WO2016087460A1 - Quantitating analytes in a flow cell via electrical measurements - Google Patents

Abstract

A method of detecting an analyte includes the steps of: decorating an analyte bound to an object with one or more conductive detection probes; causing the object to move through a microfluidic channel, the microfluidics channel having a pair of contacts; measuring an electrical parameter within a circuit coupled to the contacts that is completed when the object passes between the pair of opposing contacts; and determining an electrical property of the object based on the measured electrical parameter, wherein the electrical property indicates a presence of the analyte.

Description

QUANTITATING ANALYTES IN A FLOW CELL VIA ELECTRICAL MEASUREMENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to US Provisional Patent Application No. 62/085,749, filed December 1, 2014, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[2] This application relates to methods for quantitating analyte levels using electrical measurements such as conductance.

INTRODUCTION

[3] Over the past two decades the biomedical research field has witnessed technological breakthroughs that have enabled quantification of analytes such as proteins, DIMA, or RNA on very large scales.

[4] In assays that have been developed prior to that, readouts largely consisted of optical signals, whether by optical density (Enzyme Linked Immunosorbent Assay, ELISA), fluorescence intensity (Polymerase Chain Reaction, PCR) or the image of a gel (Sanger sequencing). Notably, optical readouts have remained the norm for a host of high throughput profiling technologies, such as microarrays that rely on sequences bound on a solid substrate to capture by hybridized complementary labeled sequences in solution. Radioactive labels that were initially used were soon replaced by fluorescent labels that could be excited by a laser and imaged using a high resolution scanner/imager.

[5] Thus, correlating fluorescence intensity with the amount of bound analytes makes it possible to obtain an analog quantification of analytes present in the sample. Technologies have been introduced more recently that allow sequencing of oligonucleotides by synthesis (so called next-generation sequencing technologies). This enables actual counting of molecules that are present in a sample (after preparation of DNA libraries). Once again, the technologies currently in use generally rely on optical signals and the use of fluorescent labels.

[6] Approaches relying on optical signals often have drawbacks in certain applications. For example, the systems tend to be large, cumbersome, and expensive, as they rely on sophisticated lasers, optical systems, and imaging capture devices, and require large data storage capacity to be able to handle the production of large image files, and may not be suitable for field diagnostic applications.

SUMMARY

[7] Accordingly, described herein are methods and systems for using electrical

measurements (e.g. conductance) to quantitate analytes, for example in a flow cell which in some embodiments is implemented using a microfluidics device.

[8] In one embodiment, the invention provides a method of detecting an analyte. The method includes the steps of: decorating an analyte bound to an object with one or more conductive detection probes; causing the object to move through a microfluidic channel, the microfluidics channel having a pair of contacts; measuring an electrical parameter within a circuit coupled to the contacts that is completed when the object passes between the pair of opposing contacts; and determining an electrical property of the object based on the measured electrical parameter, wherein the electrical property indicates a presence of the analyte.

[9] In another embodiment, the invention provides a system for detecting an analyte. The system includes: a microfluidic channel having a first pair of contacts disposed adjacent thereto on opposing sides of the microfluidic channel; a first measurement circuit coupled to the first pair of contacts and configured to complete the first measurement circuit when an object having an analyte decorated with one or more conductive detection probes flows through the microfluidic channel between the first pair of contacts; and a controller in operative communication with the first measurement circuit. The controller is configured to use the first measurement circuit to measure an electrical parameter when the object passes between the first pair of contacts, and determine an electrical property of the object based on the measured electrical parameter, wherein the electrical property indicates a presence of the analyte.

[10] In still another embodiment, the invention provides a system for quantitating an analyte. The system includes: a microfluidics channel; a first measurement circuit, the first measurement circuit including a first set of contacts positioned adjacent the microfluidics channel and configured to complete the first measurement circuit when an object having an analyte decorated with one or more conductive detection reagents flows through the microfluidics channel between the first set of contacts; a second measurement circuit, the second measurement circuit including a second set of contacts positioned adjacent the microfluidics channel and configured to complete the second measurement circuit when an object having an analyte decorated with one or more conductive detection reagents flows through the microfluidics channel and contacts the second set of metal contacts; and a controller in operative communication with the first measurement circuit and the second measurement circuit. The controller is configured to: use the first measurement circuit to measure a first electrical parameter when the object passes between the first pair of contacts, use the second measurement circuit to measure a second electrical parameter when the object passes between the second pair of contacts, determine an electrical property of the object based on the first measured electrical parameter and the second measured electrical parameter, and quantitate the analyte based on the electrical property.

INCORPORATION BY REFERENCE

[11] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. BRIEF DESCRIPTION OF THE DRAWINGS

[12] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[13] FIG. 1 is a diagram illustrating the binding of a decorated analyte to an object.

[14] FIG. 2 is a diagram illustrating different binding configurations between decorated analytes to an object.

[15] FIG. 3 is a diagram illustrating a system for measuring a conductance of an analyte.

[16] FIG. 4 is a diagram illustrating a system for measuring a conductance of an analyte. [17] FIG. 5 is a diagram illustrating a system for measuring a conductance of an analyte having multiple measurement circuits.

[18] FIG. 6 is a diagram illustrating various applications of an analyte conductance measurement system.

[19] FIGS. 7A-7F are diagrams illustrating procedures for measuring multiple analytes in single cells.

[20] FIGS. 8A-8D are diagrams illustrating measurement of electrical conductance to detect and quantify the presence of analytes in cell culture supernatants.

[21] FIGS. 9A-9B are diagrams illustrating procedures for sorting of cells that, as measured by electrical conductance, are either positive or negative for a given marker.

DETAILED DESCRIPTION

[22] As discussed above, there are a number of deficiencies with current technologies for measuring analytes, many of which require sophisticated and cumbersome optical devices for obtaining measurements. While there has been some development of sample analysis technologies which do not require optical detection methods, these technologies have a number of downsides such as complex sample preparation protocols and/or the use of sophisticated detection procedures such as mass spectrometry. These other technologies, while removing the requirement for optical detection, nevertheless would not be suitable for producing a compact and portable field-deployable unit.

[23] Accordingly, provided herein are systems and methods for detecting and quantitating analytes by measuring the electrical signal generated by the flow of bound conductance objects in a microfluidics channel having one or more pairs of electrical contacts. In particular, the system may map or convert a measured electrical signal to an abundance level of an analyte in a manner that is compatible with detection in a microfluidics chip, which in some embodiments may be part of a portable field-deployable unit.

