from elastomers. In an example, the elastomers may be selected from the group consisting of Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), Polyurethane, synthetic Polyisoprene, and the like.

[00041] In the example as shown in Fig. 1(d), a bottom layer 102 forms a base of the microfluidic device 100. The bottom layer 102 provides uniformity to surface properties of a plurality of microchannel provided in an intermediate layer 104. The intermediate layer 104 is provided on the bottom layer 102 and comprises the plurality of microchannel 106, namely, the sample microchannel, diluent microchannel, carrier fluid microchannel, supplier microchannel. The plurality of microchannels 106 are for fluidic operations and geometry assisted flow control. The plurality of microchannel 106 may be fabricated by methods, such as etching, photolithography, and the like. The microfluidic device 100 also comprises a top layer 108. The top layer 108 comprises a plurality of valves 105, namely, the sample control valve l05a and the diluent control valve l05b, which assist with flow control and, thereby, serial dilution and compartmentalization. In one example, a diaphragm layer 113 may be interposed between the intermediate layer 104 and the top layer 108. When pressure is applied through the microchannels, the diaphragm layer 113 pushes upwards against the valves 105 and causes the microchannels to open; and when pressure is withdrawn from the intermediate layer 104, the diaphragm layer 113 collapses, causing the valves 105 to close.

[00042] Illustration A depicts a single valve fully open, Illustration B depicts the single valve in a neutral position, and Illustration C depicts the single valve fully closed. Various positions of the first valve and the second valve may be used to dispense the first liquid and the second liquid. For example, when the valve is fully open, the associated liquid may be dispensed and when the valve is fully closed, dispensing of the associated liquid may be stopped. [00043] Fig. 1(e) depicts another example first connection 130 to form compartments with concentration gradient, in accordance with an implementation of the present subject matter. The example first connection 130 can include the supplier microchannel l03c upstream of the first connection 130.

[00044] The first connection 130 may be used in the example where the microfluidic device is used for providing compartments with concentration gradients of discrete particles, such as nucleic acids, that can be bound to reagents. In said example, the supplier microchannel l03c may be coupled directly to the sample inlet port to receive the sample fluid with an intervening sample microchannel. Further, a diluent fluid may not be used since the concentration gradient may be obtained by using microbeads as discussed below.

[00045] In this example, the sample fluid may comprise the component to be quantified and a marker ingredient. The component, in this case, is nucleic acid. Further, to provide a concentration gradient, the sample may also include a plurality of microbeads of varying diameters and sizes. The size of the microbeads defines the number of nucleic acids which can be bound on it. In one example, the microbeads can be coated with reagents to bind to the nucleic acids. The sample fluid flows at a flow rate of‘Q’ in the supplier microchannel l03c and arrives at the first connection 130. At the first connection 130, the carrier phase flowing in the carrier phase microchannel 109 meets the sample fluid to form compartments. The compartments so formed may have different numbers and different sizes of microbeads enclosed in the carrier phase, thereby, providing compartments with varying concentrations of the nucleic acids.

[00046] To have a governing parameter that includes interaction between the two phases (sample fluid and the carrier phase) along with their individual properties, the capillary number (defined by carrier phase microchannel) is combined with the length ratio. For sake of reference, this capillary number is called modified capillary number (Can). The modified capillary number is proposed to define the compartment generation frequency and volume for a multi-phase system. Ca_L is a dimensionless number that describes the relative dominance between tendency of continuous phase to deform the interface and tendency of dispersed phase along with interfacial tension to resist deformation. Ca_L combines the flow pressure ratio between the dispersed and continuous phases with the viscous and inertial properties of the system to demonstrate how compartment generation in different flow regimes (from dripping to squeezing) follows an inverse power law in suction driven microfluidics.

[00047] The geometry of the device makes the two fluids (sample fluid and the carrier phase) interact at the required flow rate ratios resulting in compartment generation. As the geometry is integrated in the design, each design is designated to generate a range of compartment dimensions, which can be fine -tuned by varying the suction volume.

[00048] A low Ca_L represents squeezing regime of compartment generation with high compartment volume sensitivity and low-pressure consumption. On the other hand, dripping regime is observed at high Ca_L values that signifies low compartment volume sensitivity and high generation efficiency and pressure consumption. The transition between squeezing to dripping regime corresponds to the change in slope and was observed at Ca_L ~ 10 ¹ for most two-phase fluid systems.

