Library-scale single-molecule force spectroscopy on a chip

Abstract

Methods and devices for spatially-multiplexed, simultaneous microfluidic, single-molecule force spectroscopy are provided. Devices include a plurality of microchannels formed on or in the substrate, where each microchannel is sized for immobilizing a respective bead-conjugated molecule. Each microchannel has an image channel such that collectively all image channels spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel. A lead-in channel assembly controllable with valves is positioned to introduce parallelized pressurized surface chemistry to the microchannels and inlet valves the image channels may be used to ensure homogenous surface chemistry and individualized introduction of the respective bead-conjugated molecules for simultaneous imaging while molecule cross contamination between microchannels.

Description

Patent Application 33929 / 70818 LIBRARY-SCALE SINGLE-MOLECULE FORCE SPECTROSCOPY ON A CHIPCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 741,698, filed January 3, 2025, which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA290563 and GM130332 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] The background description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventor, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.

[0004] Engineering the mechanosensitivity of biomolecules is an unexplored frontier for biotechnology. Many biological processes are force sensitive, such as T cell signaling. The misregulation of mechanosensation can lead to disorder and disease. However, we do not yet understand how mechanosensitivity is encoded in force-responsive molecules. This knowledge gap persists mainly due to the low throughput of current single-molecule force measurements: all current methods profile a single sequence at a time. Low-throughput force spectroscopy technology severely bottlenecks the discovery and engineering of new mechanosensitive molecules and their therapeutic potential.

[0005] There is a need for mechanisms and techniques for examining mechanosensitivity of biological samples.Patent Application 33929 / 70818 SUMMARY OF THE INVENTION

[0006] In example, a microfluidic platform for performing simultaneous single molecule analysis, the platform includes: a substrate; a plurality of microchannels formed on or in the substrate, each microchannel sized for immobilizing a respective bead-conjugated molecule, each microchannel comprising an image channel extending between a microchannel inlet and a microchannel outlet, wherein each image channel extends parallel to an axis defining a direction of microfluidic flow, wherein the plurality of image channels are spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel; a lead-in channel assembly positioned between a platform inlet and the plurality of microchannels for providing a parallelized surface chemistry to the plurality of microchannels through a simultaneous force exertion across the plurality of microchannels; an image area inlet valve assembly positioned to simultaneously release the parallelized surface chemistry into the plurality of image channels; and an image area outlet valve assembly positioned to selectively isolate each microchannel from molecule cross contamination.

[0007] In another example, a method for simultaneous single-molecule microfluidic imaging, the method includes: providing a surface chemistry to a general inlet of a lead-in channel assembly comprising a plurality of lead in microchannels each fluidically coupled to a respective image channel of a plurality of image channels, the plurality of image channels extending parallel to an axis defining a direction of microfluidic flow and the plurality of image channels being spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel; retaining, via an inlet valve at an inlet to each image channel, a portion of the surface chemistry to generate a pressurized buildup in the portion of the surface chemistry; simultaneously depressurizing the inlet valve at each image channel to simultaneously pattern each image channel with the surface chemistry;

[0008] repressurizing the inlet valve at each image channel; providing to each lead in microchannel of the lead-in channel assembly a respective molecule for imaging at a respective image channel, wherein each respective molecule is retained at each respective image channel via the repressurizing of the inlet valve; in response to depressurizing the inlet valve, selectively, at each image channel, opening an outlet valve of the image channel whilePatent Application 33929 / 70818 maintaining an outlet valve of each other image channel closed until each image channel contains the respective molecule in isolation; and performing a simultaneous microfluidic imaging of the image area to capture image data of each respective molecule in the respect image channels.

[0009] In yet another example, a microfluidic platform for performing simultaneous single molecule analysis, the platform includes: a substrate; a lead-in channel assembly formed on the substrate and comprising a plurality lead-in microchannels in a fan-in configuration, each lead-in microchannel having (i) a first switchable state in which an isolated surface chemistry is fed through the lead-in microchannel to a plurality of image microchannels and (ii) a second switchable state in which a different isolated molecule is fed through each different lead-in microchannel to the plurality of image microchannels; and a micro valve system having a first switchable state in which surface chemistry fed through the lead-in microchannels is pressurized against entering the plurality of image microchannels, a second switchable state in which surface chemistry fed through the lead-in microchannels is released into the plurality of image microchannels to uniformly pattern the surface chemistry in each image microchannel, and a third switchable state in which the different isolated molecules are fed into each different image microchannel for binding to the surface chemistry while preventing molecule cross contamination between image microchannels, wherein the plurality of image microchannels are formed on the substate and spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The figures described below depict various aspects of the system and methods disclosed herein. It should be understood that each figure depicts an embodiment of a particular aspect of the disclosed system and methods, and that each of the figures is intended to accord with a possible embodiment thereof. Further, wherever possible, the following description refers to the reference numerals included in the following figures, in which features depicted in multiple figures are designated with consistent reference numerals.Patent Application 33929 / 70818

[0011] This 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 United States Patent and Trademark Office upon request and payment of the necessary fee.

[0012] FIGS. 1A - 1C illustrate a spatially multiplexed, simultaneous microfluidic, singlemolecule force spectroscopy device, in accordance with an example. FIG. 1A illustrates a top view of the device. FIG. IB illustrates a top view of image channels confined to an imaging area of the device of FIG. 1A. FIG. 1C illustrates inlet and outlet connections of the device of FIG. 1A.

[0013] FIG. 2 illustrates fluorescence images captured of an example operation of the device of FIG. 1A, comparing the effects of creating a heterogeneous surface chemistry in the image channels to creating a homogenous surface chemistry, showing an increased overall yield of single molecule event capture, in accordance with an example.

[0014] FIG. 3 illustrates a florescence image captured for image channels, confined to an image area of an example spatially multiplexed, simultaneous microfluidic, single-molecule force spectroscopy device, showing concentration of molecules in respective image channels, in accordance with an example.

[0015] Fig. 4 illustrates a process for performing simultaneous single-molecule microfluidic imaging using the device of FIG. 1A, in accordance with an example.

[0016] FIG. 5 illustrates a further florescence image captured for image channels, confined to an image area of an example spatially multiplexed, simultaneous microfluidic, single-molecule force spectroscopy device, and indicating concentrations of Fab tuned density of beads immobilized by dig-bio deoxyribonucleic acid (DNA), per image channel, in accordance with an example.

[0017] FIG. 6 is a table indicating differences in throughput in various force spectroscopy techniques compared to the present techniques, in accordance with an example thereof.DETAILED DESCRIPTION

[0018] The present techniques describe spatially multiplexed, simultaneous microfluidic, single-molecule force spectroscopy devices (termed herein [SM]3FS). The [SM]3FS devicesPatent Application 33929 / 70818 herein dramatically increase the throughput of force spectroscopy measurements. In various examples, the [SM]3FS devices herein rely on the combination of a number of advances, including novel multichannel microfluidic devices that enable location-to-molecule indexing and high-throughput microfluidic force spectroscopy to exert force across many molecules and measure their responses in a single field of view. Together, in various examples, the [SM]3FS techniques here represent novel technologies and techniques for simultaneous, multiplexed force spectroscopy.

[0019] In various examples, [SM]3FS devices herein use programmable microfluidics to encode molecular identity by position in a device. For example, in some examples, multichannel devices are described that include multiple channels patterned with unique molecules, creating a channel-to-molecule index, for sample examination. For example, in an illustrated example, a multichannel device having 16 channels (e.g., image channels) is described - although, any number of parallels channels may be configured to implement devices according to the present techniques. As described, in various examples, the [SM]3FS devices herein may include binary valves that ensure a robust channel-to-molecule pairing with undetectable cross contamination. Further, the [SM]3FS devices herein may use microfluidic force spectroscopy to exert force on many molecules and measure their responses in a single field of view.

