WO2022178039A1

WO2022178039A1 - Direct digital label-free identification, characterization and quantification of proteins

Info

Publication number: WO2022178039A1
Application number: PCT/US2022/016671
Authority: WO
Inventors: Kiana ARAN; Irina M. Conboy; Michael J. Conboy; Alexander A. KANE
Original assignee: Keck Graduate Institute of Applied Life Sciences; University of California Berkeley; University of California San Diego UCSD
Current assignee: Keck Graduate Institute of Applied Life Sciences; University of California Berkeley; University of California San Diego UCSD
Priority date: 2021-02-16
Filing date: 2022-02-16
Publication date: 2022-08-25
Anticipated expiration: 2023-08-16

Abstract

Mass-electrometry utilizes liquid gated field effect transistors in a facile and miniaturized electrophoresis system to identify and measure the levels of proteins, based on their molecular weight and charge that are determined with molecular precision, digitally and in real-time wherein a mass-electrometry chip comprises an array of transistors, each comprising of a channel between two electrodes with a gate contact to modulate the electronic response of the channel; and an electrophoresis or focusing gel comprising gel-separated proteins.

Description

Direct Digital Label-free Identification, Characterization and Quantification of Proteins

[001] Introduction

[002] Proteomic technologies can be used to identify markers for cancer diagnosis, to monitor disease progression, and to identify therapeutic targets. Persistent challenges include developing simple, fast, low cost, and scalable methods for large scale protein purification with a reasonable degree of separation, and coupling them to fine resolution sample detection.

[003] Gel-based microfluidic protein separations show promise in overcoming shortcomings of technologies like 2D-PAGE and liquid chromatography-mass spectroscopy (LC-MS). Graphene field-effect transistors (gFETs) have become attractive for sensing applications due to their rapid response, high sensitivity, good reproducibility and real-time monitoring compared with several other detection techniques.

[004] Relevant Literature

[005] Y. Zang, D. Huang, C. a. Di et ak, “Device engineered organic transistors for flexible sensing applications,” Advanced Materials, vol. 28, no. 22, pp. 4549-4555, 2016.

[006] Y. Zhang, Y.-W. Tan, H. L. Stormer et ak, “Experimental observation of the quantum Hall effect and Berry's phase in graphene,” nature, vol. 438, no. 7065, pp. 201, 2005.

[007] B. Thakur, et ak, “Rapid detection of single E. coli bacteria using a graphene-based field- effect transistor device,” Biosensors and Bioelectronics, vol. 110, pp. 16-22, 2018.

[008] Summary of the Invention

[009] The invention provides novel devices and related process, termed mass-electrometry for separation and identification of proteins, their levels and modifications. Mass-electrometry utilizes field effect transistors in a facile and miniaturized electrophoresis system to identify and measure the levels of proteins with molecular precision, digitally and in real-time.

[010] In addition to being direct and more accurate than previous methods. Mass-electrometry is faster and requires significantly smaller starting material, hence enabling proteomics on tissue sub-regions and single cells. Unlike PAGE, mass-electrometry is direct and digital and it has multi-characteristic profiling similar to mass-spectroscopy; yet in contrast to mass spectrometry, mass-electrometry does not require protein digestion or a massive starting samples, and it is much faster.

[Oil] Mass-electrometry uses an AC signal readout for fast multi-characteristic profiling (2D PAGE) and label-free identification of any and all proteins, converted to digital information.

This process and device enable the, molecular accuracy and real-time resolution of proteins' identity and level (without secondary steps, optical comparisons or digesting proteins into smaller parts). In contrast to conventional 2-D PAGE and single cell capillary-based Western analysis (Nat Methods. 2014 Jul; 11(7): 749-755.), Mass Electrometry has orders of magnitude better resolution, distinguishing the single amino acid differences between the proteins, and thus allowing one to resolve the proteins of similar molecular weight and charge. The assay time and the required starting sample of M.E.Chip are significantly reduced, while the, accuracy and the dynamic range of detection are significantly improved, as compared to prior methods. Larger number of proteins can be separated and analyzed in a smaller system.

[012] The invention employs of polyacrylamide gel properties and graphene based field effect transistors to fabricate high resolution and digital identification of wide range of proteins separation in short time to represents both advantages of high resolution in separation and fast sensing and nominate it as mass electrometery.