[00049] Along with flow properties, the Ca_L also accounts for geometrical parameter like length ratio. It is expected to serve as a design parameter that defines microchannel geometry for suction microfluidics -based compartment generation. As an optimized regime, i.e. low compartment volume sensitivity and pressure consumption but high generation frequency, this study suggests a narrow range of 0.1 < Ca_L < 0.2. These properties of Ca_L are harnessed to design suction-based multi -operational microfluidic devices for compartment generation. The Ca_L can be computed based on the Equation:

[00050] Fig. 2 depicts a top view illustrating an example microfluidic device 200, in accordance with an implementation of the present subject matter. The microfluidic device 200 can include a plurality of microchannel 106. The microfluidic device 200 can comprise a carrier inlet port 202 associated with carrier phase microchannel 109, a sample inlet port 204 associated with sample microchannel l03a, a diluent inlet port 206 associated with the diluent microchannel l03b, and an outlet port 208. Flow passages associated with the carrier inlet port 202, the sample inlet port 204, the diluent inlet port 206, and the outlet port 208 can include valves. An enlarged view of the valve l05a is shown in Fig. 2(a).

[00051] Flow passages associated with the sample inlet port 204 and diluent inlet port

206 merge forming a y-junction, in an example, i.e., sample microchannel l03a and the diluent microchannel 103b merge to form a bifurcation junction 101. The y-junction is as shown in Fig. 2(b). The y-junction extends into the supplier microchannel l03c which then merges with the carrier phase microchannel 109 extending from carrier inlet port 202 to form a first connection 107 (refer Fig. 1(a)). The first connection 107 and the bifurcation junction 101 may be the t-junction and the y-junction are shown in Fig. 1(a) and 2(b). However, as will be understood, the y-junction, i.e., the bifurcation junction 101 may be not be provided and the microfluidic device may have only the supplier microchannel l03c extending from the sample inlet port 204. In said example, the diluent inlet port 206 may also be absent. Alternately, the supplier microchannel l03c may extend from the sample microchannel l03a and the diluent microchannel l03b may be closed. At the first connection 107 compartments of the sample fluid are formed in the carrier phase are formed.

[00052] Downstream of the first connection 107 forms the downstream microchannel 111. The downstream microchannel 111 can include a plurality of loops for visualization of the compartments during flow of the compartments through the downstream microchannel 310. In one example, the plurality of loops may behave as a reservoir 210. The reservoir 210 comprises, in an example, six equi-length microchannels in series as shown in Fig. 2(c). However, other examples of reservoirs are also explained with reference to Fig. 3. The reservoir 210 may be used to collect the sample fluid. In one example, the reservoir 210 may also be used for on-chip amplification of nucleic acids. In said example, the reservoir 210 can be associated with an isothermal heating stage. The isothermal heating stage can be used for maintaining a temperature for isothermal amplification of nucleic acids. It is to be understood that any number of microchannels and any length may of microchannels may be provided for the amplification and the example of Fig. 2(c) is not to be construed as limiting.

[00053] In an example, the sample contains nucleic acids as the components that are to be amplified and quantified, and sample fluid includes amplification reagents, such as enzymes, nucleotides, and the like, and the carrier phase may be an oil infused with a surfactant. The oil may be selected from fluorocarbon oil, hydrocarbon oil, silicon oil, mineral oil, or a combination thereof, and the surfactant may be selected from Perfluoropolyether (PFPE), Polysorbate 20, Polysorbate 80, or a combination thereof.

[00054] A suction unit is coupled to the outlet port 208 to provide suction force to draw the sample fluid and the carried from respective inlet ports through the bifurcation junction 101 and the first connection 107 to form compartments. The suction unit also helps in drawing the compartments out into the reservoir. The suction unit is controllable to apply the suction force based on the capillary number of the microchannel downstream of the first connection and resistance of the carrier phase microchannel 109, the supplier microchannel l03c, the sample microchannel l03a, and the diluent microchannel l03b to form compartments of substantially equal size.

