Carbon-based field effect transistor combined with microfluidics for single-cell analysis and application, method for detecting surface membrane protein of single cell

Abstract

The application provides a single cell analysis chip combining a carbon-based field effect transistor and microfluidics, comprising a plurality of single cell capture units; wherein the single cell capture unit comprises a sensing unit and a PDMS microfluidic channel unit arranged correspondingly to the sensing unit; the sensing unit is a carbon-based field effect transistor, and the PDMS microfluidic channel unit comprises a circular capture trap chamber vertically and directly above a sensing area of the carbon-based field effect transistor and a narrow channel; a first end of the circular capture trap chamber is connected with a first end of a first main channel, a second end of the circular capture trap chamber is connected with a first end of a second main channel through the narrow channel, and the first main channel and the second main channel are connected through an arc-shaped side channel; after cells are captured through the narrow channel, subsequent cells can flow into the next PDMS microfluidic channel unit through the arc-shaped side channel. The chip can separate and capture single cells, and achieve the purpose of detecting the surface proteins of single cells.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics and sensor technology, and more specifically to a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics, its application, and a method for detecting single-cell surface membrane proteins. Background Technology

[0002] In recent years, the incidence and mortality rates of cancer in my country have been rising continuously, ranking among the highest in the world, seriously threatening the lives and property of our citizens. In order to reduce the incidence and mortality rates of cancer and safeguard the health of citizens, the most effective method at present is to conduct early screening.

[0003] Currently, the most common and effective early screening method is screening for circulating tumor cells (CTCs). However, traditional screening methods involve mass screening of cells, which masks cellular heterogeneity, a crucial factor in cancer detection. Therefore, we need to analyze individual cells to study their heterogeneity, identify cell subtypes, and better tailor subsequent targeted treatments.

[0004] Analysis of single cells mainly includes the analysis of intracellular and extracellular proteins. Intracellular protein analysis requires cell lysis, which can easily lead to protein loss and result in errors, and the procedure is relatively cumbersome. Extracellular protein analysis, on the other hand, does not require lysis, is simple to perform, preserves cell viability, and offers high accuracy.

[0005] Currently available methods for analyzing single-cell surface proteins mainly include surface-enhanced Raman spectroscopy, enzyme-catalyzed reactions based on silicon nanowires, and microfluidic methods based on fluorescence analysis. While these methods can detect cell surface proteins, they suffer from drawbacks such as low detection accuracy and the inability to isolate individual cells. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a single-cell analysis chip that combines carbon-based field-effect transistors with microfluidics. The high sensitivity and high stability of carbon-based field-effect transistors enable the analysis of protein content on the surface of single cells, while microfluidic channels enable the separation and capture of single cells, thereby achieving the purpose of detecting proteins on the surface of single cells.

[0007] According to a first aspect of the present invention, a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics is provided, comprising a plurality of single-cell capture units; wherein, the single-cell capture unit comprises a sensing unit and a PDMS microfluidic channel unit corresponding to the sensing unit;

[0008] The sensing unit is a carbon-based field-effect transistor. The carbon-based field-effect transistor includes silicon and silicon dioxide stacked in sequence, and carbon nanotubes deposited on the surface of silicon dioxide as a substrate. The substrate is provided with a source electrode and a drain electrode arranged opposite to each other and separated from each other. A dielectric layer composed of yttrium oxide and hafnium oxide deposited in sequence is provided on the carbon nanotube channel layer between the source electrode and the drain electrode.

[0009] The PDMS microfluidic channel layer unit covers the sensing unit and is used to transmit the liquid to be detected, including a circular trap chamber and a narrow channel located directly above the sensing area of ​​the carbon-based field-effect transistor.

[0010] The first end of the circular capture trap chamber is connected to the first end of the first main channel, and the second end of the circular capture trap chamber is connected to the first end of the second main channel through a narrow channel. The first main channel and the second main channel are connected by an arc-shaped side channel, which allows subsequent cells to flow into the next PDMS microfluidic channel unit after the narrow channel captures cells.

[0011] The single-cell analysis chip has an inlet at the starting end for the liquid to be tested to enter, and the inlet is connected to the second end of the first main channel of the PDMS microfluidic channel unit at the starting end; the single-cell analysis chip has an outlet at the ending end for the liquid to be tested to flow out, and the outlet is connected to the second end of the second main channel of the PDMS microfluidic channel unit at the ending end.

[0012] In an optional implementation, the single-cell analysis chip contains at least four single-cell capture units.

[0013] In an optional implementation, the circular capture trap chamber is a circular channel with a radius r of 30 μm.

[0014] In an optional implementation, the first main channel and the second main channel are rectangular channels of equal width, with the width D1 being 3 to 4 times that of the target cell.

[0015] In an optional implementation, the narrow channel is a rectangular channel with a width D2 that is 2 / 3 times that of the target cell.