[013] Mass electrometry (ME) can separate proteins in a few seconds while PAGE needs at least an hour for separation due to its size. A PAGE gel is typically 10x7 Cm in size while the gel in ME is typically less than 1 cm² in size. The chips effectively replace macroscale optics with microscale surface electronics, to detect protein position using muliplexed spatial array of graphene electronic (gFET) biosensors.

[014] In an aspect the invention provides a mass-electrometry chip comprising: (a) an array of transistors, each comprising of a channel between two electrodes with the gel analyte providing the gate contact to modulate the electronic response of the channel; and (b) an electrophoresis or focusing gel comprising gel-separated proteins; wherein the chip is configured so that the array provides a digital signal readout for fast multi-characteristic profiling and/or label-free identification of the proteins.

[015] In embodiments:

[016] the transistors are graphene field-effect transistors (gFET);

[017] the transistors are silicon-based transistors;

[018] the chip is configured to include Y x X distances: chip: 5-40 mm x 5-40 mm; gel area: 5- 40 mm x 5-20 mm; gel height: 50-1000 pm; gel volume: 50-500 pL; isoelectric focusing area: 1- 10 mm x 1-20 mm; gel electrophoresis area: 5-20 mm x 2-20 mm; cover glass: thickness 20-200 um;

[019] the chip is configured to include Y x X distances: chip: 29.75 mm x 23.75 mm; gel area: 23 mm x 14 mm; max gel height: 700 pm; max gel volume: 225 pL; isoelectric focusing area: 5 mm x 11 mm; gel electrophoresis area: 16 mm x 8 mm; cover glass: thickness 170 um;

[020] the chip is configured with a power supply supplying 10-300 V output range x 2 channels; E fields: IEF: 0.91 - 27 V/mm; GE: 0.63 - 19 V/mm, wherein typical experiments use 10s of V/mm; [021] the chip is configured to include channel length /_c¾ = 100, 200 or 400 to 2000, 1000 or 600 um; channel width w_Ch = 1, 5 or lOum to 30, 50 or 100 um; window length l_w = 100, 200 or 400 um to 2000, 1000 or 600 um; window width w_w = 10, 50 or 80 um to 120, 200 or 500 um; array parameters: x-spacing Ax_a = 100, 300 or 600 um to 3000, 1500 or 1000; y-spacing Ay_a = 100, 400 or 800 to 3000, 2000 or 1200 um;

[022] the chip is configured to include channel length /_c¾ = 550 um; channel width w, i, = 20 um; window length l_w = 550 um; window width w_w = 100 um; array parameters: x-spacing Ax_a = 880 um; y-spacing Ay_a = 1080 um;

[023] the array comprises 80-480 transistors, including examples at 80, 160, 240 and 360 transistors;

[024] the chip comprises dimensions of 5-40 mm x 5-40 mm, and comprising 80-480 gFET channels divided into 2 groups, left and right, wherein for each side, the gFETs each have a unique source electrode and share a common drain electrode unique to each side of the chip, and two larger counter electrodes, and 2 smaller reference electrodes are positioned on the upper and lower sides of the chip and each pair correlate to either the left or right side of the chip, and distributed along the lateral ends of the chip, are 4 columns of contact pads spaced 0.20- 2.00 mm apart at center;

[025] the chip comprises dimensions of 24mm by 30mm, and comprising 80 gFET channels divided into 2 groups of 40 transistors, left and right, wherein for each side, the gFETs each have a unique source electrode and share a common drain electrode unique to each side of the chip, and two larger counter electrodes, and 2 smaller reference electrodes are positioned on the upper and lower sides of the chip and each pair correlate to either the left or right side of the chip, and distributed along the lateral ends of the chip, are 4 columns of 22 contact pads spaced 1.27mm apart at center;

[026] the chip is configured for separate control of isoelectric focusing (IEF) and gel electrophoresis (GE);

[027] the chip is configured to localize proteins during gel electrophoresis;

[028] the chip is configured to localize proteins to a pixel of area: /_c¾ * (Ay_a - w_Ch) = 0.10, 0.20 or 0.40 to 2.0, 1.0 or 0.80 mm²;