[00055] The suction unit, therefore, combines resistances in flow microchannels to control the flow rates when a suction force is applied at the outlet. It provides driving force that induces the fluids flow based on a ratio between the flow rates of carrier phase and the first liquid that insures the compartment generation. Negative pressure or suction through the outlet port 208 is used as the driving force for all operations. [00056] Fig. 3 depicts yet another microfluidic device 300 for on-chip amplification and quantification of nucleic acids, in accordance with an implementation of the present subject matter. The microfluidic device 300 comprises a sample inlet port 302 associated with a supplier microchannel 304. The sample inlet port 302 is for inlet of the sample fluid comprising the component to be quantified and a marker ingredient. As will be understood, the sample fluid may also include PCR mix and plurality of microbeads to form a concentration gradient. It is to be understood that the sample inlet port 302 may be associated with a sample reservoir from which the sample fluid can be drawn into the microfluidic device 300.

[00057] The device 300 also comprises a carrier inlet port 306 associated with a carrier phase microchannel 308. The device 300 depicts two carrier inlet ports and microchannels, namely, carrier inlet ports 306-1, 306-2 and two carrier phase microchannels 308-1, 308-2. However, any number of the carrier inlet ports 306 and associated microchannels 308 may be provided.

[00058] The carrier phase microchannel 308 joins the supplier microchannel 304. Each carrier phase microchannel 308 joins the supplier microchannel 304 at a first connection 309 as shown in Fig. 3. However, it is to be understood that any other configuration may also be used. At the first connection 309, sample fluid form compartments due to being interspersed with the carrier phase. These compartments pass through the downstream microchannels 310. The downstream microchannel 310 can include a plurality of loops for visualization of the compartments during flow of the compartments through the downstream microchannel 310. However, any other configuration may also be used, for example, to increase residence time.

[00059] The microfluidic device 300 also comprises a reservoir 312. A first end of the reservoir 312 can be coupled to the downstream microchannel 310 to receive the compartments. A second end of the reservoir 312 can comprise a filter 314 to prevent the compartments in the reservoir from leaving the reservoir 312. The compartments are parked in the reservoir 312 for amplification and quantification. Inset 317 shows the compartments parked in the reservoir 312 and the filter 314 in greater detail.

[00060] The second end of the reservoir 312 can be coupled to an outlet passage 316 which is coupled to an outlet port 318. The outlet port 318 can be used for providing suction to cause the sample fluid and the carrier phase to move through the supplier microchannel, the carrier phase microchannel; and to cause the compartments to move from the downstream microchannels through the reservoir 312. As explained previously, the modified capillary number can be used for determining the suction applied for the size and number of compartments to be formed.

[00061] In operation, on application of suction, the compartments are formed which are then transported via the downstream microchannel 310 to the reservoir 312. The reservoir 312 can be separated from the microfluidic device 300 and can be placed on an isothermal heating stage that can maintain the temperature of the reservoir at 37°C for amplification of the nucleic acids. Alternately, for real-time amplification, the microfluidic device 300 can be associated with the isothermal heating stage. An imaging unit can be provided to detect the presence or absence of nucleic acid in a compartment, for example, based on a fluorescent marker, color of the fluorescent marker. As will be understood, real-time visualization of the amplification can also be performed. The imaging unit can also, based on the intensity and dilution of the marker, quantify and average the nucleic acids and, thereby, quantify the nucleic acids. In one example, Poisson distribution may be used by the imaging module to determine and quantify the nucleic acids.

[00062] Fig. 4(a) depicts an example apparatus 400 for quantifying an average concentration of a component in a sample fluid, in accordance with an implementation of the present subject matter. The apparatus 400 comprises a microfluidic device 402, a suction unit 402A, a reservoir 404, and an imaging unit 406. The imaging unit 406 is to quantify the concentration of the component in the sample fluid. [00063] The microfluidic device 402 may be microfluidic device 200 and/or 300 with the first connection 107 and optionally the bifurcation junction 101 and as explained previously. The suction unit may be coupled to the outlet port of the microfluidic device 402 to generate and draw the compartments formed by the microfluidic device. As will be understood from the previous explanation, the reservoir 404 may be integrally formed with the microfluidic device 402 or may be separately formed from the microfluidic device 402. The compartments formed can be parked in the reservoir for isothermal amplification and visualization. In one example, the apparatus 400 can also include an isothermal heating stage. The reservoir can be placed on the isothermal heating stage for a pre-determined time for amplification of the components in the compartment. The pre-determined time may be determined based on the time required for the amplification to reach completion based on the type of component to be amplified. In one example, the pre-determined time may be 30 minutes when the component is nucleic acid.