[0016] In an optional implementation, the arc-shaped side channel is an arc-shaped channel with a width D3 that is 2 to 3 times that of the target cell.

[0017] In an optional implementation, the design principle for the height H of the PDMS microfluidic channel unit is that H must be greater than the diameter of one cell and less than the diameter of two cells.

[0018] In a second aspect of the present invention, an application of the aforementioned carbon-based field-effect transistor combined with microfluidics in single-cell surface protein analysis is provided.

[0019] According to a third aspect of the present invention, a method for detecting single-cell surface membrane proteins is provided, employing a single-cell analysis chip combining the aforementioned carbon-based field-effect transistor and microfluidics, comprising the following steps:

[0020] S1. The polydopamine solution is injected into the PDMS microfluidic channel through the injection port using a syringe pump, so that the polydopamine solution fills all the channels of the microfluidic chip and incubates in the entire PDMS microfluidic channel of the chip, as well as on the dielectric layer of the carbon-based field-effect transistor to form a polydopamine film.

[0021] S2. Take the cell suspension to be tested and flow it into the chip after being processed in step S1 from the injection port through the injection pump. The cells are captured into a single-cell state by the narrow channel.

[0022] S3. After the cells are captured in step S2, the chip is placed in a cell culture incubator for culture. The transfer characteristic curve of the chip after cell capture is compared and analyzed with the initial chip transfer characteristic curve. The changes in the current and threshold voltage of the device transfer characteristic curve after cell capture are observed to detect whether the chip responds to the cells.

[0023] S4. After confirming that the chip responds to cells, BSA blocking solution is injected into the chip through the injection port using a syringe pump and incubated at room temperature to reduce non-specific adsorption on the cell surface. After incubation, the transfer characteristic curve of the chip is measured as the detection baseline.

[0024] S5. Gold nanoparticles modified with the assembled Sgc8 aptamer are injected into the chip after step S4 via the injection port using a syringe pump. The chips are then incubated at room temperature. After incubation, the transfer characteristic curve of the chip is measured and compared with the detection baseline. The change in the chip transfer characteristic curve is equivalent to the content of PTK7 protein on the surface of a single cell.

[0025] In an optional embodiment, the incubation conditions for the polydopamine layer are: incubation at 25°C and 60% humidity for 30 minutes; the thickness of the resulting polydopamine film is 3 nm.

[0026] In an optional implementation, the injection flow rate in steps S2 and S4 is 0.1–0.5 μL / min.

[0027] The single-cell analysis chip proposed in this invention, which combines carbon-based field-effect transistors with microfluidics, can simultaneously separate and capture multiple single cells and subsequently culture them for extended periods. This ensures that the target cell is a single cell and does not affect cell viability. The microchamber design avoids cross-contamination and solution evaporation during subsequent testing. Furthermore, the high sensitivity and stability of the carbon-based field-effect transistors allow for the detection of even very low changes in cell surface protein content.

[0028] The present invention proposes a method for detecting single-cell surface membrane proteins using a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics. By optimizing the flow rate and narrow-band channel size, it achieves high capture efficiency and can separate and fix single cells. The method is simple, efficient, low-cost, highly sensitive, has a low detection limit, a large response, and causes minimal damage to cells, allowing for long-term cell culture observation. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of the carbon-based field-effect transistor of the present invention.

[0030] Figure 2 This is a schematic diagram of the structure of the single-cell analysis chip combining carbon-based field-effect transistors and microfluidics of the present invention.

[0031] Figure 3 This is a schematic diagram of the structure of the PDMS microfluidic channel unit of the present invention.

[0032] Figure 4 This is a diagram showing the internal structural dimensions of the PDMS microfluidic channel unit of the present invention.

[0033] Figure 5 This is a flowchart illustrating the fabrication process of an exemplary carbon-based field-effect transistor of the present invention.

[0034] Figure 6 This is an exemplary flowchart of the preparation process of the PDMS microfluidic channel of the present invention.

[0035] Figure 7 This is an illustration of the effect of capturing a single cell using a single-cell analysis chip that combines a carbon-based field-effect transistor with microfluidics, as exemplified by this invention.

[0036] Figure 8 This is a schematic diagram of the principle of cell surface protein detection using a carbon-based field-effect transistor according to the present invention.

[0037] Figure 9 This is a schematic diagram of the principle of cell surface protein detection using a carbon-based field-effect transistor according to the present invention.

[0038] Figure 10 This is a graph showing the effect of PDMS microchannel microstructure size and cell flow rate on cell capture effect according to an embodiment of the present invention; wherein, a is a graph showing the effect of microfluidic chips with five different narrow-band channels of 5-9 μm on cell capture effect at a flow rate of 0.1-0.5 μL / min; b is a graph showing the effect of microfluidic chips on cell capture effect at a flow rate of 0.1 μL / min and a narrow-band channel width of 7 μm.