[029] the chip is configured to localize proteins to a pixel of area: /_c¾ * (Ay_a - w_Ch) = 0.58 mm²; [030] the chip is configured such that proteins crossing between channels will not be detected; [031] the chip comprises liquid gated field effect transistors in a facile and miniaturized electrophoresis system configured to identify and measure the levels of proteins, based on their molecular weight and charge that are determined with molecular precision, digitally and in real time; [032] the gel is configured with distinct isoelectric focusing (IEF) and gel electrophoresis (GE) areas, the gel overlays the array, wherein the chip is configured to provide direct digital label- free identification, characterization and quantification of the proteins, and real-time instantaneous fine resolution and sample detection of the proteins’ velocity and location; and/or [033] the chip is operatively connected to one or more electronic readers for gFET measurements;

[034] In an aspect the invention provides a method of using a subject chip, comprising separating the proteins in the gel and sensing the separated proteins with the array to provide a digital signal readout for multi-characteristic profiling and/or label-free identification of the proteins.

[035] In embodiments:

[036] a liquid-gate voltage is swept from -200 - -50mV to + 200 - +50mV at a speed of 0.1 - lHz, while a voltage of 10 - 200mV is swept between the drain and source electrodes; or [037] a liquid-gate voltage is swept from -lOOmV to + lOOmV at a speed of 0.3Hz, while a voltage of 50mV is swept between the drain and source electrodes.

[038] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[039] Brief Description of the Drawings

[040] Fig. 1. Device Assembly: spacer, PDMS glue, glass cover piece top, and glass bottom. [041] Fig. 2. Electronics Assay: gel casting, anode and cathode and protein ladder electrophoresis; optional additional layers: PDMS glue between spacer and top glass, bottom glass cover piece, PDMS glue between spacer and bottom glass.

[042] Fig. 3. Electrophoresis experiment optical measurement and COMSOF simulation (without gFET), lOkD, 5V.

[043] Fig. 4. Electrophoresis experiment optical measurement and COMSOF simulation (without gFET), TV.

[044] Fig. 5A-C. gFET design, fabrication, reading and assembly (pogo pins, electrical box, CNC milling for holders, and agile system)

[045] Figs. 6A-B. gFET electrical validation: Current measurement of sensing protein ladder on gFET, and Source-Drain Resistance measurement of with perpendicular electric field.

[046] Fig. 7A-B. Electrophoresis experiment electrical sensing of protein ladder (ME chip design)

[047] Fig. 8. Chip Overview - 80 gFET array design with separate IEF and GE gel areas. [048] Fig. 9. Overview of design and implementation of mass electrometry on a chip measurement platform.

[049] Description of Particular Embodiments of the Invention

[050] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. [051] Example: Design and implementation of mass electrometry on a chip measurement platform.

[052] ME typically uses two dimensional separations. In a first dimension separation takes place based on charge. A potential is applied through the gel with higher porosity (without denaturation, e.g. without SDS) to separate proteins based on charge. The detection region contains electrodes and graphene based FET sensors to separate proteins based on size (with denaturation, with SDS). To functionalize the surface of graphene, we employ polymers compatible for integration with graphene without affecting its properties: different polyacrylamide gels were synthesized with different porosity and size to provide sufficient separation and detection by the multiplex sensor. Polyacrylamide gel can be synthesized on the surface of graphene using a compatible cross-linker such as pyrenebutyric acid. Porosity of the polyacrylamide gel is tunable, such as by varying the percentage and volume of acrylamide/ bis acrylamide, percentage of initiator, polymerization time and type of cross linker. The parameters in the synthesis of the polymer are optimized for sensor response.

[053] The ME chips generally provide an array of 80-480 transistors, including examples at 80, 160, 240 and 360 transistors, typically with n+2 (e.g. 88) connections In embodiments the wafer and chip were designed for easy and quick fabrication 6 chips per wafer.

[054] The chip allows for separate control of IEF and GE. Y x X distances: Chip: 29.75 mm x 23.75 mm; Gel Area: 23 mm x 14 mm; Max gel height: 700 pm; Max gel volume: 225 pL; Isoelectric focusing area: 5 mm x 11 mm; Gel Electrophoresis area: 16 mm x 8 mm; Cover glass: Thickness 170 um. Power Supply (BioRad PowerPac Basic): 10-300 V output range x 2 channels; E fields: IEF: 0.91 - 27 V/mm; GE: 0.63 - 19 V/mm; typically experiments use 10s of V/mm. Device and array geometry designed to localize proteins during gel electrophoresis. Channel length /_c¾ = 550 um; channel width w, i, = 20 um; window length l_w = 550 um; window width w_w = 100 um; array parameters: x-spacing Ax_a = 880 um; y-spacing Ay_a = 1080 um. Proteins can be localized to a pixel of area: /_c¾ * (Ay_a - w_Ch) = 0.58 mm² , e.g.; proteins crossing between channels will not be detected