[00064] The imaging unit 406 can include a stage 406a to receive the reservoir 404; an image capturing device 406b, and a processor 406c. For real time visualization of components, the stage 406a can be associated with a thermal stage for maintaining temperature during isothermal amplification. The image capturing device 406b is to capture an image of the compartments in the reservoir 404. The image capturing device 406b can be used for real-time capture of the amplification or for end-point capture of amplification. The image capturing device 406b may a camera, a detector for detection of infrared, visible and fluorescent light and the like.

[00065] The image capturing device 406b can capture an image of the compartments in the reservoir 404. The imaging unit 406 can communicate the captured image to the processor 406c to determine the concentration of the components, i.e., the nucleic acids. The processor 406c can determine, from the image, distribution of compartments with components in each compartment and a parameter of the marker ingredient in each compartment. The parameter may be, for example, fluorescence if the marker ingredient is a fluorescent dye.

[00066] The processor 406c can also ascertain a dilution factor of the sample fluid for each compartment based on the parameter of the marker ingredient. The processor 406c can also quantify the concentration of the component in the sample fluid for each compartment based on the distribution of compartments and the dilution factor. The processor 406c can also quantify the average concentration of the component in the sample fluid based on averaging the estimates of concentration.

[00067] In one example, the marker ingredient is a plurality of microbeads of varying diameter and the compartments comprise components, i.e., nucleic acids bound to the one or more microbeads. In said example, the parameter of the marker ingredient is the size of the microheads. The processor 406c can correlate the size of the microheads with the number of components bound to it and to determine the dilution factor and the number of molecules of the component. In another example, the marker ingredient is a fluorescent dye and the parameter is intensity of fluorescence. The processor 406c can correlate the intensity of the fluorescence with the dilution factor.

[00068] For example, if a compartment has a nucleic acid, then a fluorescence marker associated with it will fluoresce and will indicate the presence of the nucleic acid in the compartment and vice versa, i.e., after amplification, the compartment either give a fluorescent signal“1” if it contains any copy from the initial concentration or a signal “0” for empty compartment; thus, this distribution transforms the quantification into a digital format. The dilution factor that corresponds the switch from“1” to“0” indicates the initial concentration in the sample. In one example, based on the image captured by the imaging unit 406, the processor 406c can quantify the nucleic acids in a compartment based on intensity of the marker (corresponding to the dilution factor), and the distribution of on/off compartments. Further, based on quantification in each compartment, the average can be computed for final quantification of nucleic acids. [00069] The microfluidic device of the present subject matter allows for measuring concentration of multiple molecules with a wide dynamic range. An integrated programmable serial dilution mechanism generates compartments that contain a distribution of molecular concentrations as low as zero or one copy number per compartment along with required amplifying reagents and fluorescent markers. For on- chip isothermal amplification (RPA, LAMP, NASBA, HAD, RCA, and the like) the compartments travel in a designated microchannel that provides the required temperature and residence time tuned for particular type of amplification. For off-chip amplification, the compartments are chemically barcoded at the time of generation which correlates each compartment to its constituent sample concentration. The real time detection mode further enhances the resolution and dynamic range of the detection. Being high throughput, the device generates enough statistics to account for statistical nature of low copy molecules loading into compartments and thus, provides a highly accurate quantification of initial molecular concentration. The quantification can be multiplexed wherein it requires addition of molecule specific fluorescent marker for the detection of multiple target molecules for both on-chip and off-chip operation modes.

[00070] Fig. 4(b) depicts an example method 400b for quantifying an average concentration of a component in a sample fluid, in accordance with an implementation of the present subject matter. The order in which the method 400b are described is not intended to be construed as a limitation, and any number of the described method blocks can be combined in any order to implement the methods or an alternative method. Additionally, individual blocks may be deleted from the method 400b without departing from the scope of the subject matter described herein. Furthermore, the method 400b may be implemented in any suitable hardware, computer readable instructions, firmware, or combination thereof.