[24] The use of microfluidics chips in conjunction with an electrical signal readout provides solutions to the limitations of known systems, including challenges posed both by sample handling, bulkiness, and cost of the instrumentation as well as assay costs inherent in current assays that rely on optical readout. The advantages of the present invention make it particularly useful in developing countries where field applications are the most widespread, or as an in-home diagnostic and health monitoring device. Though "lab-on-a-chip" applications allow for the manipulation of minute amounts of liquids (e.g. in the nanoliter range) or individual cells, and such applications have been developed extensively, the optical read-outs that are typically used in such devices have largely prevented their use in field applications. The present invention takes advantage of highly-controllable liquid flow, which provides the ability to seamlessly transfer and mix reagents, carry out incubation steps, and make measurements within the confines of a programmable microfluidic chip. In addition, it provides for analyte detection using a relatively small device. Such systems provide a solution for fast, affordable point of care diagnosis via portable handheld devices.

[25] In preferred embodiments, the disclosed system performs the following operations: decorating analytes that are physically bound to an object (e.g. a bead or a cell) with metal nanoparticle-labeled detection reagents, flowing the object through a microfluidics channel lined with a series of pairs of contacts connected to an external circuit and separated only by the gap formed by this channel (e.g. the object flowing past the contacts effectively creates a series of electrical switches); measuring the electrical current passing through the circuit at the moment when the object flows through the channel between the electrical contacts, and determining the electrical conductance of the object. From these measurements, quantitative values corresponding to absolute or relative amounts or concentrations of the analyte being measured can be produced (e.g. by comparing to signals obtained using a control sample or analyte).

[26] Analyte: The systems and methods provided herein allow for the measurement of a wide variety of different types of analytes, including, but not limited to 1) RNA species, including messenger RNA (mRNA), non coding RNAs (such as microRNAs, long non coding RNAs) that can be present in cell preparations or in body fluids; 2) Proteins, whether at the cell surface, intracellular, or in solution (e.g. body fluids, culture supernantants or cell lysates); 3) DNA variants, specifically for the determination of Single Nucleotide Polymorphisms (SNPs); 4) As described below, viable strategies can also be implemented for the detection in body fluids of metabolites such as glucose or lipids. The system can also be used for counting cells, which is an important parameter for the assessment of the health of an individual.

[27] Object: The object flowing through the microfluidic chip can be a live or fixed cell (e.g. blood leukocytes such as T-cells, B-cells, Monocytes, Neutrophils; or circulating cancer cells; or cellular suspensions obtained from buccal swabs), a bead (also referred to as microbead or microsphere) or rod (cylindrical bead), or any object of a fixed dimension or range of dimensions that can be transported in a fluidic stream and aligned so that it passes in single file past the electrical contacts for detection of changes in electrical currents. In general, the object can be made of any material having an intrinsic conductance which differs from that of the material coupled with the detection probe. Binding of the detection probe will modify the net conductance of the object such that the measured difference can be correlated with abundance of the analyte that is bound to the surface of the object.

[28] The object that is moved past the detector may be made of a material with low conductance. Suitable objects include microbeads or microspheres which are commercially available may be made from a wide range of materials such as polystyrene (used in the multiplexing assay developed by Luminex Corp. (Dunbar and Jacobson 2007)), nitrocellulose, polymethylmethacrylate, poly(D,L-lactide-co-glycolide), poly(E-caprolactone), ceramics, latex or silica (the later being used in the BeadArray and VeraCode technology commercialized by lllumina inc. (Lin et al. 2009)). Commercial sources for the procurement of microbeads or microspheres include: Phosphorex, Hopkinton MA; Bangs Laboratories, Fishers, IN; and

Cospheric, Santa Barbara CA. The beads can be further "functionalized" by chemical treatment to enable covalent attachment of proteins, peptides, nucleic acids and other ligands (e.g. by attachment to commercially-available products such as Carboxyl Latex Beads, from Life Technologies, Carlsbad, CA, Catalog number C37278).

[29] Detection probes: Detection probes bind the analyte and "decorate" objects with material that is designed to modify its conductance. In various embodiments, particles of conductive materials (e.g. metals such as gold or silver) are used in combination with objects that have low intrinsic conductance (e.g. silica or latex beads). In other embodiments, detection probes may be made from organic-inorganic nanostructures, also called hybrid nanoparticles (Moghimi et al. 2014). In addition, cell-associated analytes, whether present inside the cell or at its surface, can be recognized and bound by antibodies conjugated with metal particles. Such reagents, which in some cases were originally developed for applications such as mass cytometry, are commercially available (e.g. from DVS Sciences, a subsidiary of Fluidigm

Sciences, Sunnyvale, CA). The detection probe could also be an aptamer, which includes oligonucleotide or polypeptide capture sequences (Millward et al. 2012) (Taylor Al, Arangundy- Franklin S 2014) that can be conjugated with metal particles (Zhang et al. 2013).

Complementary oligonucleotide sequences could be used to bind cellular DNA or RNA as in an in situ hybridization assay (Kwon 2013). These sequences could be directly conjugated with metal particles or could be biotinylated and subsequently bound by streptavidin conjugated to the metal particles (Ocana and del Valle 2014). Detection could also occur with the use of a metal-conjugated antibody or aptamer specific for double stranded DNA or DNA- NA hybrid molecules (Hu et al. 2014).

[30] Capture probes: In certain embodiments, when measuring analytes in solution an additional capture step may be necessary in order to immobilize the analyte on the object prior to binding with the detection probe. This is achieved using a capture probe. In various embodiments, antibodies or aptamers can be used for protein capture, with a secondary antibody or aptamer used subsequently for detection.

[31] In some embodiments, the use of separate capture probes may not be necessary. For example, detection of antibodies in serum constitutes a unique case given the inherent binding properties of the analyte that is being measured, i.e. the serum antibodies. In embodiments such as this, a strategy for detection may include covalently binding proteins to the object (e.g. carboxyl beads) such that antibodies specific for the protein would bind to the object. A secondary antibody conjugated to metal particles, for instance a mouse anti-human or goat anti-human antibody, could then be used as the detection probe. In other embodiments, synthetic oligonucleotide sequences could be bound to the object to capture complementary biotinylated sequences. Strepatvidin coupled with metal particles could then be used for detection. [32] The technology platform described herein can be leveraged to implement a range of clinical assays that could be broadly implemented, for example at a patient's bedside. The combination of microfluidics technology and electrical measurements facilitates the

development of portable (including in-home) devices which provide robust results for routine assays faster and at a lower cost than current technologies. Regular health exams and tests can help identify health issues early, before the onset of clinical symptom and when chances for treatment and cure are higher. Therefore, screening tests constitute a basic public health tool that can be used to identify unrecognized health conditions.

[33] More specialized tests may be run for patients who present with symptoms, the results being useful for investigating disease etiology and monitoring response to treatment or disease recurrence.