[0020] In various examples, the multichannel devices herein, including those having high numbers of channels such as 16 channels or more, have channels that are positioned in close proximity for simultaneous imaging of samples. Flow across an imaging region (also termed an image area) applies force to immobilized molecules, and their respective responses are measured in parallel across the image channels making up the imaging region. As a multichannel device, the [SM]3FS devices facilitate molecule patterning, force exertion, and measurement all within one field of view (FOV), such as one FOV of a microfluidic imager, such as a force spectroscopy imager.

[0021] To validate [SM]3FS techniques in various examples herein, the force extension curves of a set of 15 deoxyribonucleic acid (DNA)molecular variants were measured in parallel with the multichannel device; and in total, [SM]3FS devices measured 10,000 individual DNA tethers at 50-nm and sub-pN resolutions. This validation demonstrated that the present techniquesPatent Application 33929 / 70818 expand the throughput of force spectroscopy from 1 unique molecule per experiment to 15. The present techniques, of course, may be extended to other throughput examples. For example, the present techniques may be expanded to use [SM]3FS devices to measure molecules, including DNA, proteins, or ribonucleic acid (RNA). The parallel image channels of the [SM]3FS devices may be linear channels along an axis coinciding with a direction of microfluidic flow. In other [SM]3FS devices, the image channels may be arranged radially to allow an angle-to-molecule index measuring up to ~100 molecules simultaneously. Spatial indexing can increase molecular throughput 10-fold (up to 100-fold with radial indexing) compared to existing technologies.

[0022] The [SM]3FS techniques herein may be used in various different applications.Examples include high-throughput force spectroscopy equipment and kits that use cell surface chemistry reagents (e.g., buffers, patterning reagents, passivation reagents, protein attachment chemistries, DNA attachment chemistries), surface-modified glass, valved polydimethylsiloxane (PDMS) chips, flow controller, beads, and pressure control systems (for microfluidic valves and force application). Other examples include force-activated therapeutics development, where, for example, companies can use the present techniques to engineer and / or discover mechanosensors (DNA, RNA, or protein) for force-dependent function.

[0023] There are numerous technical advantages achievable with the present techniques. We list some of the more valuable ones from a design perspective and from an application of use perspective. The present techniques, for example, allow for: rapid measurement of many unique molecules under force; and an ability to efficiently engineer force-responsive molecules. Other advantages include the ability to generate training data (for artificial intelligence (Al) and / or other machine learning (ML) architectures) for predicting biomolecule dynamics under force. The datasets for training Al may be from measurements at zero force, but a substantial fraction of cellular processes happen under force or involve changes in conformation that can now be detected with single-molecule methods like [SM]3FS, thus provided for greatly enhanced training data. These and other advantages will be apparently to others, such as force spectroscopy system designers, who can offer [SM]3FS devices as a product for labs and companies to perform multiplexed profiling of force-responsive molecules. Further,Patent Application 33929 / 70818 biotechnology and pharmaceutical laboratories can use [SM]3FS to develop and engineer mechanoresponsive proteins for force-sensing applications, or to find drugs that modulate mechanosensitivity.

[0024] There are numerous technical advantages that [SM]3FS techniques herein provide over single-molecule force spectroscopy technologies (SMFS). Existing SMFS technologies measure one unique molecule per experiment, whereas various examples herein describe [SM]3FS devices that are able to measure many unique molecules in parallel. The architecture of various [SM]3FS devices herein positions many image channels into a single field of view for parallel force exertion and molecule tracking. Thus, in various examples, the [SM]3FS devices herein use a novel multichannel device to scale up the throughput of traditional SMFS experiments 15-fold and a radial device design could achieve a 100-fold enhancement in throughput over existing SMFS technologies.

[0025] In various examples, [SM]3FS devices may use parallelized surface chemistry to quicken experimental throughput and reduce experiment-to-experiment variability. Current SMFS technologies require lengthy surface chemistry preparations for each measurement, which limits throughput and also results in batch-to-batch variation. The present techniques, however, may use a similar chemical approach for patterning surfaces, but instead of measuring one unique molecule per prepared surface, the [SM]3FS devices may measure many unique molecule samples, quickening experimental throughput, reducing reagent usage, and reducing variability. Additionally, in various examples, the [SM]3FS devices may be configured with one or more internal control channels to ensure high-fidelity measurements. For example, one channel may be reserved as a negative control for testing the quality of surface chemistry within the chip. Existing technologies do not have built-in controls for validating the quality of their prepared surfaces. The [SM]3FS techniques lower the barrier for use of non-expert users compared to existing SMFS technologies (optical trapping, AFM, magnetic tweezers, centrifugal force spectroscopy, or acoustic force spectroscopy). In some examples, [SM]3FS devices in accordance with the present techniques may be implemented with only a standard epifluorescent microscope and a pressure controller -an advantage (compared to IR lasers, high NA objectives, cantilevers, and other custom-built equipment for existing SFMSPatent Application 33929 / 70818 technologies). Force application may also be simplified: force may be directly proportional to pressure applied by the pressure driven controller, allowing easy manipulation by nonspecialist. Data analysis involves only X-Y particle tracking, which is easier than other SMFS techniques that require Z-position tracking or a QPD for position detection. In sum, the [SM]3FS techniques herein allow for more efficient, accurate, and higher-throughput measurements than existing SMFS technologies.

[0026] The [SM]3FS techniques herein provide numerous advantages over an existing multiplexed force spectroscopy technique, such as, the method descried at doi.org / 10.1038 / nmeth.3099) which uses a programmable microfluidic chip to pattern an array of proteins for atomic force microscopy (AFM) measurements. Their approach demonstrates spatial multiplexing, but their method measures only one molecule at a time using a custom total internal reflection fluorescence-atomic force microscopy (TIRF-AFM) force spectroscopy setup, which was too inefficient to leverage desired throughput. As a result, the authors measured the behavior of only four proteins under force. In comparison, the present techniques have are able to apply force simultaneously across many samples and measure 10,000 single-molecule responses of 15 unique molecules in a 10-minute experiment. That is, the [SM]3FS techniques herein are (by comparison) simpler, cheaper, faster, and do not involve a custom TIRF-AFM setup that is limited to experts.Example [SM]3FS Device and Methods of Use

[0027] FIGS. 1A illustrates the resulting [SM]3FS device 100, in an example.

[0028] In the illustrated example, the [SM]3FS device 100 is a microfluidic platform capable of performing simultaneous single molecule analysis, for example, performing various methods described herein, including methods described in reference to FIG. 4, described further herein. As illustrated, the device 100 includes a plurality of microchannels formed on or in a substrate 104. In various examples, the width (across the substrate 104) and height (extending fully or partially above or below an upper surface of the substrate 104) is set such that each microchannel 102 is sized for immobilizing a respective bead-conjugated molecule. In various examples, microchannel widths can vary reasonably from 1-micron to 1-mm. The heights mayPatent Application 33929 / 70818 vary from a 1:10 to 1:1 ratio of the width. In an example experiment, the microchannel width was 150 microns, and they were 15 microns in height. As shown in FIG. IB, each microchannel 102 has a portion thereof that forms an image channel 102a extending between a respective microchannel inlet 102b and a microchannel outlet 102c. The image channels 102a may be parallel to one another, as shown, and may each extend parallel to an axis, F, defining a direction of microfluidic flow. The number of image channels 102a will depend upon the number of microchannels 102, and all or some portion of those image channels 102a are formed to be spatially confined within an image area 106 bounded by a field of view (FOV) of a microfluidic imager (not shown). This allows the sample fluid in each of the image channels to be simultaneously captured in a single image capture (or over a series of single image captures or video frames) of a standard microfluidic imager, such as a force spectroscopy imager. While the dimensions of the image area 106 may vary, in various examples, the image area corresponds to a FOV that is 100 mm2or less, and preferably 10 mm2or less, In yet other examples, larger FOVs may be used, namely 1000 mm2or less, such as preferably from 600 mm2to 900 mm2and more preferably from 700 mm2to 800 mm2

[0029] The microfluidic platforms herein may have linear or curvilinear shapes, when viewed from a top view. That is, while FIG. 1 illustrates linear shaped microchannels that extend parallel to a linear flow direction, F, in other examples, microchannels and their corresponding image channels may be curvilinear and thus, for example, parallel to a flow direction indicated by a radial axis. In some examples, such curvilinear shapes may be "S" shaped curves, or other multiple radius having curved shapes, any of which may be desired to control (i.e., slow) flow rates through the image area. In such examples, the microchannels may still be parallel to each other, while also being parallel to the curvilinear axis corresponding to the flow direction. While the device 100 is shown with 16 microchannels, the device may include any number of microchannels and preferably at least 10 microchannels.