[0039] Figure 11The graph shows the detection results of single-cell surface protein content in an embodiment of the present invention; wherein, a and b are the transfer characteristic curves tested in Example 4, a is a logarithmic coordinate and b is a linear coordinate; c and d are the transfer characteristic curves of the control group using aptamers with full T sequences, c is a logarithmic coordinate and d is a linear coordinate. Detailed Implementation

[0040] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0041] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0042] carbon-based field-effect transistors

[0043] Combination Figure 1 The carbon-based field-effect transistor of the exemplary embodiment shown includes a silicon substrate 110, a silicon dioxide dielectric layer 120, a carbon nanotube (CNT) channel layer 130, a drain electrode 141, a source electrode 142, and a dielectric layer 150.

[0044] Combination Figure 1 The silicon dioxide dielectric layer 120 is located on the first surface of the silicon substrate 110. As an optional example, the silicon dioxide dielectric layer 120 is used as the deposition substrate for CNTs, while achieving electrical insulation between the CNTs and the silicon substrate 110.

[0045] like Figure 1 For example, CNTs are located on the side of the silicon dioxide dielectric layer 120 away from the silicon substrate. The CNTs are randomly laid out on the upper surface of the silicon dioxide dielectric layer 120, and the diameter of each individual carbon nanotube is controlled between 1 and 3 nm.

[0046] Drain electrode 141 and source electrode 142 are located on CNT, arranged opposite to each other and spaced apart, so that the length of CNT channel layer 130 formed between the source and drain electrodes is maintained at about 20 μm and the width is maintained at about 40 μm.

[0047] The drain electrode 141 and the source electrode 142 are preferably titanium palladium gold electrodes, with thicknesses of 0.6 nm, 20 nm and 60 nm, respectively.

[0048] The dielectric layer 150 is located on the side above the CNT channel layer 130 away from the silicon substrate 110, and the dielectric layer should completely cover the CNT channel region between the drain electrode 141 and the source electrode 142. The dielectric layer 150 is preferably composed of yttrium oxide and hafnium oxide deposited sequentially, with thicknesses of 3 nm for yttrium oxide and 10 nm for hafnium oxide.

[0049] As is understandable, carbon-based field-effect transistors also include metal leads that guide the drain and source electrodes to the corresponding output electrodes, as well as corresponding passivation layers for protection. As this is prior art, it will not be described in detail here.

[0050] Single-cell analysis chip combining carbon-based field-effect transistors and microfluidics

[0051] The aforementioned carbon-based field-effect transistor is used as the sensing unit of a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics. Figure 2 , Figure 3 and Figure 4 The exemplary embodiment shown includes a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics, comprising multiple single-cell capture units; wherein each single-cell capture unit includes a sensing unit 210 and a PDMS microfluidic channel unit 220 corresponding to the sensing unit.

[0052] The PDMS microfluidic channel layer unit 220 covers the sensing unit 210 and is used to transfer the liquid to be detected, including a circular trap chamber 221 and a narrow channel 222 located directly above the sensing region of the carbon-based field-effect transistor.

[0053] Combination Figure 2 and Figure 3 As shown, the first end of the circular capture trap chamber 221 is connected to the first end of the first main channel 223, and the second end of the circular capture trap chamber 221 is connected to the first end of the second main channel 224 through the narrow band channel 222. The first main channel 223 and the second main channel 224 are connected by an arc-shaped side channel 225, which is used to capture cells through the narrow band channel 222, and subsequent cells can flow into the next PDMS microfluidic channel unit through the arc-shaped side channel 225.

[0054] The single-cell analysis chip has an inlet 240 at the starting end for the liquid to be tested to enter, and the inlet 240 is connected to the second end of the first main channel 222 of the starting end PDMS microfluidic channel unit; the single-cell analysis chip has an outlet 250 at the ending end for the liquid to be tested to flow out, and the outlet 250 is connected to the second end of the second main channel 223 of the ending end PDMS microfluidic channel unit.

[0055] In an optional embodiment, the single-cell analysis chip includes at least four single-cell capture units, and the spacing between adjacent single-cell capture units is preferably 500 μm to ensure that single cells are captured. During the delivery of the liquid to be tested, multiple cells may be captured after passing through the single-cell capture unit at the front end. As the path lengthens, the capture gradually stabilizes. Therefore, if there are too few single-cell capture units, it may be impossible to effectively capture single cells. Preferably, there are 10 single-cell capture units.

[0056] Combination Figure 3 , Figure 4 As shown, in an optional embodiment, the circular trap chamber 221 is a circular channel with a radius r of 30 μm, used to fix single cells and for subsequent cell culture, and is aligned with the carbon-based field-effect transistor channel sensing area at the bottom to ensure that the cell is located exactly in the device channel area when it is captured.