[34] I. Cell Counting: Routine blood tests include complete blood count (CBC) and white blood cell count (WBC). Elevated CBC also can for instance be an indicator of a bacterial infection. CBC and WBC are currently determined using side and forward scatter of single cells flowing through microfluidic channel in front of a laser beam. Leveraging the technology disclosed herein, cell counts can be determined as shifts in electrical conductance that are detected when cells flow between the contacts. To improve performance, the intrinsic conductance of the cells can be enhanced through the use of a detection probe binding ubiquitously to the cells' surface (e.g. antibodies to the hematopoietic marker CD45 can be used to label leukocytes). The WBC assay determines the overall abundance of leukocytes and that of various subsets. In various embodiments this could be achieved through the use of specific cell markers that are restricted to individual leukocyte subpopulations. Counts would be obtained for each population in separate channels using different staining reagents. Other populations such as CD4 T-cells, which numbers are determined routinely for the monitoring of HIV infection, may require the use of multiple markers. Multiple markers could be employed simultaneously (e.g. two peaks of conductance could be observed, the lower one for single positive cells and the higher one for double positive cells), or sequentially (successive sorting of positive and double-positive populations). Telomerase length measurement could also be achieved for the diagnosis of telomeropathies which are caused by excessive telomere erosion. In certain embodiments telomere measurements could be based on Flow-FISH technology, which is a fluorescence in situ hybridization assay coupled with flow cytometry (Gutierrez- Rodrigues et al. 2014), using metal particle conjugates instead of fluorophores and an electric readout generated when cells in suspension pass in a single file past an array of detectors.

[35] II. Serum Protein Measurements: Many routine assays measure serum proteins, with measurement generally based on the principle of the Enzyme Linked Immunosorbent Assay (ELISA) to detect proteins, which can be either antigens or antibodies (Hornbeck 2001). Such assays may be used for the diagnosis and management of 1) Infectious diseases, with infectious makers such as hepatitis A, B, C antigens and antibodies, HIV antigen (p24) or antibodies, Syphilis (antibody to T. pallidum), Leptospirosis (IgM), Melioidosis titer (Indirect

heamaglutination test; antibody to LPS), Typhoid fever (O, H or Vi antigens), or dengue antibody (NS1 antigen etc.); 2) Diabetes mellitus: HbAlc (gylcated hemoglobin) and insulin; 3) Autoimmune diseases: Systemic Lupus Erythematosus (anti-DNA antibodies), rheumatoid factor (IgM rheumatoid factor); and 4) Cancer: with tumor markers such as Carcinoembryonic antigen (CEA), Alpha-fetoprotein ((AFP) hepatocellular carcinoma)), Prostate specific antigen ((PSA) Prostate cancer screening), CA 125 (acute pancreatitis), CA19-9 (Cholangiocarcinoma), CA15-3 (Breast cancer). The technology platform disclosed herein is suitable for the detection of soluble proteins, for example by using capture probes or immobilization of protein substrate on the surface of beads. Subsequently, detection probes bind the analytes that are captured on the bead surface (Zhang, Birru, and Di 2014).

[36] III. Nucleic Acids: A number of tests are performed in which the analytes being tested are nucleic acids. For example, polymerase Chain Reaction (PCR) is routinely used for the detection of pathogen-derived genetic material, either DNA or RNA (e.g. respiratory

pathogens), which includes testing for the presence of the material in the sample (e.g. derived from a virus or bacterial pathogen). Detection of PCR products is also feasible using the methods and systems disclosed herein, via capture by a complementary sequence immobilized on microbeads and detection using either streptavidin or anti dsDNA antibodies conjugated with metal particles, as described above. A similar strategy can be used for genotyping. The use of genotyping tests in clinical settings has also become widespread, for perinatal screening applications as well as for detection of risk variants for diseases such as breast cancer (e.g. BRCA genes) or drug metabolism (CYP450). Other tests that are currently available or under development rely on the quantitation of RNA abundance in tissues such as blood or tumors (e.g. Oncotype DX, Allomap) (Nguyen et al. 2014; Pankla et al. 2009; Sarwal and Sigdel 2013).

[37] IV. Metabolic Panels: Metabolic panels are important components of routine screening / diagnostic assays that may be carried out using the methods and systems disclosed herein. These panels include the measurement of soluble proteins that can be assayed using strategies that are described above (e.g. for liver function: alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, total protein, and bilirubin; for Kidney function: creatinine). Other important parameters in standard metabolic panels are not proteins but instead are metabolites including small molecules (<1500 Da) found body fluids. While a few metabolites are routinely quantified, such as lipids (cholesterol, triglycerides, HDL cholesterol, LDL cholesterol), or glucose, thousands of other serum metabolites have been identified and catalogued and may be of diagnostic value (Psychogios et al. 2011). Viable strategies exist that would enable the use of the technology platform disclosed herein for the quantitation of metabolites such as glucose (widely used as a marker for the diagnosis and management of diabetes). Assays have been developed that rely on glucose binding proteins purified from bacteria that allow assessment of blood glucose levels (Kanjananimmanont et al. 2014; Veetil, Jin, and Ye 2010). Similarly, lipid binding proteins have been identified and used in assay development (Bandyopadhyay and Bong 2011). Thus, these and other binding proteins could be incorporated into the disclosed methods and systems as a way to implement small molecule detection systems to use as part of a metabolic panel.

[38] In some embodiments, a microfluidics system may facilitate carrying out different washing and staining steps in the chip using nanoliter scale reagent volumes. Thus, the system enables sample sparing, does not require hands-on processing, and reduces costs. Multiple microfluidic systems may also be parallelized, enabling the detection of tens, hundreds, or potentially thousands of analytes in as many independent reactions, among other things.

[39] As described herein, the system relies on the detection of electrical signals (rather than relying on an optical signal, which requires expensive and cumbersome equipment and an abundance of the analyte being assayed). This occurs as an object (e.g. a cell or bead) that is "decorated" by the detection reagent (e.g. metal nanoparticle-labeled antibody or

complementary sequence) passes between contacts separated by the microfluidics channel, thereby closing the circuit. The contacts operate as a switch, with the presence of the decorated object between the contacts serving to close the circuit. The amount of electric charge that will flow through the circuit at that moment will be proportional to the amount of metal particles bound to the object.

[40] The greater the amount of analytes detected on the object (e.g. cell or bead), the greater the amount of detection probe (e.g. gold nanoparticles) will be present on its surface, and the greater its conductivity. Measuring electrical parameters such as the current and/or voltage through the circuit can be used to determine the conductivity of the object closing the switch. In other words, the electrical conductance (or other parameter) of the object will be quantified and correlated with the concentration of the analyte being measured. In some embodiments, each object will flow through a series of switches connected to circuits with increasing current and/or voltage. This will constitute a "conductivity profile" for each object and allow measurement of analytes through an especially wide range of concentrations: high abundance will confer high conductivity on the object, with the highest resolution obtained when lower currents or voltages are applied, while low abundance will confer low conductivity on the object, with the highest resolution obtained when higher currents or voltages are applied.