[00071] A person skilled in the art will readily recognize that steps of the method 400b can be performed by programmed computers. Herein, some examples are also intended to cover program storage devices and non-transitory computer readable medium, for example, digital data storage media, which are computer readable and encode computer-executable instructions, where said instructions perform some or all of the steps of the described methods. The program storage devices may be, for example, digital memories, magnetic storage media, such as magnetic disks and magnetic tapes, hard drives, or optically readable digital data storage media.

[00072] The method 400b, at block 410, comprises providing the sample fluid at a sample inlet port and a carrier fluid at a carrier inlet port of a microfluidic device. The microfluidic device may be microfluidic device 200, 300. The sample inlet port and the carrier inlet port may be the sample inlet port 204, 302 and carrier inlet port 202, 306, respectively. As explained previously, the microfluidic device 200, 300 comprises outlet port 208, 318.

[00073] At block 412, the method 400b comprises applying a suction force to the outlet port of the microfluidic device by a suction unit coupled to the outlet port. The suction port is to draw the sample fluid and the carrier fluid from respective inlet ports through the first connection to form the compartments and to draw the compartments out from the outlet port. The first connection may be connection 107. The suction force applied is controlled based on a function of the capillary number of a downstream microchannel and resistance of the carrier phase microchannel, supplier microchannel and downstream microchannel to form the compartments of substantially equal size. The downstream microchannel, carrier phase microchannel, and supplier microchannel may be downstream microchannel 111, carrier phase microchannel 109, 308, and the supplier microchannel 304, l03c.

[00074] At block 414, the method 400b comprises collecting the compartments of the sample fluid in a reservoir. The reservoir may be reservoir 210, 312. At block 416, the method 400b comprises quantifying the concentration of the component in the sample fluid by an imaging unit. The imaging unit may be imaging unit 406 comprising the stage 406a, image capturing device 406b, and processor 406c. [00075] For quantifying the concentration of the component, the method 400b, at block 416a comprises receiving the reservoir at a stage of the imaging unit. In one example, the reservoir is received at stage 406a. In one example, the method 400b, comprises placing the reservoir on an isothermal heating stage for a pre -determined time for amplification of the component in the compartments prior to transfer to the stage 406a of the imaging unit 406.

[00076] At block 416b, the method 400b comprises capturing an image of the compartments in the reservoir by an image capturing device of the imaging unit. The image may be captured by image capturing device 406b. At block 416c, the method 400b comprises determining from the image, by a processor of the imaging unit, distribution of compartments that include the component, for example, above a threshold level, and a parameter of the marker ingredient in each compartment. The determination may be performed by processor 406c of the imaging unit 406. At block 4l6d, the method 400b comprises ascertaining, by the processor, a dilution factor of the sample fluid for each compartment based on the parameter of the marker ingredient.

At block 4l6e, the method 400b comprises obtaining, by the processor, estimates of concentration of the component in the sample fluid from each compartment based on the distribution of compartments and the dilution factor. At block 4l6f, the method 400b comprises quantifying, by the processor, the average concentration of the component in the sample fluid based on averaging the estimates of concentration.

[00077] Fig. 4(c) depicts a method 400c for dilution of sample fluid, in accordance with an implementation of the present subject matter. At block 450, the method 400c comprises providing a diluent fluid at a diluent inlet port of the microfluidic device. The diluent inlet port may be diluent inlet port 206. As explained previously, the microfluidic device can comprise the bifurcation junction 101 formed by intersection of the sample microchannel l03a and the diluent microchannel l03b. The sample microchannel l03a can extend from the sample inlet port 302, 204 to the bifurcation junction and the diluent microchannel l03b can extend from the diluent inlet port 206 to the bifurcation junction 101 to obtain diluted sample fluid at the bifurcation junction 101. The supplier microchannel l03c can extend from the bifurcation junction 101 to the first connection 107 and can supply the diluted sample fluid from the bifurcation junction 101 to the first connection 107.

[00078] The method 400c, at block 452, comprises controlling a supply of the sample fluid to the bifurcation junction by a sample control valve disposed in the sample microchannel. The sample control valve may be sample control valve l05a.

[00079] The method 400c, at block 454, comprises controlling a supply of the diluent fluid to the bifurcation junction by a diluent control valve disposed in the diluent microchannel. The diluent control valve may be diluent control valve l05b. The sample control valve l05a and diluent control valve l05b are controllable to vary the flow rates of the sample fluid and the diluent fluid to provide a constant flow rate of the diluted sample fluid at varying dilution levels.