[0030] Returning to FIG. 1A, in the illustrated example, the device 100 further includes a lead-in channel assembly 108 positioned between a platform inlet 110 and the plurality of microchannels 102 for providing surface chemistry to the microchannels 102. For example, the lead-in channel assembly 108 may be positioned to provide a parallelized surface chemistry toPatent Application 33929 / 70818 the microchannels 102 through a simultaneous force exertion across those microchannels 102. The platform inlet 110 may be general inlet that receives surface chemistry and feeds the same to a plurality of lead-in channels 108a forming the assembly 108. Such feeding may be controlled by a platform inlet valve 112. In various examples, the surface chemistry may contain one or more of a buffer, a patterning reagent, a passivation reagent, a protein, an antibody, an attachment chemistry, or a DNA attachment chemistry. Other example surface chemistries are provided herein.

[0031] In the illustrated example, the device 100 further includes an image area inlet valve assembly 114a positioned to controllably, simultaneously release the parallelized surface chemistry into the image channels 102a. An image area outlet valve assembly 114b may also be used and positioned to controllably retain parallelized surface chemistry in the image channels 102b. Collectively, the image area valve assemblies 114a and 114b formed the image valve 114 described herein. Further, device 100 includes an outlet valve assembly 116 positioned to selectively isolate each microchannel 102 from cross contamination from other microchannels. The outlet valve assembly 116 may be formed of multiple outlet valves 116a-116d, in some examples, as shown.

[0032] The image area inlet valve assembly 114a may be at (e.g., adjacent to, formed on, integrally formed with) the microchannel inlet 102b of each microchannel 102, for simultaneously controlling opening and closing of each image channel 102a of the microchannels 102. In some examples, the image area inlet valve assembly 114a is positioned at (e.g., adjacent to, formed on, integrally formed with) a distal end of the lead-in channel assembly 108. That is, the image area inlet value assembly 114a may be positioned upstream of the microchannel inlet 102b.

[0033] To introduce surface chemistry to each microchannel simultaneously, in some examples, the image area inlet valve assembly 114 (114a and / or 114b) may be formed a single controllable micro valve, such as a (de)pressurizable, deformable channel spanning the plurality of microchannelsln response to a pressurization or depresserization force, the valve is selectively opened or closed, simultaneously for each of the microchannels, and thus for eachPatent Application 33929 / 70818 of the respective image channels. . In some examples, the valve assemblies 114a and 114b are formed of corresponding micro valves, one at each of the microchannels 102.

[0034] Further, devices herein have an outlet valve assembly 116 that controls flow through an outlet end of the image channels. As illustrated in FIG. IB, the outlet valve assembly 116 may be formed of a plurality of micro valves 116a-116d, such that each image channel 102a has a respective micro valve 116 associated therewith. The micro valves 116 may be positioned at portions of the image channel that are downstream of the image area. Further, in some examples, the micro valves 116a-116d are longitudinally offset from adjacent micro valves along the axis, F. For example, as shown, each image channel has its micro valve positioned at a different distance from the microchannel inlet than the adjacent image channels. In the illustrated example, the micro valves 116 are positioned in a repeating cascading manner, such that each adjacent image channel 102a is associated with one of the 116a, 116b, 116c, or 116d micro valves.

[0035] As illustrated in FIG. 1A, in some examples, the lead-in channel assembly 108 includes lead in microchannels 120 each having an inlet end 120a for receiving a fluid. Each lead in microchannel 120 is fluidly coupled to the microchannel inlet 102b of a respective one of the microchannels 102 for feeding fluid into the respect microchannel. For example, each lead in microchannels 120 may be fluidly coupled to the platform inlet 110 for receiving the surface chemistry received at the platform inlet 110, as controlled by the platform inlet valve 112 that controllably releases surface chemistry into the device 100. To prevent backflow into the platform inlet 110, in the illustrated example, the lead-in channel assembly 108 further includes one or more backflow valves 121 which may be controllable valves or micron sized check valves that prevent backflow into the platform inlet 110.

[0036] In the illustrated example, the lead in microchannels 120 are spatially arranged in a fan-in pattern from an outer region 124 coinciding with inlets for the lead in microchannels 120 to an inner region 126 where each lead in microchannel 120 is coupled to respective microchannel 102. While lead in microchannels 120 are illustrated as linear in the fan-in pattern, in other examples, these lead in microchannels may be curvilinear or take on another shape.Patent Application 33929 / 70818

[0037] While surface chemistry is introduced into each of the lead in microchannels 120 through the shared platform inlet 110, to introduce specific fluids into each respective microchannel 120, each microchannel 120 is configured with a lane inlet 122 and a three-way lane valve 123 that controls the flow state of the microchannel. The lane inlets 122 receive respective fluids, such as different bead-conjugated molecules that are to be introduced to microchannels 102 and imaged across the image channels 102a of the image area 106. In various examples, the lane inlets receive respective molecules, which may include the same DNA in each lead in microchannel, different DNA, the same proteins in each lead in microchannel, different proteins, the same RNA in each lead in microchannel, different RNA, or other molecules to be imaged by the microfluidic imager.

[0038] To control fluid flow in the lead in microchannels 120, e.g., controllably flowing surface chemistry or bead-conjugated molecules therethrough, each lead in microchannel 120 may include a lane valve 123. In the illustrated example, the lane valves 123 are three-way valves having a first position that opens a flow path from the platform inlet 110 to the respective lead in microchannel 120 while closing a flow path from the respective lane inlet 122 to the respective lead in microchannel 120. The three-way valves have a second position that opens the flow path from the lane inlet 123 to the respective lead in microchannel 120 while closing the flow path form the platform inlet 110 to the same and a third position that closes both the flow path from the platform inlet 110 and the flow path from the lane inlet 123.

[0039] The channels of the device 100 may be formed of any suitable material for allowing surface chemistry, bead-conjugated molecules, or other sample fluids herein. For example, the microchannels 102, the lead-in channel assembly 108, the image area valve assemblies 114a and 114b, and the outlet valve assemblies 116a-116d may all be formed of a silicone polymer, preferably polydimethylsiloxane.

[0040] In various examples, devices herein may be configured for throughput operations that allow for more efficient removal of fluid imaged in the image area 106. For example, the device 100 is configured such that subsets of microfluidic outlets of microchannels may be paired with one another to form a plurality of platform outlets for expelling one or more bead-conjugated molecules. In the illustrated example, microchannel outlets are pared to form four fluid outletsPatent Application 33929 / 70818 130, each receiving fluid from four respective microchannels. The four-way parings each correspond to a different output valve assembly combination, A, B, C, and D.

[0041] Further, in some examples, one or more of the microchannels may be configured as a baseline channel that does not receive a fluid sample. For example, one or more valves forming the image area inlet valve assembly 114a and one or more valves forming the image area outlet valve assembly 114b are configured to control one or more of the plurality of microchannels 120 as a negative microchannel that does not receive the surface chemistry or a respective molecule.