[41] In some embodiments, the system described herein may be incorporated into handheld microfluidics devices capable of performing various diagnostic assays on small samples (e.g. a few microliters of blood) including, for example, plasma protein quantification, DNA SNP analysis, quantitation of immune cell populations, RNA profiling, and so on.

[42] Thus, in various embodiments, a method of quantitating an analyte may include decorating an analyte bound to an object with one or more metal detection reagents, causing the object to move through a microfluidics channel, the microfluidics channel having one or more pairs of opposing metal contacts, measuring an electrical parameter (e.g. current) within a circuit that includes one or more set of opposing metal contacts, and determining a property (e.g. conductance) of the object based on the measured electrical parameter, such as determining an absolute or relative amount of the analyte based on the measured electrical parameter of the object.

[43] The disclosed methods may be carried out by various apparatuses and systems. A microfluidic chamber includi ng one or more microfluidic channels may be made using a number of microfabrication techniques, which traditional ly have encom passed a range of MEMS techniques including photolithography and soft lithography and more recently have included 3D printing technology, as known to those skilled in the art. The microfluidic channel may have an approximately circular cross-section and a width or diameter between about 0.1 μιη and about 100 μιη, in particular about 0.1 μιη, about 1 μιη, about 2 μιη, about 5 μιη, about 10 μητι, about 15 μιτι, about 20 μιτι, about 30 μιη, about 40 μιτ, about 50 μιη, about 60 μιη, about 70 μιη, about 80 μνη, about 90 μηη, or about 100 μιτι, although other intermediate

widths/diameters are also possible. In general, the decorated object (whether a spherical object or a rod-shaped object) flowing through the microfluidic channel has a diameter that is somewhat smaller than the diameter of the channel itself, so that the object can flow through the channel without getting stuck but still have a tight enough fit so that it moves closely past (and in some embodiments, comes into contact with) the electrical contacts. Thus, in various embodiments the width or diameter of the object is less than the width or diameter of the microfluidic channel through which the object flows by about 0.1 μιτι, about 1 μηη, about 2 μητι, about 3 μιη, about 4 μιτι, about 5 μιη, or about 10 μιη. In certain embodiments, the microfluidic chamber also includes suitable fluid handling capabilities such as pumps, reservoirs, and/or vacuum lines.

[44] In various embodiments, the electrical contacts are located in the wall of the microfluidic channel such that conductive portions of the electrical contacts are directly exposed to the channel. In other embodiments, the electrical contacts may be recessed from the microfluidic channel and include a conductive, semi-conductive, or insulating coating on the electrical contacts, such that the coating is exposed to the microfluidic channel. In some embodiments the coating may be permeable to the fluid in which the objects are bathed in order to facilitate electrical measurements. [45] In certain embodiments, the solution (which may be salt-based solution) in which the object moves through the microfluidic channel has an electrical conductivity which ranges from about 0.5 mS/cm to about 200 mS/cm, and in various embodiments is about 1 mS/cm, about 5 mS/cm, about 10 mS/cm, about 20 mS/cm, about 50 mS/cm, about 100 mS/cm, about 125 mS/cm, or about 150 mS/cm. In some embodiments, the conductivity of the solution in which the objects are bathed is substantially lower than the expected conductivity of the decorated objects so that a significant difference in conductivity will be observed when the objects pass between the pairs of contacts.

[46] In various embodiments, a suspension of objects (e.g. cells or beads) is transferred to a flow cell which produces a stream of liquid which carries and aligns the objects so that they enter and move through the microfluidic channel(s) in single-file order so that each object can pass individually between the pairs of electrical contacts. In some embodiments, the concentration of objects in the suspension is adjusted in order to ensure that objects are not passing between the electrical contacts too close to one another, so that there is enough temporal and physical spacing between subsequent objects to permit each object's electrical parameters to be measured separate from the other objects in the sample.

[47] In various embodiments, the invention includes a system for carrying out the methods disclosed herein, including a microfluidic chamber having one or more pairs of electrical contacts and circuits coupled to the contacts, along with a controller in communication with the circuits. The controller includes a processor and may be part of a computer system which includes input and output and memory. The system may include one or more computer systems in communication with one another through various wired and wireless

communication means, which may include communications through the Internet. Each computer system may include an input device, an output device, a computer-readable medium, and a processor. Possible input devices include a keyboard, a computer mouse, a touch screen, and the like. Output devices may include a cathode-ray tube (CRT) computer monitor, a liquid- crystal display (LCD) or LED computer monitor, and the like. Computer-readable media include various types of memory such as a hard disk, RAM, flash memory, and other transient and non- transient magnetic, optical, physical, or electronic memory devices. The processor (whether part of a computer system or the controller) may be any typical computer processor for performing calculations and directing other functions for performing input, output, calculation, and display of data in the disclosed methods and systems. Implementation of the system includes generating a set of instructions and data that are stored on one or more of the storage media and operated on by the controller. The data associated with the system can include numerical and other types of data. In certain embodiments, the invention includes a computer- readable medium having instructions for carrying out embodiments of the present invention.

[48] In one embodiment, the system may include a web page for facilitating input, control, analysis, and other functions. In other embodiments, the system may be implemented as a locally-controlled program on a local computer system and may or may not be accessible to other, external computer systems. In still other embodiments, the system may include modules which provide access to portable devices such as laptops, tablet computers, and smart phones.

[49] In certain embodiments, the controller applies a current or voltage to the pairs of electrical contacts and measures an electrical parameter as objects pass between the pairs of contacts. In some embodiments the controller applies a DC voltage to the electrical contacts and measures current levels as the objects flow through the microfluidic channel past the pairs of contacts. The current measurements can then be used to determine an electrical property of the objects, for example the conductance levels associated with the objects, which in turn can be related to levels of analyte. In various embodiments, calibration or standard curves are produced under controlled conditions using objects having known levels of analytes attached thereto in order to relate calculated conductance levels to analyte levels. In some

embodiments, the measured electrical parameters may be related directly to analyte levels, for example measured current levels could be related to analyte levels based on predetermined standards. In particular embodiments, a determination may be made of simply whether the analyte is present or not rather than determining a specific analyte level.

[50] In some embodiments, AC voltages (e.g. ranging from about 10 Hz to about 100 MHz) may be applied to the pairs of electrical contacts so that impedance levels associated with the objects may be determined, where the impedances can be related to analyte levels using calibration curves, as discussed above. [51] In various embodiments, AC or DC voltages may be applied to the pairs of contacts in a range of about 1 mV to about 100 V. In certain embodiments, a microfluidic channel may include more than one pair of electrical contacts along a length thereof (e.g. as shown in FIG. 5), each with a circuit coupled thereto. In such embodiments, the controller may apply a different level of voltage or current to each pair of electrical contacts and measure separate electrical parameters from each pair of contacts associated with a given object flowing through the microfluidic channel. The electrical parameters associated with a given object which are measured at the different pairs of contacts can be combined to determine an electrical property of the object and hence to determine an analyte level (or to determine the presence or absence of the analyte). For example, each of the pairs of contacts may have an increasingly higher voltage (e.g. in lV-100 V increments) or current applied, in a linear or non-linear fashion, so that the object is exposed to different current or voltage levels as it progresses through the microfluidic channel. The electrical parameters that are measured or electrical properties that are determined will therefore benefit from the wider dynamic range afforded by the wider range of electrical values applied to each object.