[00080] The present subject matter will now be illustrated with working examples, which are intended to illustrate the working of disclosure and not intended to be taken restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. It is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary depending on the process and inputs used as will be easily understood by a person skilled in the art.

EXAMPLES

EXAMPLE 1 : VALVE BASED SERIAL DILUTION

[00081] Fig. 5(a) depicts formation of compartments in the absence of valves, in accordance with an implementation of the present subject matter. As can be seen, each compartment as shown in Fig. 5(a) has the same concentration. [00082] Fig. 5(b) depicts formation of compartments in the presence of valves, in accordance with an implementation of the present subject matter. As can be seen, a concentration gradient is seen in the series of compartments of Fig. 5(b). The concentration of each compartment and the concentration gradient of the series of compartments will depend on the actuation of the valves 502 and 504. The actuation of the valves 502 and 504 to obtain a uniform flow rate is as explained previously.

EXAMPLE 2: DETECTION OF PRESENCE OR ABSENCE OF NUCLEIC ACIDS IN A

COMPARTMENT

[00083] Fig. 6 illustrates a schematic of series of compartments which may or may not comprise nucleic acids, in accordance with an implementation of the present subject matter. Series 601 depicts the schematic of the compartments and Series 602 depicts marker characteristics of each of the compartments. As can be seen from 601-1, if the compartment has any nucleic acid, there will be a marker output, i.e., if the marker is a fluorescent dye, then the compartment will fluoresce. As can be seen from 601-3, in the absence of the fluorescent dye, there will be no marker output. Further, the present subject matter, is also sensitive to a single nucleic acid strand in a compartment as shown by 60l-n and the associated output 602-n. EXAMPLE 3: INTENSITY STUDY AND DISTRIBUTION STUDY

[00084] Fig. 7(a) illustrates graphs indicating rate kinetics of amplification in compartments, in accordance with an implementation of the present subject matter. Illustration A indicates rate kinetics for 100 copies per compartment; Illustration B indicates rate kinetics for 10 copies per compartment; Illustration C indicates 1 copy per compartment; and Illustration D indicates rate kinetics for 0 copy per compartment at different periods of time, namely, 0, 10, 30, 60, and 90 mins of isothermal amplification. As can be seen, intensity was seen to be highest in compartments with 100 copies. This decreased with decrease in number of copies per compartment. This indicated that the light intensity of the end product is a function of the initial number of nucleic acids in each compartment.

[00085] Fig. 7(b) depicts a correlation between copy number in bulk and copy number per compartment, in accordance with an implementation of the present subject matter. As can be seen there is a good correlation with correlation coefficient R=0.988.

EXAMPLE 4: QUANTIFICATION OF ON AND OFF COMPARTMENTS

[00086] Fig. 8 depicts an example graph for quantification of ON and OFF compartments, i.e., determining distribution of the compartments with the component, in accordance with an implementation of the present subject matter. As will be understood, ON compartments are the compartments which contain nucleic acids and OFF compartments are the compartments which do not contain nucleic acids.

[00087] To quantify the ON and OFF compartments, after 30 minutes of isothermal amplification, the average intensity of all the compartments can be calculated. The average can be a threshold. The number of compartments above the threshold are detected. The fraction of positive compartments will be equal to the number of compartments above the threshold divided by the total number of compartments analyzed.

[00088] The microfluidic device of the present subject matter, therefore, allows for measuring concentration of multiple molecules with a wide dynamic range. The microfluidic device also allows for serial dilution of solutions with high accuracy. Further, the microfluidic device provides for on-chip amplification and both on-chip and off-chip detection of nucleic acids in real-time, therefore, combining the effects of both qPCR and compartment PCR. Further, as the microfluidic device works based on isothermal amplification, it also reduces the requirement of thermal cycling as required in traditional PCR.

[00089] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible. As such, the scope of the present subject matter should not be limited to the description of the preferred examples and implementations contained therein.