[0042] Fig. 4 illustrates a process 200 for performing simultaneous single-molecule microfluidic imaging using a [SM]3FS device, such as the device 100 shown in FIG. 1. At a block 210, surface chemistry is provided to a general inlet of a lead-in channel assembly formed of a plurality of lead in microchannels each fl uidica lly coupled to a respective image channel portion of a microchannel. The image channels may extend parallel to an axis, F, defining a direction of microfluidic flow. Further, the image channels are spatially confined to an image area bounded by a FOV of a microfluidic imager for simultaneously imaging each image channel. At a block 220, a portion of the surface chemistry is retained, via an inlet valve at an inlet to each image channel, at a microchannel inlet to generate a pressurized buildup in the portion of the surface chemistry for release into each image channel. That pressurized buildup results from keeping the inlet valves at the inlet of the image channel from opening, for example. At a block 230, the inlet valves or the entire inlet valve assembly is controlled to simultaneously depressurize thereby simultaneously patterning each image channel with the surface chemistry. After each image channel has fully received the surface chemistry, at a block 240 the inlet valve for each image channel is repressurized. With the surface chemistry provided through each image channel from an inlet thereof to an outlet, at a block 250, each lead in microchannel of a lead-in channel assembly is provided with a respective molecule for imaging at a respective image channel. With the inlet valves repressurized, each respective molecule is retained at the inlet of each respective image channel. At the block 260, the method 200, in response to depressurizing the inlet valve, selectively, at each image channel, opens an outlet valve of the image channel while maintaining the outlet valves of each other image channel closed untilPatent Application 33929 / 70818 each image channel contains the respective molecule in isolation. That is, depressurizing an inlet valve for an image channel and opening an outlet valve of the same image channel, while maintaining all the inlet values pressurized and outlet valves closed, allows for selective introducing the respective molecule into each image channel, while avoiding cross contamination of either forward flow or backflow of molecules from other image channels from entering that channel.

[0043] At the block 270, after each image channel has been introduced with a respective molecule, a microfluidic imager performs simultaneous microfluidic imaging of the image area to capture image data of each respective molecule in the respect image channels at the same time, generating images such as those shown in FIGS. 2 and 3.

[0044] In some examples, prior to repressurizing the inlet valve at a block 240, a passivation agent is provided to the platform inlet. In some examples, under a pressurized control, beads may thus be passed through each image channel, where the pressurized control allows beads to bind to the surface chemistry along the entire length of each image channel.

[0045] In some examples, prior to providing the surface chemistry to the general inlet, at block 210, a backflow valve at the general inlet may be pressurized, and / or a lane valve at a lane inlet of each lead in microchannel may be pressurized, and / or the inlet valve at each image channel may be pressurized.

[0046] To control provisional of respective molecules at the block 250, in some examples the positions of each lane valve may be selectively controlled between a second position that opens the flow path from the lane inlet to the respective lead in microchannel while closing the flow path form the general inlet to the respective lead in microchannel and a third position that closes both the flow path from the platform inlet and the flow path from the lane inlet. For example, providing each lead in microchannel with a respective molecule may include opening the respective lane valve from the third position to the second position. Furthermore, the lane valve positions may be opened and maintained open to generate different pressures within different lead in microchannels. Thus, the process 200 can allow for not only introducing different molecules into different image channels, but also different pressure states within eachPatent Application 33929 / 70818 image channel. Indeed, such variability allows for introducing the same molecules into different image channels and maintaining them within those channels at different pressure levels based on the amount of molecule introduced as determined by the amount of molecule fluid flow and pressure buildup resulting from the inlet valve operation.

[0047] In some implementations of the block 230, simultaneously depressurizing the inlet valves at each image channel is performed after sufficient pressurized buildup in the portion of the surface chemistry to uniformly pattern the entire length of each image channel with the surface chemistry.

[0048] To create a baseline for comparison of molecule capture across the image channel, in some examples the process 200 further includes operating the [SM]3FS device with one or more image channels as a negative control image channel in the image area. These negative control image channels do not receive the surface chemistry or a respective molecule. For example, subsequent to the block 270, in analyzing the image data captured over the image area, the microfluidic imager or other computing system may analyze the image data captured from the one or more negative control image channels for identifying an indication in the image data of molecule cross contamination between the image channels. If the image data indicates the presence of a molecule in a negative control image channel, there has been cross contamination and the entire image data captured may be scraped and the device flushed for a subsequent fluid sample imaging.

[0049] To implement the block 270, in some examples, the process 200 includes applying a uniform force in-plane to the image area and measuring, as the image data, a fluorescence data or brightfield data of particle displacement in-plane over the image area. The process 200 may further include, at the block 720 or subsequent thereto, analyzing the fluorescence data or brightfield data to determine a responsiveness of each respective molecule in reach respective image channel to the applied uniform force.

[0050] We now describe an example [SM]3FS device and methods of use, in accordance with an example.Patent Application 33929 / 70818 Example Protocol (Method) Overview

[0051] We describe the following example method of fabrication of an [SM]3FS device in accordance with the present techniques: 1. Wafer and PDMS device fabrication; 2. Experiment setup (includes surface chemistry); 3. Experiment measurement; and 4. Data analysis.1. [SM]3FS wafer device and PDMS device fabrication

[0052] Overview: We used standard photolithography to pattern wafers with our device designs. We made multichannel PDMS devices using published Fordyce lab two-layer device fabrication protocols In particular, in this example, we applied a protocol following the supplementary methods of Markin et al. under "Photolithographic mold production" and "PDMS device fabrication": DOI: 10.1126 / science.abf8761.2. Experiment setup (includes surface chemistry)

[0053] Overview: This protocol details the preparation of the [SM]3FS devices for force spectroscopy experiments. The general workflow involved 1) patterning surface chemistry for immobilizing molecules across the entire device, 2) checking surface chemistry quality, 3) patterning unique molecules in each channel, and 4) attaching beads for force spectroscopy measurements.Protocol for performing sample analysis using the [SM]3FS device 100 included:1. Fill all valve control lines (including the control lines for each of the platform inlet valve 112, the backflow valves 121, the lane valves 123, the image area valve assembly 114, outlet valve assembly 116) with water at 10 PSI. Once full, change pressure to 25 PSI. 2. Once full, depressurize all control lines fed in step 1.3. Tie Tygon tubing and plug into the device outlets 130.4. Prepare anti-dig Fab. 1 uL of Fab + 19 uL of 0.1% tween20 in PBS + 20 uL PBS.

[0054] The Fab antibody concentration used here (5% v / v relative to tween) was important for zero background binding. We collected antibody titration data showing that 25% v / v Fab results in an optimal number of single molecule observations for 1-micron beads, and up to 33% Fab is tolerated before the surface begins to nonspecifically bind beads in the absence of DNA. We developed Monte Carlo simulations to help predict how changes in concentrationPatent Application 33929 / 70818 affect surface patterning. Our simulations showed that surface patterning density of single molecules resembles first-order kinetics and helps guide experimental design.5. Pressurize the backflow valves 121, the image area valves 114, and the lane inlets 122.6. Plug in Fab solution to the platform inlet 110. Pressurize to 750 mbar with Fluigent MFCS.7. Open the backflow valve(s) 121 and flow antibody solution to edge of the image area 106 (the image valve 114 remains closed).8. Depressurize the image valve 114 and pattern all of the image area 106, simultaneously, i.e., all of the image channels 102a.