[52] FIG. 1 is a diagram illustrating the binding of a decorated analyte to an object. The system described herein utilizes a conductor material (depicted as a filled circle), such as a gold or other metal, as the probe for detection. In FIG. 1, the probe conjugated to the conductor is shown to bind the analyte at the surface of a cell, which serves as the solid substrate in this embodiment.

[53] FIG. 2 is a diagram illustrating different configurations for binding decorated analytes to an object. In certain embodiments, the systems and methods described herein can be used to measure analytes in solution; to facilitate quantitation of the analyte levels, a capture probe is used to capture the analyte onto a solid substrate (such as a microbead, as shown in FIG. 2).

[54] FIG. 3 is a diagram illustrating a system for measuring an electrical parameter associated with an object having analyte and detection probes coupled thereto. The system described herein includes a solid substrate of a defined size (e.g. microbead or cell) coupled to conductor material (e.g. metal particle conjugated to a detection probe) that flows through a microfluidics channel and passes between contacts connected to an electrical circuit. The solid substrate thus acts as a switch. Measuring electrical parameters such as the amount of current that passes through the circuit as well as the difference in potential between the two contacts as the object passes between them allows the determination of a property such as the conductance of the object, which would be proportional to the amount of conductor material bound to it and hence to analyte concentration.

[55] FIG. 4 is a diagram illustrating a system for measuring an electrical parameter (e.g. conductance) of an object having analyte and detection probes coupled thereto. The system described herein includes flowing objects (e.g. microbeads) through a microfluidics channel, such that the objects pass between pairs of contacts, each pair being connected to an independent electrical circuit. The electrical properties (e.g. conductivity) of the object (e.g. microbead) will depend on the amount of conductor particles (e.g. gold) that are bound to it, which in turn depends on the amount of analyte present. For example, the graph depicts changes in current that would be measured as the microbeads flow through the microfluidic channel between the contacts.

[56] FIG. 5 is a diagram illustrating a system for measuring an electrical parameter (e.g. conductance) of an object having analyte and detection probes coupled thereto, the system including multiple measurement circuits. The system disclosed herein includes solid substrates (e.g. microbeads) flowing through a microfluidic channel, which pass between a series of pairs of contacts, each connected to an independent electrical circuit. For example, in FIG. 5 there are six pairs of contacts and six independent circuits, numbered 1-6. In various embodiments, the electrical measurements in the pairs of contacts may be varied from one pair to the next in order to improve the accuracy of the measurements, for example by increasing the dynamic range. In one embodiment, for example, increasingly higher voltage may be applied between the contacts of circuits 1 through 6 in order to obtain more accurate current measurements for objects having very large or very small amounts of detection probe material. In this

embodiment, the bar graphs indicate the amount of current measured as the beads pass between each of the pairs of contacts for the six different circuits. In the example shown in FIG. 5, no conductive material is bound to bead A, since no analyte is present at its surface, whereas large amounts of conductive material are present on the surface of bead B and a lesser amount of conductive material is present at the surface of bead C. By including multiple pairs of contacts, each pair providing different electrical parameter levels, this facilitates more accurate electrical measurements at the lower and higher ends of the measurement ranges (e.g. current measurements, as shown in FIG. 5).

[57] Therefore, in some embodiments, a system for quantitating analytes includes a microfluidics channel, a first measurement circuit, and a second measurement circuit. The first measurement circuit includes a first pair of opposing metal contacts positioned proximate to the microfluidics channel, generally on opposite sides of the channel, and configured to complete the first measurement circuit when an object having an analyte decorated with one or more metal detection reagents flows through the microfluidics channel and between the contacts. The second measurement circuit includes a second set of metal contacts positioned proximate to the microfluidics channel and configured to complete the second measurement circuit when an object having an analyte decorated with one or more metal detection reagents flows through the microfluidics channel and contacts the second set of metal contacts.

Alternatively, the system may include only one or many measurement circuits.

[58] In some embodiments, the object and its associated detection probes make contact with the first set of metal contacts to help complete a circuit. In other embodiments, the object and its associated detection probes do not touch the pair of contacts but pass sufficiently close to the contacts to facilitate detection of a change in an electrical parameter. In still other embodiments, no direct contact occurs between the object and its associated detection probes and the pair of contacts but electrical communication occurs between the pair of contacts and the object and its associated detection probes via the solution (e.g. a salt solution) in which the objects are flowing.

[59] FIG. 6 is a diagram illustrating various applications of a measurement system such as that disclosed herein, including applications that combine the functionalities and capabilities of a microfluidics chip and an electronic microchip. The functionalities shown diagrammatically in FIG. 6 may be implemented on a single device or using several interconnected devices.

Microfluidics applications allow separation of blood into a cellular fraction (FIG. 6, black arrow pointing left) and a plasma fraction (FIG. 6, black arrow pointing right). The cellular fraction can be further utilized for "Cellular Profiling" and "Genetic Profiling" (FIG. 6, grey boxes and arrows). Under "Genetic Profiling", DNA/cDNA libraries are prepared as the cells undergo different incubation steps with appropriate reagents (depicted as a circle labeled "Library Prep"), as done for instance on a Fluidigm CI chip (Pollen et al. 2014). The nucleic acid preparation can then be aliquoted and incubated in separate chambers for each separate analyte with appropriate capture and detection reagents (each depicted as a circle associated with lanes A-D).

[60] For signal detection, solid substrates (e.g. microbeads) bound to the analyte and detection probe flow single-file between a series of pairs of contacts connected to circuits on which varying amounts of currents are applied. In this example each analyte measured has a dedicated "detection channel". A similar process applies for Serum and Cellular profiling (the other two gray boxes and arrows) using the appropriate reagents for each application. In the case of cellular profiling additional separation steps can be envisioned and are depicted, in which cells can be sorted in a negative (circle with - sign) and positive fraction (circle with + sign) based on the signal acquired for each individual cell, (depicted by the histograms). Cells could be further separated into positive and double positive fractions after staining with additional reagents and detection. This would for instance allow determination of "CD4 counts" of HIV patients (CD4 T cells can be accreted identified using CD3 and CD4 as markers). Cells in a microfluidics chips can also be incubated using appropriate cell medium, thus making it possible to envision applications where live sorted cells could then be stimulated and subsequently subjected to genetic profiling within the same microfluidic chip (black arrow pointing up, with cells being channeled towards the Genetic profiling workflow). Of course, other applications may utilize the described system and methods. In particular, the following are three (l-lll) embodiments using the disclosed systems and methods to measure analytes, stimulate cells in situ and sort object such as cells:

[61] I. Multiple analyte measurement in single cells

[62] Cellular phenotyping often requires assessing expression levels of multiple markers, for example when analyzing blood leukocyte populations. Thus in various embodiments the disclosed methods and systems can be used for determination of abundance of multiple analytes expressed at the surface of individual cells. In one particular embodiment for clinical applications, would be the measurement of CD4 T cell counts in patients with HIV, a parameter that is used by clinicians to make treatment decisions. Cellular phenotyping is also widely used in research settings, especially in the field of immunology.