[0057] In an optional implementation, the first main channel 222 and the second main channel 223 are rectangular channels of equal width, with a width D1 that is 3 to 4 times that of the target cell, to prevent cells from clogging the main channels and affecting the capture and fixation of subsequent single cells; in an exemplary embodiment, D1 is 50 μm.

[0058] In an optional embodiment, the narrow channel 224 is a rectangular channel with a width D2 that is 2 / 3 times that of the target cell, used to trap a single cell at the exit of the circular capture trap chamber 221; in an exemplary embodiment, D2 is 5 to 9 μm, particularly preferably 7 μm, and the length of the narrow channel 224 is preferably 15 μm;

[0059] If D2 is less than 5μm, it is easy to cause blockage and affect subsequent tests; if D2 is greater than 9μm, the cells cannot be fixed in the circular single-cell capture trap and the cells will flow away directly from the narrow main channel as the solution is injected; the current optimal size is 7μm.

[0060] In an optional implementation, the arc-shaped side channel 225 is an arc-shaped channel with a width D3 that is 2 to 3 times that of the target cell. After the narrow-band channel 224 captures the cell, subsequent cells can flow into the next capture unit through the second main channel. In an exemplary embodiment, D3 is 35 μm and the length of the arc-shaped side channel is designed to be 750 μm.

[0061] In an optional implementation, the height H of the PDMS microfluidic channel layer unit is designed to be greater than the diameter of one cell but less than the diameter of two cells to prevent cells from stacking up and being trapped in a circular single-cell trap. In an exemplary embodiment, H is 18 μm.

[0062] In an optional embodiment, both the inlet 240 and the outlet 250 are circular in shape with a radius of 1 mm, which is used to subsequently drill an inlet / outlet with a radius of 0.4 mm at the center. This shape and size can prevent the circular inlet / outlet from being drilled off-center.

[0063] Design method for the dimensions of each part in PDMS microchannel unit

[0064] Because the fluid resistance along the straight channel is smaller than that along the annular channel (narrow channel resistance < arc-shaped side channel resistance), the cell flow in the channel will be carried to the circular trap chamber and fixed at the junction of the narrow channel and the circular trap chamber, which will greatly increase the fluid resistance of the narrow channel. The subsequent cell flow will bypass the narrow main channel and flow through the arc-shaped side channel.

[0065] The pressure drop across the narrow main channel and the curved side channel is the same, according to Darcy's law:

[0066]

[0067] Its pressure drop is in the form of:

[0068]

[0069] Where Δp is the pressure drop, l is the length of the microchannel, d is the height of the microchannel, V is the flow velocity, λ is the Darcy friction factor, and ρ is the liquid density. Transforming the formula, we get:

[0070]

[0071] Where P is the perimeter of the microchannel, L is the length of the microchannel, A is the cross-sectional area of ​​the microchannel, μ is the fluid viscosity, Q is the volumetric flow rate Q=V×A, and C(α) is a dimensionless parameter related to the aspect ratio.

[0072] Δp is applied to the narrow main channel and the curved side channel respectively. Since the pressure difference is the same in both paths, Q1 / Q2 can be expressed as:

[0073]

[0074] Substitute the numerical calculations and design the channel size according to the size of the cells to be captured. Here, Q1 represents the volumetric flow rate at both ends of the narrow channel and Q2 represents the volumetric flow rate at both ends of the arc-shaped side channel. When the value of Q1 is greater than the value of Q2, it means that the volumetric flow rate in the narrow channel is greater and the flow rate is faster. The mainstream direction is the direction of the narrow channel, and the cells will be carried to the circular capture trap chamber along with the mainstream.

[0075] Once the cells are captured and fixed, the main flow direction changes to an arc-shaped side channel, and the cells will flow into the next capture unit along the arc-shaped side channel.

[0076] Fabrication method of single-cell analysis chip combining carbon-based field-effect transistors and microfluidics

[0077] The single-cell analysis chip combining carbon-based field-effect transistors and microfluidics of the present invention is formed by placing the carbon-based field-effect transistor array and the PDMS microfluidic channel layer into a plasma cleaner for plasma treatment, and then aligning and bonding them using a high-precision multifunctional two-dimensional material transfer platform.

[0078] Combination Figure 5 and Figure 6 As shown in the exemplary embodiment of the present invention, the fabrication process of the single-cell analysis chip combining carbon-based field-effect transistors and microfluidics is as follows:

[0079] (1) Fabrication of carbon-based field-effect transistor array

[0080] like Figure 5 As shown, a layer of carbon nanotubes is deposited on a silicon wafer. Figure 5 Part a), and then using LOR and S1813 photoresist, the metal electrode is prepared through steps such as homogenization, photolithography, development, and deposition. Figure 5 (part b);

[0081] Secondly, a channel window is created, and carbon nanotubes outside the channel are etched. Figure 5 Part c), then 3nm yttrium oxide is deposited to form yttrium oxide ( Figure 5 The d-part), followed by the growth of a 10 nm thick hafnium oxide layer on the yttrium oxide surface using ALD (to form) Figure 5 (part e).