[055] Example: Graphene-based gel electrophoresis for high resolution and digital identification of proteins

[056] In an embodiment the invention provides a battery-operated and portable protein analysis electronic chip for protein separation and identification. The system termed, Mass Electrometry (ME), combines on-chip 2D-GE with SDS-PAGE and can electronically detect protein molecules utilizing an array of graphene field effect transistors (gFETs) in a real-time format. The ME-chip is engineered using established methods for fabrication of gFETs, to build a total sample-to-answer protein analysis system with a digital readout. A proof-of-concept ME system has 10 rows of 8 transistors, each capable of detecting one protein based on size and charge, significantly expanding the capacity of commonly used gel-based assays while reducing the cost and time of protein analysis. The device miniaturizes the traditional 2D-GE, thereby significantly reducing assay time, number of reagents, and system size while also increasing throughput and protein separation resolution. Additionally, the system has an integrated software analysis tool and provides one-step sample to answer system vs multi-step 2D-GE system, improving the efficiency of the process significantly.

[057] An advantage of our device is the use of gFET sensors because of its ability to perform label-less detection of analyte and its compact system size, which reduces complexity and cost associated with expensive fluorescent labels and bulky optical equipment. Furthermore, our use of gFET technology provides continuous monitoring of the protein migration and reduces the risk of sample loss due to over-run of electrophoresis. Real-time data acquisition is an advantage over others that utilize optics for data collection because it reduces the risk of obtaining ambiguous results. Furthermore, information gathered from gFETs can be utilized to identify unknown proteins in biological samples as well since the data collected from these sensors can be translated to protein velocity. Information about an unknown protein’ s speed can be used to estimate its mass and therefore size. This simplifies the process of identifying the unknown protein through the knowledge of its charge and size. The physical and chemical properties of gFET facilitate uses in biosensors through mechanical strength and pliability, conductivity and tunable band gap, specific surface area for bio molecule detection and tunable optical properties. The two-dimensional structure of graphene has a number of benefits over bulk semiconductors, such as silicon, used in standard FETs. Because most semiconductor transistor sensors are three- dimensional, electric charge changes at the surface of channel and do not always penetrate deeper into the body of the channel. This can dramatically limit the response sensitivity of the device. On the other hand, as the graphene in a gFET is only one carbon atom thick, the entirety of the channel is exposed to the analyte.

[058] Overall, the gFET-ME system is very versatile, specific, and reliable. Utilizing sensors that continually collect data over imaging techniques that rely on exact protein placement provide a great advantage in obtaining reliable data that can be utilized effectively. Data collected on the movement of individual proteins throughout the gel can be used to understand their many properties that distinguish them including charge and mass. Such properties would be more difficult to quantify using imaging techniques due to the low sensitivity of such techniques. Conversely, the high sensitivity of the gFET-ME system to moving charge allows for accurate data acquisition and protein identification.

[059] Spacer and Cover Piece Assembly

[060] The construction of the ME prototype includes spacer and cover piece fabrication, structural assembly, and gel fabrication. The spacer was fabricated from low density polyethylene. This material is compatible with the polyacrylamide gel and its malleability makes it resistant to permanent deformation. The part was designed in computer assisted design (CAD) software, and was fabricated through computer numerical control (CNC) milling (Roland Model MDX-40A). The purpose of the spacer was to define the thickness of the gel along the z-axis. There are clearly defined alignment marks on the spacer to ensure that the cover piece is placed in a reproducible location to minimize assay-to-assay variability. For the reproducible placement of the spacer onto the chip, an aluminum alignment structure was fabricated with CNC milling. Positive stops ensure that the spacer and ME chip sit securely in a cavity cut out from the alignment structure. A cover piece was fabricated from a glass slide that was cut to size using a glass cutter and further refined using a Dremel fitted with a diamond bit (7150, Dremel) (Dremel 3482, USA). Once the individual components of the ME were manufactured, the structure was assembled using a custom milled alignment guide. The different parts were adhered together using the silicone-based elastomer, liquid polydimethylsiloxane (PDMS) (Ellsworth 184 SIL ELAST KIT 3.9KG) as an adhesive. The PDMS was prepared as a 10:1 base to curing agent weight ratio. After the PDMS was applied to the chip, it was incubated at 60°C for 2 hours to cure.