Claims

I/We claim:

1. An apparatus for quantifying an average concentration of a component in a sample fluid, the apparatus comprising:

a microfluidic device comprising:

a sample inlet port for inlet of the sample fluid, the sample fluid comprising the component to be estimated and a marker ingredient; a carrier inlet port for inlet of a carrier fluid;

an outlet port for outlet of compartments of the sample fluid interspersed with the carrier fluid;

a first connection formed at an intersection of a carrier phase microchannel and a supplier microchannel, wherein the carrier phase microchannel is to carry the carrier fluid from the carrier inlet port to the first connection and the supplier microchannel is to supply the sample fluid to the first connection, wherein the sample fluid is partitioned into the compartments interspersed with the carrier fluid at the first connection, wherein the compartments have varying concentrations of the component and corresponding concentrations of the marker ingredient; and

a downstream microchannel extending from the first connection to the outlet port to allow outflow of the compartments; a suction unit coupled to the outlet port of the microfluidic device to provide a suction force to draw the sample fluid and the carrier fluid from respective inlet ports through the first connection to form the compartments and to draw the compartments out from the outlet port, wherein the suction unit is controllable to apply the suction force based on a function of the capillary number of the downstream microchannel and resistance of the carrier, supplier, and downstream microchannels to form the compartments of substantially equal size; a reservoir to collect the compartments of the sample fluid; and an imaging unit to quantify the concentration of the component in the sample fluid, the imaging unit comprising:

a stage to receive the reservoir;

an image capturing device to capture an image of the compartments in the reservoir; and

a processor to process the image to determine the concentration of the component, wherein the processor is to:

determine, from the image, a distribution of compartments that include the component above a threshold level and a parameter of the marker ingredient in each compartment;

ascertain a dilution factor of the sample fluid for each compartment based on the parameter of the marker ingredient; obtain estimates of concentration of the component in the sample fluid from each compartment based on distribution of compartment and the dilution factor; and

quantify the average concentration of the component in the sample fluid based on averaging the estimates of concentration.

2. The apparatus as claimed in claim 1 , wherein the marker ingredient is a plurality of microbeads of varying diameters, the compartments comprise component bound to one or more microbeads, the parameter of the marker ingredient is the size of each bead of the plurality of microbeads, and the processor is to correlate the size of the microbeads with the component bound to the microbeads to determine the dilution factor and the number of molecules of the component.

3. The apparatus as claimed in claim 1 comprising an isothermal heating stage, wherein the reservoir is to be placed on the isothermal heating stage for a pre determined time for amplification of the component in the compartments prior to transfer to the imaging unit.

4. The apparatus as claimed in claim 1, wherein the microfluidic device comprises:

a diluent inlet port for inlet of a diluent fluid;

a bifurcation junction formed by intersection of a sample microchannel and a diluent microchannel, the sample microchannel extending from the sample inlet port to the bifurcation junction and the diluent microchannel extending from the diluent inlet port to the bifurcation junction to obtain diluted sample fluid at the bifurcation junction, wherein the supplier microchannel extends from the bifurcation junction to the first connection and supplies the diluted sample fluid from the bifurcation junction to the first connection; a sample control valve disposed in the sample microchannel to control a supply of the sample fluid to the bifurcation junction; and

a diluent control valve disposed in the diluent microchannel to control a supply of the diluent fluid to the bifurcation junction, wherein the sample control valve and diluent control valve are controllable to vary the flow rates of the sample fluid and the diluent fluid to provide a constant flow rate of the diluted sample fluid at varying dilution levels.

5. The apparatus as claimed in claim 3, wherein the marker ingredient is a fluorescent dye, the parameter of the marker ingredient is intensity of fluorescence, and wherein the processor is to correlate the intensity of the fluorescent dye with the concentration of the component to determine the dilution factor and the number of molecules of the component.

6. The apparatus as claimed in claim 1, wherein the image capturing device one of: a camera and a detector for detection of infrared, visible and fluorescent light and the like.

7. The apparatus as claimed in claim 1, wherein the carrier fluid is an oil infused with a surfactant, wherein the oil is selected from fluorocarbon oil, hydrocarbon oil, silicon oil, mineral oil, or a combination thereof, and the surfactant is selected from Perfluoropolyether (PFPE), Polysorbate 20, Polysorbate 80, or a combination thereof.

8. The apparatus as claimed in claim 1, wherein the component is nucleic acids, cells, proteins and particles.

9. The apparatus as claimed in claim 3, wherein the diluent fluid is a fluid which is miscible with the sample fluid.

10. The apparatus as claimed in claim 1, wherein the downstream microchannel includes a plurality of loops for visualization of the compartments during flow of the compartments through the downstream microchannel.