[63] The following is a description of procedures for measuring multiple analytes in single cells (FIGS. 7A-7F):

[64] A. Background signal measurement (FIG. 7A): Cells are flowed in a single file through a microfluidics channel. The first cell passes in front of contacts before entering the first chamber. This cell conductance measurement allows determination of the background signal for this individual cell.

[65] B. Cell staining for Analyte 1 (FIG. 7B): Detection probe reagents for analyte 1 are flowed through the first chamber; followed by a wash solution to eliminate excess reagent.

[66] C. Analyte 1 detection (FIG. 7C): The cell passes in front of a second set of contacts. The comparison of cell conductance measurement between the first and second set of contacts (background and signal, respectively) allows determination of expression levels of the first analyte. A second cell advances through the channel and starts the same sequence with first determination of background signal as described in A.

[67] D. Elution of the detection probe (FIG. 7D): The cell enters the second chamber and is stripped of the bound detection probes, for example using a low pH / high salt concentration solution.

[68] E. Background signal measurement (FIG. 7E): The cell flows in front of a third set of contacts in order to measure background signal following elution.

[69] F. Cell staining for Analyte 2 (FIG. 7F): As described in step B for analyte 1 detection probe reagents for analyte 2 are flowed through the third chamber; followed by a wash solution to eliminate excess reagent. Subsequent steps described above are repeated to allow determination of expression levels of the second analyte and possibly a third and a forth analyte, etc.

[70] II. Cell stimulation in situ [71] In various embodiments, the methods and systems disclosed herein can be used to perform cell stimulation in situ (on the chip) and measurement of analytes released in culture supernatants.

[72] FIGS. 8A-8D illustrate an embodiment of the disclosed technology in which

measurement of electrical conductance is used to detect and quantify the presence of analytes in cell culture supernatants.

[73] In FIG. 8A, one cell or multiple cells are captured in a chamber containing culture medium and maintained at 37°C; stimuli are flowed along with the culture medium. In FIG. 8B, after a period of incubation analytes are produced by the cell(s) in the supernatant. In FIG. 8C, culture supernatants are flowed into a different chamber for staining and subsequent detection. Detection probes are bound to latex beads that serve as the solid substrate. In FIG. 8D, actuating/opening the valves allows the medium to flow and signal to be detected when latex beads that are "decorated' with the antigen and detection probes pass in front of the contacts.

[74] III. Cell sorting

[75] Cells can be isolated and sorted based on specific cell surface or intracellular markers. For example, sorting of cells can be performed using flow cytometry, which employs fluorescent probes as a readout. FIGS. 9A-9B illustrate procedures using the methods and systems disclosed herein for sorting of cells that are either positive or negative for a given marker as measured by electrical conductance. This enables the isolation of cells with a specific function or characteristic: for instance, it is possible to target specific markers for the isolation of rare circulating cancer cells in cancer patients. These cells can be further characterized via genomics or proteormics profiling and can be used to provide information on patient status and/or prognosis. Furthermore, research applications are particularly wide ranging and isolated cells can be subjected to various treatments (e.g. exposed to immune stimulators) in situ (on the chip) and their molecular characteristics and that of culture supernatants can be further investigated in downstream assays (also on the chip).

[76] Procedures for sorting cells are shown in FIGS 9A-9B. Cells are flowed in a single file through a microfluidics channel with signal measured in the form of electrical conductance before (background) and after (signal) staining with probes specific for a marker of interest labeled with conductive elements. When signal is detected, the cells flow through and are collected in a chamber for further analysis or experimentation (FIG. 9A). When signal is not detected, valves are actuated, allowing for diversion of the flow and collection of the cells in a second holding chamber that contains cells negative for the marker of interest (FIG. 9B).

EXAMPLES

[77] The application may be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the application. The following examples are presented in order to more fully illustrate embodiments and should in no way construed, however, as limiting the broad scope of the application. While certain embodiments of the present application have been shown and described herein, it will be obvious that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the embodiments; it should be understood that various alternatives to the embodiments described herein may be employed in practicing the methods described herein.

[78] Example 1: Sample preparation method for cellular profiling assays

[79] The sample preparation steps described here can be performed at a lab bench, in which case the cellular suspension is flowed through the detection device. The device may include a flow cell that produces a liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file between the electrical contacts.

[80] In the present methods, a signal is detected as the objects (e.g. cells) coupled with conductor material (e.g. metal particles) pass between contacts of an electrical circuit.

[81] In systems in which mass spectrometry has been used as a detection system, cells are labeled with antibodies or other detection probes coupled with chelating polymers containing highly-enriched metal isotopes (referred to as CyTOF Mass Cytometers) (Leipold and Maecker 2012; Newell et al. 2012). Thus a similar labeling protocol can be used in various embodiments in a manner similar to the protocols used for CyTOF applications and make use of the same reagents: the following protocol adapted from (Newell et al. 2012) and illustrates the sample preparation procedure. Although the procedure uses cells as the objects, in other embodiments other types of objects could be used with suitable modifications of the procedure below:

[82] For surface (extracellular) staining:

1. Cells will be transferred to 96-well plates (or tubes), washed, and resuspended in

cytometry buffer (PBS + 0.05% sodium azide + 2 mM EDTA + 2% fetal calf serum).

2. Cells will then be stained for 30 min on ice with a prepared cocktail of metal-conjugated surface-marker antibodies at concentrations found to be effective in prior antibody tests.

3. After surface staining, cells will be washed 3x in cytometry buffer and resuspended in PBS.

4. The cell suspension will then be ready to be aspirated in the flow cells that will generate a liquid stream and align the cells so that they pass single-file between the electrical contacts of the detector.

[83] For intracellular staining:

1. Cells will be transferred to 96-well plates (or tubes), washed, and resuspended in

cytometry buffer (PBS + 0.05% sodium azide + 2 mM EDTA + 2% fetal calf serum) + 2% paraformaldehyde

2. After overnight fixation at 4°C, the cells will be washed 2x in 1 x intracellular staining permeablization buffer and stained with an intracellular antibody on ice for 45 min, washed 2x in cytometry buffer, and labeled for 20 min at room temperature with 250 nM iridium interchelator suspended in PBS + 2% paraformaldehyde.