[061] Gel Electrophoresis Assembly

[062] The gel was prepared according to a standard gel protocol (BIORAD) to formulate a 37:1 acrylamide/bis-acrylamide gel, with adaptations: (1) a stacking gel was not cast and (2) the gel was cast in a nitrogen gas filled vacuum chamber. In brief, the glass substrate was cleaned using Acetone, Isopropyl alcohol (IP A), Deionized (DI) water, and dried with N2 gas. The gel was prepared by mixing 187.5 pL 40% 37:1 Acrylamide/ Bis solution (Sigma- Aldrich a6050) with 10 pL 10% SDS solution and 250 pL 1.5M Tris-HCL (pH 8.8, BioRad, 1610798). 548 pL of diH20 was added to make approximately 1 mL of gel. The gel precursor solution was then degassed using a vacuum desiccator for roughly 15 minutes. Following degassing, 0.5 pL tetramethylethylenediamine (TEMED, BioRad 161-0801) and 5 pL of fresh 10% ammonium persulfate (APS, BioRad 1610700) were added to the gel solution and mixed in a manner that did not introduce air into the gel precursor. The precursor gel solution was then pipetted into the cavity atop the ME chip, as defined by the spacer and glass cover piece. The gel was introduced from the bottom of the chip and it was filled, leaving a 1mm gap at the top of between the liquid front and the edge of the cover piece. Finally, the gel was cured at approximately 25°C for 120 minutes, in a nitrogen filled chamber at a slight downward slope. The gel is made so that the top and bottom of the gel have openings that can accept the Tris Glycine SDS (TGS) buffer for the gel, and also allow for any gases formulated during electrophoresis to vent.

[063] Once the structure of the ME chip and gel was cast, the gel electrophoresis procedure began. To ran gel electrophoresis, two sections of 3cm platinum 30-gauge wires were bent into a hook-shape with a straight edge and connected to a benchtop power supply using alligator clips. The anode was placed at the top of the chip and the cathode was positioned at the bottom in a mirrored orientation. The cathode platinum wire was placed so that it did not contact the platinum counter electrode at the bottom of the chip.

[064] Time and Separation Validation

[065] A separate, modified experiment was conducted to estimate how long it would take electrophoresis to ran in the specific geometry of the ME chip. This experiment would also demonstrated proper separation of proteins. The first step of this validation process was fabricating the modified ME chip, using conventional procedure except the silicon chip substrate was replaced with a glass slide. Then, a 10kda-250kda protein ladder was loaded on top of the gel, in the well that was formed below the glass slide, at the interface between the air and gel. The bottom reservoir was filled with TGS running buffer. The cathode electrode was then submerged into the running buffer and the anode electrode was placed in contact with the protein ladder. 5V of DC potential was applied to the gel. After 20 minutes, the anode reservoir at the top of the chip was washed 3 times with TGS buffer, and then filled with TGS buffer. The buffer in both reservoirs was replaced every 30 minutes and the amount of time required for electrophoresis to ran was recorded. The completion of electrophoresis was marked by the dye front reaching the bottom of the gel. This would also verify that the system is capable of separating proteins of different sizes. At the end of this validation process, the identification of distinct protein bands was visually confirmed.

[066] Electronics [067] The device uses two Agile R100 system (Nanomed) electronic readers for the gFET measurements. Each R100 is capable of reading 40 transistors, therefore, a total of 80 transistors are utilized for readings. The readers control either the left or right side of the chip, and compile measurements to a computer through a USB cable connection.

[068] During the experiment, a liquid-gate voltage is swept from -lOOmV to + lOOmV at a speed of 0.3Hz. Meanwhile, a voltage of 50mV is swept between the drain and source electrodes. Prior to the introduction of the analyte, a calibration step collects the initial background readings until an equilibrium is reached. This equilibrium point is then set as the zero point. The units of measurements are in BU (biological unit). 10 BU is equivalent to 1% variation in the current or capacitance of the channel.

[069] The R100 has two cable connections: a USB cable and a 68 pin VHDCI connector that leads to a breakout box. The breakout box houses two 68 pin VHDCI breakout boards, one for each R100. The 68 individual pins from the connector correspond with the other 68 pins on the breakout board. Metal clamps can be attached to screw terminals on the board, which contacts the wire to the corresponding pins of the connector.