[0082] (2) Preparation of PDMS microfluidic channel layer

[0083] like Figure 6 As shown, an SU-8 mold with a thickness of 18 μm was prepared by ultraviolet lithography, and then a PDMS microfluidic channel layer was formed by pouring a mixture of PDMS and PDMS prepolymer onto the SU-8 mold and drying and curing it.

[0084] (3) Cover the prepared PDMS microfluidic channel layer over the carbon-based field-effect transistor array and position the circular trap chamber vertically directly above the sensing area of ​​the carbon-based field-effect transistor.

[0085] In an exemplary embodiment of the present invention, an application of the aforementioned carbon-based field-effect transistor combined with microfluidics single-cell analysis chip in single-cell surface protein analysis is also provided.

[0086] Methods for detecting single-cell surface membrane proteins

[0087] Combination Figure 7As shown, a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics is used to detect single-cell surface membrane proteins. Taking 10 single-cell capture units as an example, the detection process includes the following steps:

[0088] S1. The polydopamine solution is injected into the PDMS microfluidic channel through the injection port using a syringe pump, so that the polydopamine solution fills all the channels of the microfluidic chip and incubates in the entire PDMS microfluidic channel of the chip, as well as on the dielectric layer of the carbon-based field-effect transistor to form a polydopamine film.

[0089] S2. Take the cell suspension to be tested and flow it into the chip after being processed in step S1 from the injection port through the injection pump. The cells are captured into a single-cell state by the narrow channel.

[0090] S3. After the cells are captured in step S2, the chip is placed in a cell culture incubator to allow the cells to fully bind with polydopamine. The transfer characteristic curve of the chip after cell capture is compared and analyzed with the initial chip transfer characteristic curve. The changes in the current and threshold voltage of the device transfer characteristic curve after cell capture are observed to detect whether the chip responds to the cells.

[0091] S4. After confirming that the chip responds to cells, BSA blocking solution is injected into the chip through the injection port using a syringe pump and incubated at room temperature to reduce non-specific adsorption on the cell surface. After incubation, the transfer characteristic curve of the chip is measured as the detection baseline.

[0092] S5. Gold nanoparticles modified with the assembled Sgc8 aptamer are injected into the chip after step S4 via the injection port using a syringe pump. The chips are then incubated at room temperature. After incubation, the transfer characteristic curve of the chip is measured and compared with the detection baseline. The change in the chip transfer characteristic curve is equivalent to the content of PTK7 protein on the surface of a single cell.

[0093] In an optional embodiment, the incubation conditions for the polydopamine layer are: incubation at 25°C and 60% humidity for 30 minutes; the thickness of the resulting polydopamine film is 3 nm.

[0094] In an optional implementation, the injection flow rate in steps S2 and S4 is 0.1–0.5 μL / min.

[0095] In an optional embodiment, the polydopamine solution is prepared by mixing Tris buffer solution at pH 8.5 with polydopamine powder at a volume ratio of 2 mg: 1 mL.

[0096] In an optional embodiment, the cell suspension is a solution prepared by mixing RPMI-1640 (containing double antibiotics) basal culture medium with fetal bovine serum and cells at a volume ratio of 10:1.

[0097] Detection mechanism such as Figure 8 , Figure 9 As shown, Figure 8 In this invention, the sensing unit captures the cell to be tested, and then, as... Figure 9 As shown, the captured PTK7 protein on the surface of A549 cells specifically binds to the self-assembled Sgc8 aptamer gold nanoparticles. However, the concentration of the cell surface protein is very low, and the response may be small. Therefore, the signal is amplified by the gold nanoparticles at the other end of the aptamer. When the aptamer binds to the target protein, since the aptamer itself is negatively charged, this negative charge is conducted to the cell membrane surface, increasing the amount of negative charge on the cell membrane surface. Since the carbon-based field-effect transistor is a P-type device, the charge carriers are positively charged holes. The large amount of negative charge on the cell membrane surface will generate an electrostatic induction in the channel, inducing a large amount of positive charge, increasing the concentration of positive charge, which manifests as an increase in current and a decrease in the absolute value of the threshold voltage.

[0098] The following will provide exemplary experiments and comparisons of the single-cell analysis chip combining carbon-based field-effect transistors and microfluidics, along with their detection effects. Of course, the embodiments of this invention are not limited thereto.