11. A method for quantifying an average concentration of a component in a sample fluid, the method comprising:

providing a sample fluid at a sample inlet port and a carrier fluid at a carrier inlet port of a microfluidic device, the microfluidic device comprising:

the sample inlet port for inlet of the sample fluid, the sample fluid comprising the component to be estimated and a marker ingredient;

the carrier inlet port for inlet of the carrier fluid;

a downstream microchannel extending from the first connection to the outlet port to allow outflow of the compartments;

applying a suction force to the outlet port of the microfluidic device by a suction unit coupled to the outlet port to draw the sample fluid and the carrier fluid from respective inlet ports through the first connection to form the compartments and to draw the compartments out from the outlet port, wherein the suction force applied is controlled based on a function of the capillary number of the downstream microchannel and resistance of the carrier, supplier, and downstream microchannels to form the compartments of substantially equal size;

collecting the compartments of the sample fluid in a reservoir; and quantifying the concentration of the component in the sample fluid by an imaging unit, the quantifying comprising:

receiving the reservoir at a stage of the imaging unit;

capturing an image of the compartments in the reservoir by an image capturing device of the imaging unit;

determining from the image, by a processor of the imaging unit, distribution of compartments that include the component above a threshold level and a parameter of the marker ingredient in each compartment; ascertaining, by the processor, a dilution factor of the sample fluid for each compartment based on the parameter of the marker ingredient;

obtaining, by the processor, estimates of concentration of the component in the sample fluid from each compartment based on the distribution of the compartments and the dilution factor; and

quantifying, by the processor, the average concentration of the component in the sample fluid based on averaging the estimates of concentration.

12. The method as claimed in claim 11, wherein the marker ingredient is a plurality of microbeads of varying diameters, the compartments comprise component bound to one or more microbeads, the parameter of the marker ingredient is the size of each bead of the plurality of microbeads, and the processor is to correlate the size of the microbeads with the component bound to the microbeads to determine the dilution factor and the number of molecules of the component.

13. The method as claimed in claim 11, comprising placing the reservoir on an isothermal heating stage for a pre-determined time for amplification of the component in the compartments prior to transfer to the imaging unit.

14. The method as claimed in claim 11, comprising:

providing a diluent fluid at a diluent inlet port of the microfluidic device, wherein the microfluidic device comprises a bifurcation junction formed by intersection of a sample microchannel and a diluent microchannel, the sample microchannel extending from the sample inlet port to the bifurcation junction and the diluent microchannel extending from the diluent inlet port to the bifurcation junction to obtain diluted sample fluid at the bifurcation junction, wherein the supplier microchannel extends from the bifurcation junction to the first connection and supplies the diluted sample fluid from the bifurcation junction to the first connection; controlling a supply of the sample fluid to the bifurcation junction by a sample control valve disposed in the sample microchannel; and

controlling a supply of the diluent fluid to the bifurcation junction by a diluent control valve disposed in the diluent microchannel, wherein the sample control valve and diluent control valve are controllable to vary the flow rates of the sample fluid and the diluent fluid to provide a constant flow rate of the diluted sample fluid at varying dilution levels.

15. The method as claimed in claim 11, wherein the marker ingredient is a fluorescent dye, the parameter of the marker ingredient is intensity of fluorescence, and wherein the processor is to correlate the intensity of the fluorescent dye with the concentration of the component to determine the dilution factor and the number of molecules of the component.

16. The method as claimed in claim 11 , wherein the image capturing device is one of: a fluorescent camera, an infrared camera, and a visible light camera.

17. The method as claimed in claim 11, wherein the carrier fluid is an oil infused with a surfactant, wherein the oil is selected from fluorocarbon oil, hydrocarbon oil, silicon oil, mineral oil, or a combination thereof, and the surfactant is selected from Perfluoropolyether (PFPE), Polysorbate 20, Polysorbate 80, or a combination thereof.

18. The method as claimed in claim 11, wherein the component is nucleic acids, protein, cells, and particles.

19. The method as claimed in claim 11, wherein the diluent fluid is a fluid which is miscible with the sample fluid.

20. The method as claimed in claim 11 , comprising visualizing the compartments during flow of the compartments through a plurality of loops in the downstream microchannel.