[070] The ME chip sits on an acrylic mounting plate within a recess. Features of the recess allow for the reliable and reproducible placement. Acrylic brackets were designed to connect to the plate, which were localized with dowel pins and secured with set screws. Holes within the bracket were milled with the CNC in precise locations where pogo pins were then pushed down and pressure fit into this cavity: two rows of 22 pins for a total of with 44 pogo pins. Pogo pins were spaced 1.27mm apart on center. 26 AWG magnet wires connect to the pogo pins to trace the individual connections of the reader to the corresponding contact pads on the ME chip.

[071] gFET Fabrication

[072] The gFET has been fabricated using optimised lithographic procedures. The gFET chip has dimensions of 24mm by 30mm. The 80 gFET channels can be divided into 2 groups of 40 transistors, left and right. For each side, the individual gFET each contain a unique source electrode and share a common drain electrode unique to each side of the chip. Two large counter electrodes, and 2 small reference electrodes are positioned on the upper and lower sides of the chip and each pair correlate to either the left or right side of the chip. The surface of the chip is passivated with a layer of silicon nitride. Evenly distributed along the lateral ends of the chip, there are 4 columns of 22 contact pads spaced 1.27mm apart at the centre.

[073] Electrical and optical validation of gFET

[074] Optical inspection of the graphene was performed with scanning microscope (Nanotronic, nSpec 3D) using 20X objective with differential enhance contrast. The detailed features were inspected to identify graphene channel defects such as double layer, scratches, contamination, or tears.

[075] Device Electrical validation

[076] For the electric validation of the gFET sensing device, rigorous quality control (QC) steps were followed. In addition to optical evaluation of gFETs, drain to source resistance measurements under dry conditions were obtained. The resistance measurements would then inform on the health and locations of defective gFET sensors within the gFET array. Transfer curves measuring the Dirac point of the gFET sensing array incubated in IX PBS and 0.5M MES buffers were also be measured on randomly selected samples for quality control. Sensitivities of the gFET devices in the array were then evaluated by gauging the magnitude and the direction of shifts in Dirac points during the buffer exchange. Histograms of the electrical measurements were collected to characterize the devices within each individual batch process. Variations from batch to batch were related to observations from the optical inspection and records of manufacturing processes to identify crucial process steps that may have impacted the device performance. The QC information obtained was then used to guide the process and to maintain consistent batch to batch performance. The results successfully showed that the 80-plex chips with at least 72 working transistors (90%) within acceptable tolerance (50+20kO) in the source-to-drain resistance.

[077] Optical validation of electrophoresis

[078] To verify positive gFET detection of target proteins during electrophoresis, optical validation was acquired simultaneously to the assay to correlated with the electric acquisition. Acquisition was performed with the UVP iBox2 imaging microscope (Analytik Jena) due to its large working distance. To capture the FITC tagged proteins, an excitation (455-495nm) and emission (535-544nm) bandpass filters used. Images were captured and processed using the VisionWorks software and with a light intensity setting of 6 and a 1 second of exposure.

[079] COMSOL Analysis

[080] A simulation using COMSOL Mutiphysics applying the Finite Element Method (FEM) was created to predict and stimulate the Mass Electrometry (ME) chip.This was followed by several parametric studies to validate the model and optimize the design parameters. On verifying these numerical results, a PDMS based microfluidic based device on a glass slide/wafer was then fabricated.

Claims

CLAIMS:

1. A mass-electrometry chip comprising: an array of transistors, each comprising of a channel between two electrodes with a gate contact to modulate the electronic response of the channel; and an electrophoresis or focusing gel comprising gel-separated proteins; wherein the chip is configured so that the array provides a digital signal readout for fast multi-characteristic profiling and/or label-free identification of the proteins.

2. The chip of claim 1 wherein the transistors are graphene field-effect transistors (gFET).

3. The chip of claim 1 wherein the transistors are silicon-based transistors.

4. The chip of claim 1 configured to include Y x X distances: chip: 5-40 mm x 5-40 mm; gel area: 5-40 mm x 5-20 mm; gel height: 50-1000 pm; gel volume: 50-500 pL; isoelectric focusing area: 1-10 mm x 1-20 mm; gel electrophoresis area: 5-20 mm x 2-20 mm; cover glass: thickness 20-200 um.