3. Finally, the cells will be washed 2x in cytometry buffer, 2x in PBS, and 2x in distilled water before diluting to the appropriate concentration.

4. The cell suspension will then be ready to be aspirated in the flow cells that will generate a liquid stream and align the cells so that they pass single-file between the contacts of the detector.

[84] Notably the staining and washing steps described above could also be performed using a microfluidics chip, which would have the advantage of drastically reducing the need for hands-on sample manipulation and minimizing the volume of reagents needed, and thereby minimizing assay cost. The use of microfluidics chips for this purpose has been described earlier (Srivastava and Singh 2011).

[85] In certain embodiments, bead sensors may be combined with microfluidic elements to allow for high-throughput testing for a variety of analytes and assays, including enzymatic assays; general chemistries; protein; and antibody and oligonucleotide applications. Bead sensors are considered a viable option for clinical applications, and especially near patient sensing (Chou et al. 2012). A variety of microbead types supporting these applications can be obtained, e.g., from Luminex Corporation. In such embodiments, beads coupled with conductor material (e.g. metal particles) will be flowed through microfluidic channels so that they pass between electrical contacts of an electrical circuit. This will allow detection of soluble analytes captured and immobilized on the beads and bound by a detection probe coupled with conductive detection probes such as chelating polymers containing highly-enriched metal isotopes.

[86] Example 2: Sample preparation method for measuring soluble analytes

[87] Methods for capturing an analyte (e.g. protein, DNA or RNA) in solution and detecting its abundance are known to those skilled in the art. Further, surface staining with the cellular profiling assays described above in the sample preparation steps can be implemented within a microfluidics chips (Chou et al. 2012):

1. Resuspend the microbeads by vortex and sonication

2. Prepare a working microbead mixture by diluting the coupled microbead stocks to a final concentration in PBS-1% Bovine Serum Albumin (BSA).

3. Pre-wet a 1.2 μιη 96-well filter plate with 100 μΙ/weW of PBS-1% BSA and aspirate by vacuum manifold.

4. Aliquot 50 μΐ of the working microbead mixture into the appropriate wells of the filter plate.

5. Add 50 μί of PBS-1% BSA to each background well. Add 50 μΐ of standard or sample to the appropriate wells. Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. Cover the filter plate and incubate for 30 minutes at room temperature on a plate shaker. Aspirate the supernatant by vacuum manifold. Wash each well twice with 100 μί of PBS-1% BSA and aspirate by vacuum manifold. Resuspend the microbeads in 50 μΐ of PBS-1% BSA by gently pipetting up and down five times with a multi-channel pipettor. Dilute the detection antibody to 4 g/mL in PBS-1% BSA. Add 50 μί. of the diluted detection antibody to each well. Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. Cover the filter plate and incubate for 30 minutes at room temperature on a plate shaker. Aspirate the supernatant by vacuum manifold. Wash each well twice with 100 μί of PBS-1% BSA and aspirate by vacuum manifold. Resuspend the microbeads in 50 \xl of PBS-1% BSA by gently pipetting up and down five times with a multi-channel pipettor. The microbead suspension will then be ready to be aspirated in the flow cells that will generate a liquid stream and align the cells so that they pass single-file between the electrical contacts of the detector. 20. A read-out is obtained and conductivity profiles thus obtained are correlated with analyte concentration.

[88] REFERENCES

[89] Bandyopadhyay, Saibal, and Dennis Bong. 2011. "Synthesis of Trifunctional

Phosphatidylserine Probes for Identification of Lipid-Binding Proteins." European Journal of Organic Chemistry 2011(4):751-58.

[90] Chou, Jie et al. 2012. "Porous Bead-Based Diagnostic Platforms: Bridging the Gaps in Healthcare." Sensors (Basel, Switzerland) 12(ll):15467-99.

[91] Dunbar, Sherry A., and James W. Jacobson. 2007. "Quantitative, Multiplexed Detection of Salmonella and Other Pathogens by Luminex xMAP Suspension Array." Methods in molecular biology (Clifton, N.J.) 394:1-19.

[92] Gdowski, Andrew, Amalendu P. Ranjan, Anindita Mukerjee, and Jamboor K.

Vishwanatha. 2014. "Nanobiosensors: Role in Cancer Detection and Diagnosis." Advances in experimental medicine and biology 807:33-58.

[93] Gutierrez-Rodrigues, Fernanda, Barbara A. Santana-Lemos, Priscila S. Scheucher, Raquel M. Alves-Paiva, and Rodrigo T. Calado. 2014. "Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans." PloS one 9(ll):ell3747.

[94] Hornbeck, P. 2001. "Enzyme-Linked Immunosorbent Assays." Current protocols in immunology / edited by John E. Coligan ... [et al.] Chapter 2:Unit 2.1.

[95] Hu, Zonglin, Stephen H. Leppla, Baoguang Li, and Christopher A. Elkins. 2014.

"Antibodies Specific for Nucleic Acids and Applications in Genomic Detection and Clinical Diagnostics." Expert review of molecular diagnostics 14(7):895-916.

[96] Kanjananimmanont, Sunsanee et al. 2014. "Passive Diffusion of Transdermal Glucose: Noninvasive Glucose Sensing Using a Fluorescent Glucose Binding Protein." Journal of diabetes science and technology 8(2):291-98.

[97] Kwon, Sunjong. 2013. "Single-Molecule Fluorescence in Situ Hybridization: Quantitative Imaging of Single RNA Molecules." BMB reports 46(2):65-72. [98] Leipold, Michael D., and Holden T. Maecker. 2012. "Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer." Journal of visualized

experiments : JoVE (69):e4398.

[99] Lin, Charles H., Joanne M. Yeakley, Timothy K. McDaniel, and Richard Shen. 2009.

"Medium- to High-Throughput SNP Genotyping Using VeraCode Microbeads." Methods in Molecular Biology 496:129-42.

[100] Millward, Steven W. et al. 2012. "In Situ Click Chemistry: From Small Molecule Discovery to Synthetic Antibodies." Integrative Biology.

[101] Moghimi, Nafiseh et al. 2014. "In Situ Hybridization of Superparamagnetic Iron-

Biomolecule Nanoparticles." Journal of the American Chemical Society 136(29):10478-85.

[102] Newell, Evan W., Natalia Sigal, Sean C. Bendall, Garry P. Nolan, and Mark M. Davis. 2012.

"Cytometry by Time-of-Flight Shows Combinatorial Cytokine Expression and Virus-Specific Cell

Niches within a Continuum of CD8+ T Cell Phenotypes." Immunity 36(l):142-52.

[103] Nguyen, Michaela T. et al. 2014. "Impact of Oncotype DX Recurrence Score in the

Management of Breast Cancer Cases." Clinical breast cancer 14(3):182-90.

[104] Ocana, Cristina, and Manel del Valle. 2014. "Signal Amplification for Thrombin

Impedimetric Aptasensor: Sandwich Protocol and Use of Gold-Streptavidin Nanoparticles."