[0055] These steps (6-8) were counterintuitive and generated unexpected results, to achieve high-quality, reproducible surface chemistry. The antibody solution was loaded up until the beginning of the imaging area. The opening of the image valve initiated patterning in the imaging area in FIG. 1 (e.g., the image channels 102a). The antibody was patterned at high pressure, 750 mbar, to reduce channel-to-channel variance. The patterning was not only 16x more efficient than patterning each channel individually, but it also reduced variance of surface patterning density between channels. Not following these steps resulted in heterogeneous surface chemistry that makes comparisons between channels difficult and lowers the overall yield of single molecule events (see FIG. 2 Illustrating the unexpected effects of implementing with steps 6-8 and without).

[0056] This advance in surface chemistry allowed for selecting optimal surface chemistry conditions (in step 4) by performing a surface chemistry titration experiment (i.e. patterning a unique antibody concentration in each channel instead of a unique DNA molecule: pattern antibody through channel-specific inlets, passivate with tween, immobilize DNA, then bind beads) (see FIG. 3).

[0057] The protocol continued as follows:9. Preserve freshly patterned surface. Pressurize the image valve 114, the backflow valve 121, and the platform inlet valve 112.10. Plug in 0.1% tween20 PBS in general inlet at 550 mbar. Depressurize all valves except lane valves. Flow tween20 solution across all channels and passivate for 1 minute.11. Pressurize the platform inlet 112. Tween20 solution is still plugged in.Patent Application 33929 / 70818 12. Flow beads to quantify background binding. 1 uL of 1-micron fluorescent green, streptavidin-coated beads + 50 uL tween20 solutions.13. Flow beads at 100 mbar.14. Let beads bind at 0 mbar.15. Flush away unbound beads with the tween solution at 100 mbar. Image in GFP to quantify the amount of non-specific bead binding. For a 5% v / v Fab-patterned surface, no beads should be bound.16. Keep the platform valve 112 open, with pressurized tween solution at 550 mbar.17. Pressurize the image valve 114 and the backflow valve 121.

[0058] In this example, we determined the backflow valve 121 must be closed here for robust molecule-to-channel patterning. The backflow valve 121 ensured minimal backflow-mediated cross contamination that could occur by molecules flowing up through the inlet tree and back down through a neighboring channel.18. Make sure all white 3-way valves are closed. Pressurize flow-layer pressure sources to 6 PSI.19. Mix 1 uL of varying DNA (at 40 ng / uL of DNA) in 60 uL of 1 mM MgCI2 borate buffer per channel. Each channel has a unique molecule sequence that is to be measured in subsequent steps. Plug in 30 uL of DNA to each of the 16 channels via the lane inlets. The last channel may be a DNA (-) control.

[0059] Typically, we reserve one channel as a negative control, either a DNA molecule lacking the attachment chemistry, DNA buffer, or protein buffer such as PURExpress, to allow direct quantification of background binding signal. This is an advance over existing techniques because since efficient surface chemistry generates 16 channels in parallel, it is reasonable to sacrifice 1 of the 16 channels as a negative control. Including an on-chip negative control is not required, of course, but the ability to build in negative controls in-parallel with the experimental protocol builds confidence in observed single-molecule behavior in comparison to a null control.20. With the lane valves 123 still closed, open the flow-layer 3-way lane valves 123 and pressurize channel-specific DNA solutions.21. With the image valve 114 and backflow valves 121 closed, open the lane valves 123. 22. Bubbles will appear near the microchannel inlets 120a. Wait 15 minutes for the bubbles to be dead-end filled.Patent Application 33929 / 70818

[0060] For this experimentation we determined that the image valves 114 and backflow valves 121 were to be closed when the lane valves 123 were opened due to some air being trapped in the lane-specific inlets. It is counterintuitive to purposely introduce air into the device (because bubbles destroy surface chemistry), but by introducing air in a controlled manner, pressing the bubbles out via dead-end filling prior to flowing over the preserved image area, ensures that there are no bubbles being introduced during the molecule patterning steps 23-33.23. Close the outlet valves 116 (labeled A-D).24. Open the image valve 114.25. Open the outlet valve A. Flow across A channels for 30 seconds.26. Close the outlet valve A.27. Open the outlet valve B. Flow across B channels for 30 seconds.28. Close the outlet valve B.29. Open the outlet valve C. Flow across C channels for 30 seconds.30. Close the outlet valve C.31. Open the outlet valve D. Flow across D channels for 30 seconds.32. Close the outlet valve D.

[0061] Outlet-specific outlets helped ensure robust channel-to-molecule coupling without cross-contamination.33. Close the image valve 114.34. Let DNA bind to surface chemistry for 20 minutes.35. Flow tween solution at 550 mbar and flush out unbound DNA.3. Experiment measurement

[0062] Overview: This protocol used pressure-induced flow to exert force on immobilized beads. Bead movements under force were measured using a standard epifluorescence microscope. The general workflow involved 1) ensuring equal resistances across channels, 2) using a programmable pressure controller to exert drag force on beads, and 3) acquiring data.Patent Application 33929 / 70818 Protocol:1. Plug in 0.1% tween 20 in PBS to the platform inlet 110. Or plug in beads containing a ligand of interest can also be included if making a transient binding measurement under force.2. Ensure that there are no tubes attached to the outlets. Let outlet fluid bead on device surface. Occasionally wick up excess fluid with a kirn wipe.3. Decrease control pressure to 10 PSI to ensure that resistance across all channels is equal.

[0063] Steps 2 and 3 were useful in ensuring low channel-to-channel variance for force application. Device-to-device variance of control layer-flow layer alignment can make resistance across channels uneven. Outlet tube height also impacts resistance and thus are not used to ensure the outlet height is kept constant across channels. The device design also helped ensure equal path lengths, and therefore, resistance, and steps 2 and 3 were also valuable for maintaining equal resistance. Equal resistances mean equal forces applied to molecules.4. For a force-ramp assay, program a linear pressure ramp with a Fluigent MFCS pressure controller. Alternatively, pressure can be held constant for a force-clamp assay.5. Start recording a video using the epifluorescent microscope to image the immobilized fluorescent beads under force. Image with a 4x objective with 1 by 1 binning for maximum spatial resolution.4. Data analysis

[0064] Overview: Open-source TrackMate software tracks many bead X-Y positions overtime in the recorded videos. Bead X-Y position data were analyzed using custom python codes. The bead position was used to identify molecular identity, as encoded during the channel patterning step.

[0065] One of the main differences from other analysis tools was that the bead position encodes molecule identity. Beads were grouped based on their X-position (indicating channel identity) then analyzed as batches for their mechanical properties.

[0066] Data analysis was straightforward to analyze since molecular movements were translated into changes in X-Y position, which allowed for using a standard, commercially available epifluorescence microscope instead of custom optics typically used by traditionalPatent Application 33929 / 70818 force spectroscopy approaches. Open-source software was also sufficient for accurate subpixel X-Y localization. The ease of analysis afforded by microfluidic force spectroscopy was important for efficient data collection and analysis.

[0067] As shown, in some examples, the micro valves herein (such as the image area valve assembly 114, the outlet valve assembly 116) are controlled by an electronic switch, which pressurizes the micro valve. The micro valve may be a channel that is positioned above the flow layer (at the inlet of the image channels), and when pressurized, deforms and cuts off flow in the channel below. One control channel can control multiple flow-layer valves at the same time. Uniform operation is achieved because the same control channel spans all of the parallelized flow channels. By turning off the imaging valve electronically, all of the on-chip valves associated with the image valve control channel are opened simultaneously. For homogenous surface chemistry, the image valve is opened once the antibody solution has completely filled the fan-in shaped inlet region of the device.

[0068] Further, in various examples, the plurality of microchannels, the lead-in channel assembly, the image area inlet valve assembly, and the image area outlet valve assembly may be formed of a silicone polymer, preferably polydimethylsiloxane (PDMS). It will be appreciated that other materials that provide the following design characteristics may be used: 1) optical transparency, 2) deformability (for valves to function properly), and 3) adhesive to glass.