[0099] Example 1

[0100] Fabrication of carbon-based field-effect transistor arrays

[0101] Take a four-inch wafer with the following surface structure: the bottom layer is a silicon substrate, the top layer is silicon oxide, and a layer of carbon nanotubes is deposited on the silicon oxide. Use a diamond pen to cut a square carbon-based silicon wafer with a length and width of 1.5 cm. Then rinse the surface of the wafer with acetone alcohol and water in turn to remove impurities. After cleaning, blow it dry with a nitrogen gun.

[0102] (1) Place it on a hot plate at 115℃ and bake for 3 minutes to remove excess moisture from the surface.

[0103] (2) Use a spin coater to coat a layer of LOR photoresist on the surface of a carbon-based silicon wafer at a speed of 3000 rpm, and then immediately place it on a hot plate at 165°C for 5 minutes.

[0104] (3) Next, apply a layer of S1813 photoresist to the surface of the carbon-based silicon wafer at a rotation speed of 4000 rpm on a spin coater, and then immediately place it on a hot plate at 165°C for 5 minutes.

[0105] (4) Draw the source and drain, Mark point, lead and channel of the device in CleWin-5 software; use laser direct writing to expose the pattern, and immerse it in developer for 70s.

[0106] (5) After development, a titanium palladium gold electrode with a thickness of 0.6 / 20 / 60 nm is deposited in an electron beam coating instrument. Then, it is placed in PG for 8 hours for peeling. After peeling, the surface is rinsed with isopropanol and water.

[0107] (6) Using the same method, perform photolithography, protect the channel area of ​​the device with photoresist, and then etch away the carbon nanotubes outside the channel in a reactive ion etching machine, and remove the photoresist by soaking in PG; the channel formed between the source and drain electrodes has a length of 20 μm and a width of 40 μm.

[0108] (7) A 3nm layer of yttrium was deposited above the channel using an electron beam coating instrument, PG was removed, and then heated on a hot plate at 250°C for 30 minutes to oxidize the yttrium on the surface into yttrium oxide. A 10nm layer of hafnium oxide was deposited on the surface of yttrium oxide using an ALD (atomic deposition system). A total of 10 sensing units were prepared to obtain a carbon-based field-effect transistor array.

[0109] [Preparation of PDMS microchannels]

[0110] The PDMS microchannels are formed by casting and curing PDMS using an SU-8 template and a PDMS mold. The SU-8 template is prepared using a soft photolithography method, and the microchannel structure template is prepared according to the following steps:

[0111] (1) Spin-coat SU-82015 photoresist on the silicon wafer (rotation speed is 2500 rpm). The thickness of the photoresist after spin coating is 18 micrometers. Preheat at 65°C for 2 minutes, and then place it in a 95°C hot plate for 30 minutes.

[0112] (2) The silicon wafer coated with SU-82015 photoresist was placed in the EVG-610 ultraviolet lithography machine for exposure at a dose of 135 joules, baked at 65°C for 2 minutes, and then placed in a 95°C hot plate for 30 minutes.

[0113] (3) After the intermediate baking is completed, wait for the silicon wafer to cool to room temperature, then place it in SU-8 developer for 5 minutes. After development, rinse the surface with isopropanol and then rinse with ultrapure water.

[0114] (4) After preparing the SU-8 mold, immerse it in a solution of silanizing reagent and anhydrous ethanol at a ratio of 1:99 for 15 minutes to perform silanization treatment on the mold surface. Then rinse the surface with alcohol and water, dry it with a nitrogen gun, and heat it on a hot plate at 120°C for 15 minutes.

[0115] (5) Mix PDMS and PDMS prepolymer in a ratio of 10:1, stir with a glass rod, pour into the surface of SU-8 mold, put into a vacuum chamber and vacuum for 40-50 minutes until the surface bubbles are completely removed, then put into a hot plate at 115°C and bake for 1 hour until it is completely cured.

[0116] The obtained microchannel structures are as follows: the width of the first and second main channels is 50 μm, the width of the arc-shaped side channel is 35 μm, the diameter of the circular capture trap chamber is 60 μm, the height of the entire microchannel is 18 μm, and the width of the narrow channel is 5 μm, 6 μm, 7 μm, 8 μm, and 9 μm; a total of five channel structures.

[0117] [Chip Assembly]

[0118] The prepared PDMS microfluidic channel layer is applied over the carbon-based field-effect transistor array, and the circular trap chamber is positioned vertically directly above the sensing area of ​​the carbon-based field-effect transistor to obtain the desired chip.

[0119] Example 2

[0120] [Procedures for Single-Cell Isolation and Fixation]

[0121] Single cells were isolated and immobilized using the five carbon-based field-effect transistors combined with microfluidics single-cell analysis chips obtained after Example 1, involving the following solutions:

[0122] A polydopamine solution was prepared by mixing 2 mg of polydopamine powder with 1 mL of 10 mM Tris buffer (pH = 8.5, sterile).