Biosensors & bioelectronics 54:408-14.

[105] Pankla, Rungnapa et al. 2009. "Genomic Transcriptional Profiling Identifies a Candidate Blood Biomarker Signature for the Diagnosis of Septicemic Melioidosis." Genome biology 10(11):R127.

[106] Pollen, Alex A. et al. 2014. "Low-Coverage Single-Cell mRNA Sequencing Reveals Cellular Heterogeneity and Activated Signaling Pathways in Developing Cerebral Cortex." Nature biotechnology 32(10):1053-58.

[107] Psychogios, Nikolaos et al. 2011. "The Human Serum Metabolome." PloS one

6(2):el6957.

[108] Sarwal, Minnie, and Tara Sigdel. 2013. "A Common Blood Gene Assay Predates Clinical and Histological Rejection in Kidney and Heart Allografts." Clinical transplants 241-47. [109] Srivastava, Nimisha, and Anup Singh. 2011. "Microfluidic Devices and Methods for Integrated Flow Cytometry."

[110] Taylor Al, Arangundy-Franklin S, Holliger P. 2014. "Towards Applications of Synthetic Genetic Polymers in Diagnosis and Therapy." Curr Opin Chem Biol. 22C79-84.

[Ill] Veetil, Jithesh V, Sha Jin, and Kaiming Ye. 2010. "A Glucose Sensor Protein for

Continuous Glucose Monitoring." Biosensors & bioelectronics 26(4):1650-55.

[112] Zhang, Jiani, Bo Liu, Huixia Liu, Xiaobing Zhang, and Weihong Tan. 2013. "Aptamer- Conjugated Gold Nanoparticles for Bioanalysis." Nanomedicine (London, England) 8(6):983-93.

[113] Zhang, Yingze, Rahel Birru, and Yuanpu Peter Di. 2014. "Analysis of Clinical and

Biological Samples Using Microsphere-Based Multiplexing Luminex System." Methods in molecular biology (Clifton, NJ.) 1105:43-57.

[114] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to." As used herein, the terms "connected," "coupled," or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling of connection between the elements can be physical, logical, or a combination thereof. Additionally, the words "herein," "above," "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application. Where the context permits, words in the above Detailed

Description using the singular or plural number may also include the plural or singular number respectively. The word "or," in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list.

[115] The above detailed description of embodiments of the apparatus is not intended to be exhaustive or to limit the apparatus to the precise form disclosed herein. While specific embodiments of, and examples for, the apparatus are described above for illustrative purposes, various equivalent modifications are possible within the scope of the apparatus, as those skilled in the relevant art will recognize.

[116] While certain aspects of the apparatus are presented below in certain claim forms, the inventors contemplate the various aspects of the apparatus in any number of claim forms. Accordingly, the inventors reserve the right to add additional claims after filing the application to pursue such additional claim forms for other aspects of the apparatus.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of detecting an analyte, the method comprising:

decorating an analyte bound to an object with one or more conductive detection probes;

causing the object to move through a microfluidic channel, the microfluidics channel having a pair of contacts; and

measuring an electrical parameter within a circuit coupled to the contacts that is completed when the object passes between the pair of opposing contacts; and

determining an electrical property of the object based on the measured

electrical parameter, wherein the electrical property indicates a presence of the analyte.

2. The method of claim 1, wherein the electrical parameter is current.

3. The method of claim 2, wherein the electrical property is conductance.

4. The method of claim 1, wherein the analyte is bound to the object using a capture probe.

5. The method of claim 4, wherein the capture probe comprises an antibody, an aptamer, or streptavidin.

6. The method of claim 1, wherein the one or more conductive detection probes comprises a metal.

7. The method of claim 6, wherein the metal comprises gold or silver.

8. The method of claim 1, wherein the microfluidic channel comprises a plurality pairs of opposing contacts along a length of the microfluidic channel.

9. The method of claim 1, wherein the object comprises a cell, a microvesicle, a microbead, or a microrod.

10. The method of claim 1, further comprising:

determining an amount of the analyte based on the determined electrical property of the object.

11. A system for detecting an analyte, the system comprising:

a microfluidic channel having a first pair of contacts disposed adjacent thereto on opposing sides of the microfluidic channel, either lining the inside or outside of the channel;

a first measurement circuit coupled to the first pair of contacts and configured to complete the first measurement circuit when an object having an analyte decorated with one or more conductive detection probes flows through the microfluidic channel between the first pair of contacts; and a controller in operative communication with the first measurement circuit, the controller configured to

use the first measurement circuit to measure an electrical parameter when the object passes between the first pair of contacts, and determine an electrical property of the object based on the measured electrical parameter, wherein the electrical property indicates a presence of the analyte.

12. The system of claim 11, wherein the electrical parameter is current.

13. The system of claim 12, wherein the electrical property is conductance.

14. The system of claim 11, wherein the analyte is bound to the object using a capture probe.

15. The system of claim 14, wherein the capture probe comprises an antibody, an aptamer, or streptavidin.

16. The system of claim 11, wherein the one or more conductive detection probes comprises a metal.

17. The system of claim 16, wherein the metal comprises gold or silver.

18. The system of claim 11, wherein the object comprises a cell, a microvesicle, a microbead, or a microrod.

19. The system of claim 11, wherein the controller is further configured to determine an amount of the analyte based on the determined electrical property of the object.

20. The system of claim 11, wherein the microfluidic channel comprises a second pair of contacts disposed adjacent the microfluidic channel on opposing sides thereof and wherein the second pair of contacts includes a second measurement circuit coupled thereto.

21. The system of claim 20, wherein the controller is further in operative

communication with the second measurement circuit, the controller being further configured to

use the second measurement circuit to measure an electrical parameter when the object passes between the second pair of contacts, and

determine an electrical property of the object based on the measured electrical parameters from the first and second.

A system for quantitating an analyte, the system comprising:

a microfluidics channel;

a first measurement circuit, the first measurement circuit including a first set of contacts positioned adjacent the microfluidics channel and configured to complete the first measurement circuit when an object having an analyte decorated with one or more conductive detection reagents flows through the microfluidics channel between the first set of contacts;

a second measurement circuit, the second measurement circuit including a second set of contacts positioned adjacent the microfluidics channel and configured to complete the second measurement circuit when an object having an analyte decorated with one or more conductive detection reagents flows through the microfluidics channel and contacts the second set of metal contacts; and

a controller in operative communication with the first measurement circuit and the second measurement circuit, the controller being configured to

use the first measurement circuit to measure a first electrical parameter when the object passes between the first pair of contacts, use the second measurement circuit to measure a second electrical parameter when the object passes between the second pair of contacts,

determine an electrical property of the object based on the first measured electrical parameter and the second measured electrical parameter, and

quantitate the analyte based on the electrical property.