[0069] Various examples are provided showing 10 or more microchannels (and image channels), such as 16 microchannels. The length of the image channels may be set by the total FOV: if the FOV is 100 mm2, at max, the channel length may be 10 mm long, for example. For a FOV that is at 1000 mm2, at max, the channel length by 100 mm long, for example. With the present techniques, we can fit many channels into a single FOV. However, the geometry of fitting all of the channel-specific inlets becomes challenging (space becomes limited on chip), with up to and including 100 image channels achievable with the present techniques.

[0070] The examples described herein include force spectroscopy, where force is exerted inplane with the image plane of the image area, and particle displacement is measured also inplane with the image plane. Captured fluorescence data or bright field day may be used toPatent Application 33929 / 70818 image in the X-Y orientation allows the FOV while not sacrificing positional resolution. By analyzing the fluorescence data or bright field data, we can determine a responsiveness of each respective molecule in each respective image channel to the applied uniform force. For example, we can measure displacement in response to force (for instance, DNA unfolding, DNA stretching). We can also measure bond lifetimes, i.e., how long two molecules interact with one another under force. We can perform force ramp and force clamp assays.

[0071] As noted above, FIG. 3 is a fluorescence image of 16 image channels, confined to an image area, of an example [SM]3FS device, for example the device 100, showing . FIG. 5 illustrates a florescence image of 16 image channels, confined to an image area, of an example [SM]3FS device, showing, as with the image of FIG. 3, the concentration of molecules in the respective image channels. FIG. 5 further indicates the concentrations of Fab tuned density of beads immobilized by dig-bio DNA, per image channel, in an example use of the [SM]3FS device. FIG. 6 is a table indicating differences in throughput in various force spectroscopy techniques compared to [SM]3FS. As listed, the techniques herein are able to image and analyze multiple sequences at one time, over a sufficiently large FOV (e.g., 10 mm2to 20 mm2), with a sufficiently fine resolution (e.g., 20 nm - 100 nm, 30 nm - 100 nm, 40 nm - 100 nm, 50 nm - 100 nm, etc.), thereby tracking over 10,000 beads per image capture.

[0072] FIG. 1C illustrates inlet and outlet connections for various features of the [SM]3FS device 100, thus illustrating an example of microfluidic connections to the device 100 during operation. Various respective valves are not shown. In FIG. 1C, each lane inlets 122 for example is connect to a respective controllable microinjector 150 providing a molecule for imaging at a respective image channel, each of the outlets 130 are connected to a microinjector 160 controlled to exert a negative pressure that removes fluid from the outlets 130. The platform inlet 110 is connected to a microinjector 170 and, in the illustrated example, the valves 112, 121, 123, 114, and 116 are each connected to respective controllable microinjectors 180, 182, 184, 186, and 188, for controlling operation of the respective valves.Patent Application 33929 / 70818 Additional Considerations

[0073] Aspect 1. A microfluidic platform for performing simultaneous single molecule analysis, the platform comprising: a substrate; a plurality of microchannels formed on or in the substrate, each microchannel sized for immobilizing a respective bead-conjugated molecule, each microchannel comprising an image channel extending between a microchannel inlet and a microchannel outlet, wherein each image channel extends parallel to an axis defining a direction of microfluidic flow, wherein the plurality of image channels are spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel; a lead-in channel assembly positioned between a platform inlet and the plurality of microchannels for providing a parallelized surface chemistry to the plurality of microchannels through a simultaneous force exertion across the plurality of microchannels; an image area inlet valve assembly positioned to simultaneously release the parallelized surface chemistry into the plurality of image channels; and an image area outlet valve assembly positioned to selectively isolate each microchannel from molecule cross contamination.

[0074] Aspect 2. The microfluidic platform of aspect 1, wherein the image area inlet valve assembly is positioned at the microchannel inlet of each of the plurality of microchannels.

[0075] Aspect 3. The microfluidic platform of aspect 1, wherein the image area inlet valve assembly is positioned at the lead-in channel assembly.

[0076] Aspect 4. The microfluidic platform of aspect 1, wherein the image area inlet valve assembly comprises a single controllable micro valve, wherein the single controllable micro valve is a (de)pressurizable, deformable channel spanning the plurality of microchannels.

[0077] Aspect 5. The microfluidic platform of aspect 1, wherein the image area outlet valve assembly comprises a plurality of micro valves, each micro valve assigned to a different image channel.

[0078] Aspect 6. The microfluidic platform of aspect 5, wherein each micro valve is longitudinally offset along the axis from the micro valve assigned to an adjacent image channel.

[0079] Aspect 7. The microfluidic platform of aspect 1, wherein the lead-in channel assembly comprises a plurality of lead in microchannels each having an inlet for receiving a respectivePatent Application 33929 / 70818 molecule and each fluidly coupled to the microchannel inlet of a respective one of the plurality of microchannels for feeding the respective molecule into the respect microchannel.

[0080] Aspect 8. The microfluidic platform of aspect 7, wherein the plurality of lead in microchannels are fluidly coupled to the platform inlet for receiving the surface chemistry received at the platform inlet.

[0081] Aspect 9. The microfluidic platform of aspect 8, wherein the lead-in channel assembly further comprises a platform inlet valve at the platform inlet for controllably releasing the surface chemistry received at the platform inlet into each of the lead in microchannels.

[0082] Aspect 10. The microfluidic platform of aspect 9, wherein the lead-in channel assembly further comprises a plurality of backflow valves one for each lead in microchannel and controllable to prevent backflow into the lead in microchannel platform inlet valve for controllably releasing the surface chemistry received at the platform inlet into each of the lead in microchannels.

[0083] Aspect 11. The microfluidic platform of aspect 7, wherein the plurality of lead in microchannels are spatially arranged in a fan-in pattern from an outer region of the lead-in channel assembly coinciding with the inlets for the lead in microchannels to an inner region of the lead-in channel assembly coupled to the plurality of microchannels.

[0084] Aspect 12. The microfluidic platform of aspect 1, wherein the plurality of microchannels, the lead-in channel assembly, the image area inlet valve assembly, and the image area outlet valve assembly are formed of a silicone polymer, preferably polydimethylsiloxane.

[0085] Aspect 13. The microfluidic platform of aspect 1, wherein subsets of the plurality of microfluidic outlets are paired with one another to form a plurality of platform outlets for expelling one or more bead-conjugated molecules.

[0086] Aspect 14. The microfluidic platform of aspect 1, wherein one or more of the image area inlet valve assembly and the image area outlet valve assembly are configured to control one or more of the plurality of microchannels as negative microchannels that do not receive the surface chemistry or a respective molecule.Patent Application 33929 / 70818

[0087] Aspect 15. The microfluidic platform of aspect 1, wherein the FOV is 100 mm2 or less.

[0088] Aspect 16. The microfluidic platform of aspect 1, wherein the FOV is 10 mm2 or less.

[0089] Aspect 17. The microfluidic platform of aspect 1, wherein microfluidic imager is a force spectroscopy imager.

[0090] Aspect 18. The microfluidic platform of aspect 1, wherein the axis defining the direction if microfluidic flow is a radial axis and wherein the image channels are curvilinear parallel to the radial axis.

[0091] Aspect 19. The microfluidic platform of aspect 1, wherein the plurality of microchannels comprises at least 10 microchannels.