[0123] A cell suspension is prepared by mixing RPMI-1640 (containing double antibiotics) basal medium with fetal bovine serum and A549 cells at a volume ratio of 10:1.

[0124] The polydopamine solution used to fix single cells was infused into the microchannels via a syringe pump. Taking advantage of the high permeability of PDMS, all microchannel chambers were filled with polydopamine solution and incubated in a constant temperature and humidity chamber at 25°C and 60% humidity for 30 minutes. The channel solution was then dried with a nitrogen gun to form a polydopamine film with a thickness of 3 nm.

[0125] Cell suspensions after centrifugation were injected into five different narrow-band channels (5–9 μm) at flow rates of 0.1 μL / min, 0.3 μL / min, and 0.5 μL / min. Cells were captured as single cells by the narrow-band main channel, ultimately achieving single-cell capture within a circular trap chamber. The capture effects of different flow rates were compared, and the results are as follows: Figure 8 As shown.

[0126] The results demonstrated that, at flow rates of 0.1–0.5 μL / min, microfluidic chips with five different narrow-band channels (5–9 μm) all exhibited cell capture capabilities. Figure 10a) and the capture effect is best under the conditions of a flow rate of 0.1 μL / min and a narrow channel width of 7 μm. Figure 10 b).

[0127] The entire microfluidic chip was then placed in a cell culture incubator and cultured for 2 hours for subsequent cell culture studies.

[0128] Example 3

[0129] [Analysis of individual lung cancer cells]

[0130] A549 cells, used as model cells, were infused into 0.1×PBS solution, centrifuged, and the supernatant was discarded. The cell concentration was 2.25×10⁻⁶ cells / mL. 5 Under the condition of cells / mL, with an injection flow rate of 0.1 μL / min and a narrow channel width of 7 μm, the capture rate of this microfluidic chip for cancer cells was studied. The capture process is as described in Example 2.

[0131] Studies have found that some small lung cancer cells can flow directly through the capture traps. The first few capture units may capture multiple cells, but subsequent capture units generally capture one cell per trap.

[0132] The microfluidic chip was then placed in a cell culture incubator and cultured for 2 hours. The cells were captured in the trap by utilizing the adsorption properties of polydopamine on cell surface proteins. The transfer characteristic curve of the device was tested using a semiconductor analyzer. In order to shield the influence of non-specific adsorption on the detection results, BSA solution was introduced into the microchannel for sealing treatment and incubated at room temperature for 1 hour.

[0133] Finally, a self-assembled Sgc8 aptamer and gold nanoparticle conjugate that specifically binds to the target protein are introduced. In the control group, an aptamer with the full T sequence and gold nanoparticle conjugate are introduced. After incubation at room temperature for half an hour, the on-state current and threshold voltage changes of the two groups are compared. The difference between the current and threshold voltage changes can be equated to the content of our target PTK7 protein.

[0134] The results are as follows Figure 11 As shown, Figure 11 In a and 11b, HfO2 represents the transfer characteristic curve of the chip in Example 4. The response was tested after subsequent incubation with polydopamine, represented by PDA. Next, cell infusion was used to test the response, followed by the infusion of BSA to reduce non-specific adsorption, and the transfer characteristic curve was tested again. Finally, the specific binding of the Sgc8 aptamer to the cell surface PTK7 protein was tested by comparison. Figure 11 The protein content is determined by the changes in on-state current and threshold voltage of the BSA and Sgc8-AuNPs curves in b, where the content is determined by... Figure 11 b shows that the change in threshold voltage is 0.12V.

[0135] Figure 11 c and Figure 11 d. Aptamers with full T sequences were selected as the control group. Figure 11 As can be seen from d, the change in threshold voltage is 0.05V, which is much smaller than that in the experimental group. Therefore, the change in threshold voltage of 0.07V is equivalent to the PTK7 protein content on the surface of A549 cells.

[0136] As can be seen from the above, the advantage of the chip of the present invention is that it has a significant response to high concentrations of protein on the surface of a single cell. Therefore, it can be used to test the concentration of protein on the surface of a single cell. When the protein concentration is high, the chip of the present invention will have a significant threshold change. Conversely, when the protein concentration on the cell surface is low, the threshold voltage response is almost unchanged. The chip and detection method of the present invention provide a completely new approach to the detection of surface proteins on a single cell.

[0137] The chip and detection method of this invention can efficiently separate and fix individual cells in a large cell population, and utilize the high stability of carbon-based field-effect transistors to detect and analyze the protein content on the surface of individual cells. The fabrication method and detection are highly accurate, which is beneficial for the study of cancer cases and subsequent targeted treatment.