5. The chip of claim 1 configured to include Y x X distances: chip: 29.75 mm x 23.75 mm; gel area: 23 mm x 14 mm; max gel height: 700 pm; max gel volume: 225 pL; isoelectric focusing area: 5 mm x 11 mm; gel electrophoresis area: 16 mm x 8 mm; cover glass: thickness 170 um.

6. The chip of claim 1 configured with a power supply supplying 10-300 V output range x 2 channels; E fields: IEF: 0.91 - 27 V/mm; GE: 0.63 - 19 V/mm, wherein typical experiments use 10s of V/mm.

7. The chip of claim 1 configured to include channel length /_c¾ = 100, 200 or 400 to 2000, 1000 or 600 um; channel width w_Ch = 1, 5 or lOum to 30, 50 or 100 um; window length l_w = 100, 200 or 400 um to 2000, 1000 or 600 um; window width w_w = 10, 50 or 80 um to 120, 200 or 500 um; array parameters: x-spacing Ax_a = 100, 300 or 600 um to 3000, 1500 or 1000; y-spacing Ay_a = 100, 400 or 800 to 3000, 2000 or 1200 um.

8. The chip of claim 1 configured to include channel length /_c¾ = 550 um; channel width w, i, = 20 um; window length l_w = 550 um; window width w_w = 100 um; array parameters: x-spacing Ax_a = 880 um; y-spacing Ay_a = 1080 um.

9. The chip of claim 1 wherein the array comprises 80-480 transistors, including examples at 80, 160, 240 and 360 transistors.

10. The chip of claim 1 comprising dimensions of 5-40 mm x 5-40 mm, and comprising 80-480 gFET channels divided into 2 groups, left and right, wherein for each side, the gFETs each have a unique source electrode and share a common drain electrode unique to each side of the chip, and two larger counter electrodes, and 2 smaller reference electrodes are positioned on the upper and lower sides of the chip and each pair correlate to either the left or right side of the chip, and distributed along the lateral ends of the chip, are 4 columns of contact pads spaced 0.20- 2.00 mm apart at center.

11. The chip of claim 1 comprising dimensions of 24mm by 30mm, and comprising 80 gFET channels divided into 2 groups of 40 transistors, left and right, wherein for each side, the gFETs each have a unique source electrode and share a common drain electrode unique to each side of the chip, and two larger counter electrodes, and 2 smaller reference electrodes are positioned on the upper and lower sides of the chip and each pair correlate to either the left or right side of the chip, and distributed along the lateral ends of the chip, are 4 columns of 22 contact pads spaced 1.27mm apart at center.

12. The chip of claim 1 configured for separate control of isoelectric focusing (IEF) and gel electrophoresis (GE).

13. The chip of claim 1 configured to localize proteins during gel electrophoresis.

14. The chip of claim 1 configured to localize proteins to a pixel of area: /_c¾ * (Ay„ - w_Ch) = 0.10, 0.20 or 0.40 to 2.0, 1.0 or 0.80 mm².

15. The chip of claim 1 configured to localize proteins to a pixel of area: /,_/, * (Ay„ - w_Ch) = 0.58 mm².

16. The chip of claim 1 configured such that proteins crossing between channels will not be detected.

17. The chip of claim 1 comprising liquid gated field effect transistors in a facile and miniaturized electrophoresis system configured to identify and measure the levels of proteins, based on their molecular weight and charge that are determined with molecular precision, digitally and in real-time.

18. The chip of claim 1 wherein the gel is configured with distinct isoelectric focusing (IEF) and gel electrophoresis (GE) areas, the gel overlays the array, wherein the chip is configured to provide direct digital label-free identification, characterization and quantification of the proteins, and real-time instantaneous fine resolution and sample detection of the proteins’ velocity and location.

19. The chip of claim 1 operatively connected to one or more electronic readers for gFET measurements.

20. A method of using the chip of any of claims 1, comprising separating the proteins in the gel and sensing the separated proteins with the array to provide a digital signal readout for multi characteristic profiling and/or label-free identification of the proteins.

21. The method of claim 20, wherein a liquid-gate voltage is swept from -200 - -50mV to + 200 - +50mV at a speed of 0.1 - lHz, while a voltage of 10 - 200mV is swept between the drain and source electrodes.

22. The method of claim 20, wherein a liquid-gate voltage is swept from -lOOmV to + lOOmV at a speed of 0.3Hz, while a voltage of 50mV is swept between the drain and source electrodes.