[0092] Aspect 20. A method for simultaneous single-molecule microfluidic imaging, the method comprising: providing a surface chemistry to a general inlet of a lead-in channel assembly comprising a plurality of lead in microchannels each fluidically coupled to a respective image channel of a plurality of image channels, the plurality of image channels extending parallel to an axis defining a direction of microfluidic flow and the plurality of image channels being spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel; retaining, via an inlet valve at an inlet to each image channel, a portion of the surface chemistry to generate a pressurized buildup in the portion of the surface chemistry; simultaneously depressurizing the inlet valve at each image channel to simultaneously pattern each image channel with the surface chemistry; repressurizing the inlet valve at each image channel; providing to each lead in microchannel of the lead-in channel assembly a respective molecule for imaging at a respective image channel, wherein each respective molecule is retained at each respective image channel via the repressurizing of the inlet valve; in response to depressurizing the inlet valve, selectively, at each image channel, opening an outlet valve of the image channel while maintaining an outlet valve of each other image channel closed until each image channel contains the respective molecule in isolation; and performing a simultaneous microfluidic imaging of the image area to capture image data of each respective molecule in the respect image channels.Patent Application 33929 / 70818

[0093] Aspect 21. The method of aspect 20, further comprising, prior to repressuring the inlet valve: providing a passivation agent, for example tween20, to the general inlet, and under a pressurized control, flowing beads through each image channel, where the pressurized control allows beads to bind to the surface chemistry along the entire length of each image channel.

[0094] Aspect 22. The method of aspect 20, further comprising: prior to providing the surface chemistry to the general inlet, pressurizing a backflow valve at the general inlet, pressurizing a lane valve at a lane inlet of each lead in microchannel, and pressurizing the inlet valve at each image channel.

[0095] Aspect 23. The method of aspect 22, wherein each lane valve is a three-way valve having a first position that opens a flow path from the general inlet to the respective lead in microchannel while closing a flow path from the lane inlet to the respective lead in microchannel, a second position that opens the flow path from the lane inlet to the respective lead in microchannel while closing the flow path form the general inlet to the respective lead in microchannel, and a third position that closes both the flow path from the general inlet and the flow path from the lane inlet.

[0096] Aspect 24. The method of aspect 23, wherein providing to each lead in microchannel the respective molecule further comprises opening each lane valve from the third position to the second position.

[0097] Aspect 25. The method of aspect 23, wherein providing to each lead in microchannel the respective molecule further comprises selectively opening each lane valve from the third position to the second position to generate different pressures within different lead in microchannels.

[0098] Aspect 26. The method of aspect 20, wherein simultaneously depressurizing the inlet valve at each image channel is performed after sufficient pressurized buildup in the portion of the surface chemistry to uniformly pattern the entire length of each image channel with the surface chemistry.Patent Application 33929 / 70818

[0099] Aspect 27. The method of aspect 20, further comprising providing one or more negative control image channels in the image area, wherein the one or more negative control image channels do not receive the surface chemistry or a respective molecule.

[0100] Aspect 28. The method of aspect 27, further comprising analyzing the image data captured from the one or more negative control image channels for an indication of molecule cross contamination between the image channels.

[0101] Aspect 29. The method of aspect 20, wherein performing the simultaneous microfluidic imaging of the image area comprises: applying a uniform force in-plane to the image area; and measuring, as the image data, a fluorescence data or brightfield data of particle displacement in-plane over the image area.

[0102] Aspect 30. The method of aspect 29, further comprising analyzing the fluorescence data or brightfield data to determine a responsiveness of each respective molecule in reach respective image channel to the applied uniform force.

[0103] Aspect 31. The method of aspect 20, wherein each respective molecule comprises different DNA.

[0104] Aspect 32. The method of aspect 20, wherein each respective molecule comprises a same DNA, proteins under force, or RNA.

[0105] Aspect 33. The method of aspect 20, wherein the surface chemistry comprises one or more of a buffer, a patterning reagent, a passivation reagent, a protein, an antibody, an attachment chemistry, or a DNA attachment chemistry.

[0106] Aspect 34. The method of aspect 20, wherein the FOV is 100 mm2 or less.

[0107] Aspect 35. The method of aspect 34, wherein the FOV is 10 mm2 or less.

[0108] Aspect 36. The method of aspect 20, wherein the plurality of image channels comprises at least 10 image channels.

[0109] Aspect 37. The method of aspect 36, wherein the plurality of image channels comprises at least 16 image channels.Patent Application 33929 / 70818

[0110] Aspect 38. A microfluidic platform for performing simultaneous single molecule analysis, the platform comprising: a substrate; a lead-in channel assembly formed on the substrate and comprising a plurality lead-in microchannels in a fan-in configuration, each lead-in microchannel having (i) a first switchable state in which an isolated surface chemistry is fed through the lead-in microchannel to a plurality of image microchannels and (ii) a second switchable state in which a different isolated molecule is fed through each different lead-in microchannel to the plurality of image microchannels; and a micro valve system having a first switchable state in which surface chemistry fed through the lead-in microchannels is pressurized against entering the plurality of image microchannels, a second switchable state in which surface chemistry fed through the lead-in microchannels is released into the plurality of image microchannels to uniformly pattern the surface chemistry in each image microchannel, and a third switchable state in which the different isolated molecules are fed into each different image microchannel for binding to the surface chemistry while preventing molecule cross contamination between image microchannels, wherein the plurality of image microchannels are formed on the substate and spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel.

[0111] Unless specifically stated otherwise, discussions herein using words such as "processing," "computing," "calculating," "determining," "presenting," "displaying," or the like may refer to actions or processes of a machine (e.g., a computer) that manipulates or transforms data represented as physical (e.g., electronic, magnetic, or optical) quantities within one or more memories (e.g., volatile memory, non-volatile memory, or a combination thereof), registers, or other machine components that receive, store, transmit, or display information.

[0112] As used herein, any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0113] Some embodiments may be described using the expression "coupled" and "connected" along with their derivatives. For example, some embodiments may be describedPatent Application 33929 / 70818 using the term "coupled" to indicate that two or more elements are in direct physical or electrical contact. The term "coupled," however, may also mean that two or more elements are not in direct contact with each other, but yet still co-operate or interact with each other. The embodiments are not limited in this context.

[0114] Those skilled in the art will recognize that a wide variety of modifications, alterations, and combinations can be made with respect to the above described embodiments without departing from the scope of the invention, and that such modifications, alterations, and combinations are to be viewed as being within the ambit of the inventive concept.

[0115] While the present invention has been described with reference to specific examples, which are intended to be illustrative only and not to be limiting of the invention, it will be apparent to those of ordinary skill in the art that changes, additions and / or deletions may be made to the disclosed embodiments without departing from the spirit and scope of the invention.

[0116] The foregoing description is given for clearness of understanding; and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

Claims

1. Patent Application 33929 / 70818 WHAT IS CLAIMED:

1. A microfluidic platform for performing simultaneous single molecule analysis, the platform comprising:a substrate;a plurality of microchannels formed on or in the substrate, each microchannel sized for immobilizing a respective bead-conjugated molecule, each microchannel comprising an image channel extending between a microchannel inlet and a microchannel outlet, wherein each image channel extends parallel to an axis defining a direction of microfluidic flow, wherein the plurality of image channels are spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel;a lead-in channel assembly positioned between a platform inlet and the plurality of microchannels for providing a parallelized surface chemistry to the plurality of microchannels through a simultaneous force exertion across the plurality of microchannels;an image area inlet valve assembly positioned to simultaneously release the parallelized surface chemistry into the plurality of image channels; andan image area outlet valve assembly positioned to selectively isolate each microchannel from molecule cross contamination.

2. The microfluidic platform of claim 1, wherein the image area inlet valve assembly is positioned at the microchannel inlet of each of the plurality of microchannels.

3. The microfluidic platform of claim 1, wherein the image area inlet valve assembly is positioned at the lead-in channel assembly.

4. The microfluidic platform of claim 1, wherein the image area inlet valve assembly comprises a single controllable micro valve, wherein the single controllable micro valve is a (de)pressurizable, deformable channel spanning the plurality of microchannels.Patent Application 33929 / 70818 5. The microfluidic platform of claim 1, wherein the image area outlet valve assembly comprises a plurality of micro valves, each micro valve assigned to a different image channel.