[0138] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A single-cell analysis chip combining carbon-based field-effect transistors and microfluidics, characterized in that, It includes multiple single-cell capture units; wherein, each single-cell capture unit includes a sensing unit and a PDMS microfluidic channel unit corresponding to the sensing unit; The sensing unit is a carbon-based field-effect transistor. The carbon-based field-effect transistor includes silicon and silicon dioxide stacked in sequence, and carbon nanotubes deposited on the surface of silicon dioxide as a substrate. The substrate is provided with a source electrode and a drain electrode arranged opposite to each other and separated from each other. A dielectric layer composed of yttrium oxide and hafnium oxide deposited in sequence is provided on the carbon nanotube channel layer between the source electrode and the drain electrode. The PDMS microfluidic channel unit covers the sensing unit and is used to transmit the liquid to be detected. It includes a circular trap chamber and a narrow channel located directly above the sensing area of ​​the carbon-based field-effect transistor. The width D2 of the narrow channel is 2 / 3 times that of the target cell. The first end of the circular capture trap chamber is connected to the first end of the first main channel, and the second end of the circular capture trap chamber is connected to the first end of the second main channel through a narrow channel. The first main channel and the second main channel are connected by an arc-shaped side channel, which allows subsequent cells to flow into the next PDMS microfluidic channel unit after the narrow channel captures cells. The single-cell analysis chip has an inlet at the starting end for the liquid to be tested to enter, and the inlet is connected to the second end of the first main channel of the PDMS microfluidic channel unit at the starting end; the single-cell analysis chip has an outlet at the ending end for the liquid to be tested to flow out, and the outlet is connected to the second end of the second main channel of the PDMS microfluidic channel unit at the ending end. Microfluidic channels are used to separate and capture individual cells, and carbon-based field-effect transistors are used to analyze the protein content on the surface of individual cells, thus achieving the purpose of detecting proteins on the surface of single cells.

2. The single-cell analysis chip combining carbon-based field-effect transistors and microfluidics according to claim 1, characterized in that, The single-cell analysis chip contains at least four single-cell capture units.

3. The single-cell analysis chip combining carbon-based field-effect transistors and microfluidics according to claim 1, characterized in that, The circular trap chamber has a circular channel with a radius r of 30 μm.

4. The single-cell analysis chip combining carbon-based field-effect transistors and microfluidics according to claim 1, characterized in that, The first and second main channels are rectangular channels of equal width, with width D1 being 3 to 4 times that of the target cells.

5. The single-cell analysis chip combining carbon-based field-effect transistors and microfluidics according to claim 1, characterized in that, The width D3 of the arc-shaped side channel is 2 to 3 times that of the target cell arc-shaped channel.

6. The single-cell analysis chip combining carbon-based field-effect transistors and microfluidics according to claim 1, characterized in that, The design principle for the height H of the PDMS microfluidic channel unit is: H must be greater than the diameter of one cell and less than the diameter of two cells.

7. The application of a single-cell analysis chip combining a carbon-based field-effect transistor and microfluidics as described in any one of claims 1-6 in the analysis of surface proteins on single cells.

8. A method for detecting single-cell surface membrane proteins, characterized in that, The detection is performed using a single-cell analysis chip combining carbon-based field-effect transistors and microfluidics as described in any one of claims 1-6, comprising the following steps: S1. The polydopamine solution is injected into the PDMS microfluidic channel through the injection port using a syringe pump, so that the polydopamine solution fills all the channels of the microfluidic chip and incubates in the entire PDMS microfluidic channel of the chip, as well as on the dielectric layer of the carbon-based field-effect transistor to form a polydopamine film. S2. Take the cell suspension to be tested and flow it into the chip after being processed in step S1 from the injection port through the injection pump. The cells are captured into a single-cell state by the narrow channel. S3. After the cells are captured in step S2, the chip is placed in a cell culture incubator for culture. The transfer characteristic curve of the chip after cell capture is compared and analyzed with the initial chip transfer characteristic curve. The changes in the current and threshold voltage of the device transfer characteristic curve after cell capture are observed to detect whether the chip responds to the cells. S4. After confirming that the chip responds to cells, BSA blocking solution is injected into the chip through the injection port using a syringe pump and incubated at room temperature to reduce non-specific adsorption on the cell surface. After incubation, the transfer characteristic curve of the chip is measured as the detection baseline. S5. Gold nanoparticles modified with the assembled Sgc8 aptamer are injected into the chip after step S4 via the injection port using a syringe pump. The chips are then incubated at room temperature. After incubation, the transfer characteristic curve of the chip is measured and compared with the detection baseline. The change in the chip transfer characteristic curve is equivalent to the content of PTK7 protein on the surface of a single cell.

9. The method according to claim 8, characterized in that, The incubation conditions for the polydopamine layer were: incubation at 25°C and 60% humidity for 30 minutes; the resulting polydopamine film had a thickness of 3 nm.

10. The method according to claim 8, characterized in that, The injection flow rate in steps S2 and S4 is 0.1~0.5 μL / min.

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