6. The microfluidic platform of claim 5, wherein each micro valve is longitudinally offset along the axis from the micro valve assigned to an adjacent image channel.

7. The microfluidic platform of claim 1, wherein the lead-in channel assembly comprises a plurality of lead in microchannels each having an inlet for receiving a respective molecule and each fluidly coupled to the microchannel inlet of a respective one of the plurality of microchannels for feeding the respective molecule into the respect microchannel.

8. The microfluidic platform of claim 7, wherein the plurality of lead in microchannels are fluidly coupled to the platform inlet for receiving the surface chemistry received at the platform inlet.

9. The microfluidic platform of claim 8, wherein the lead-in channel assembly further comprises a platform inlet valve at the platform inlet for controllably releasing the surface chemistry received at the platform inlet into each of the lead in microchannels.

10. The microfluidic platform of claim 9, wherein the lead-in channel assembly further comprises a plurality of backflow valves one for each lead in microchannel and controllable to prevent backflow into the lead in microchannel platform inlet valve for controllably releasing the surface chemistry received at the platform inlet into each of the lead in microchannels.

11. The microfluidic platform of claim 7, wherein the plurality of lead in microchannels are spatially arranged in a fan-in pattern from an outer region of the lead-in channel assembly coinciding with the inlets for the lead in microchannels to an inner region of the lead-in channel assembly coupled to the plurality of microchannels.Patent Application 33929 / 70818 12. The microfluidic platform of claim 1, wherein the plurality of microchannels, the lead-in channel assembly, the image area inlet valve assembly, and the image area outlet valve assembly are formed of a silicone polymer, preferably polydimethylsiloxane.

13. The microfluidic platform of claim 1, wherein subsets of the plurality of microfluidic outlets are paired with one another to form a plurality of platform outlets for expelling one or more bead-conjugated molecules.

14. The microfluidic platform of claim 1, wherein one or more of the image area inlet valve assembly and the image area outlet valve assembly are configured to control one or more of the plurality of microchannels as negative microchannels that do not receive the surface chemistry or a respective molecule.

15. The microfluidic platform of claim 1, wherein the FOV is 1000 mm2or less.

16. The microfluidic platform of claim 1, wherein the FOV is 100 mm2or less.

17. The microfluidic platform of claim 1, wherein microfluidic imager is a force spectroscopy imager.

18. The microfluidic platform of claim 1, wherein the axis defining the direction if microfluidic flow is a radial axis and wherein the image channels are curvilinear parallel to the radial axis.

19. The microfluidic platform of claim 1, wherein the plurality of microchannels comprises at least 10 microchannels.

20. A method for simultaneous single-molecule microfluidic imaging, the method comprising:Patent Application 33929 / 70818 providing a surface chemistry to a general inlet of a lead-in channel assembly comprising a plurality of lead in microchannels each fl uidica lly coupled to a respective image channel of a plurality of image channels, the plurality of image channels extending parallel to an axis defining a direction of microfluidic flow and the plurality of image channels being spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel;retaining, via an inlet valve at an inlet to each image channel, a portion of the surface chemistry to generate a pressurized buildup in the portion of the surface chemistry;simultaneously depressurizing the inlet valve at each image channel to simultaneously pattern each image channel with the surface chemistry;repressurizing the inlet valve at each image channel;providing to each lead in microchannel of the lead-in channel assembly a respective molecule for imaging at a respective image channel, wherein each respective molecule is retained at each respective image channel via the repressurizing of the inlet valve;in response to depressurizing the inlet valve, selectively, at each image channel, opening an outlet valve of the image channel while maintaining an outlet valve of each other image channel closed until each image channel contains the respective molecule in isolation; and performing a simultaneous microfluidic imaging of the image area to capture image data of each respective molecule in the respect image channels.

21. The method of claim 20, further comprising, prior to repressuring the inlet valve:providing a passivation agent, for example tween20, to the general inlet, and under a pressurized control, flowing beads through each image channel, where the pressurized control allows beads to bind to the surface chemistry along the entire length of each image channel.

22. The method of claim 20, further comprising:prior to providing the surface chemistry to the general inlet, pressurizing a backflow valve at the general inlet, pressurizing a lane valve at a lane inlet of each lead in microchannel, and pressurizing the inlet valve at each image channel.Patent Application 33929 / 7081823. The method of claim 22, wherein each lane valve is a three-way valve having a first position that opens a flow path from the general inlet to the respective lead in microchannel while closing a flow path from the lane inlet to the respective lead in microchannel, a second position that opens the flow path from the lane inlet to the respective lead in microchannel while closing the flow path form the general inlet to the respective lead in microchannel, and a third position that closes both the flow path from the general inlet and the flow path from the lane inlet.

24. The method of claim 23, wherein providing to each lead in microchannel the respective molecule further comprises opening each lane valve from the third position to the second position.

25. The method of claim 23, wherein providing to each lead in microchannel the respective molecule further comprises selectively opening each lane valve from the third position to the second position to generate different pressures within different lead in microchannels.

26. The method of claim 20, wherein simultaneously depressurizing the inlet valve at each image channel is performed after sufficient pressurized buildup in the portion of the surface chemistry to uniformly pattern the entire length of each image channel with the surface chemistry.

27. The method of claim 20, further comprising providing one or more negative control image channels in the image area, wherein the one or more negative control image channels do not receive the surface chemistry or a respective molecule.

28. The method of claim 1 , further comprising analyzing the image data captured from the one or more negative control image channels for an indication of molecule cross contamination between the image channels.Patent Application 33929 / 7081829. The method of claim 20, wherein performing the simultaneous microfluidic imaging of the image area comprises:applying a uniform force in-plane to the image area; andmeasuring, as the image data, a fluorescence data or brightfield data of particle displacement in-plane over the image area.

30. The method of claim 29, further comprising analyzing the fluorescence data or brightfield data to determine a responsiveness of each respective molecule in reach respective image channel to the applied uniform force.

31. The method of claim 20, wherein each respective molecule comprises different DNA.

32. The method of claim 20, wherein each respective molecule comprises a same DNA, proteins under force, or RNA.

33. The method of claim 20, wherein the surface chemistry comprises one or more of a buffer, a patterning reagent, a passivation reagent, a protein, an antibody, an attachment chemistry, or a DNA attachment chemistry.

34. The method of claim 20, wherein the FOV is 100 mm2or less.

35. The method of claim 34, wherein the FOV is 10 mm2or less.

36. The method of claim 20, wherein the plurality of image channels comprises at least 10 image channels.

37. The method of claim 36, wherein the plurality of image channels comprises at least 16 image channels.Patent Application 33929 / 7081838. A microfluidic platform for performing simultaneous single molecule analysis, the platform comprising:a substrate;a lead-in channel assembly formed on the substrate and comprising a plurality lead-in microchannels in a fan-in configuration, each lead-in microchannel having (i) a first switchable state in which an isolated surface chemistry is fed through the lead-in microchannel to a plurality of image microchannels and (ii) a second switchable state in which a different isolated molecule is fed through each different lead-in microchannel to the plurality of image microchannels; anda micro valve system having a first switchable state in which surface chemistry fed through the lead-in microchannels is pressurized against entering the plurality of image microchannels, a second switchable state in which surface chemistry fed through the lead-in microchannels is released into the plurality of image microchannels to uniformly pattern the surface chemistry in each image microchannel, and a third switchable state in which the different isolated molecules are fed into each different image microchannel for binding to the surface chemistry while preventing molecule cross contamination between image microchannels,wherein the plurality of image microchannels are formed on the substate and spatially confined to an image area bounded by a field of view (FOV) of a microfluidic imager for simultaneously imaging each image channel.

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