A probe type organic electrochemical transistor sensor for detecting ATP in a single cell and a preparation method and a detection method thereof

Abstract

The application discloses a probe type organic electrochemical transistor sensor for detecting ATP in a single cell and a preparation method and a detection method thereof, wherein the probe type organic electrochemical transistor sensor comprises a probe type transistor inserted into the single cell and a probe type gate electrode; the probe type transistor is a micro double-hole capillary structure; an electrically conductive layer is arranged in each hole in the micro double-hole capillary structure, and the electrically conductive layer is connected with a metal wire and extends outwards from the hole; and an organic semiconductor thin film layer in contact with the electrically conductive layer is arranged at the bottom end of the micro double-hole capillary structure; the probe type gate electrode is a nano single-hole capillary structure; a functional layer is modified on the inner wall of the needle tip of the nano single-hole capillary structure; and the functional layer comprises an ATP aptamer combined with a first modifier, and the binding strength of the ATP aptamer and ATP is greater than the binding strength of the ATP aptamer and the first modifier. The probe type organic electrochemical transistor sensor has higher detection sensitivity for ATP in the single cell.

Description

Technical Field

[0001] This invention relates to the field of organic electrochemical transistor technology, and in particular to a probe-type organic electrochemical transistor sensor for detecting ATP in single cells, as well as its preparation method and detection method. Background Technology

[0002] The cell is the fundamental unit of biology, and the discovery and study of the mechanisms of material transport and energy conversion within a single cell will expand our knowledge of the origin and evolution of life, disease mechanisms, and other related fields. Batch cell culture experiments represent the average state of cells, but because the population average masks the differences between individual cells—these minute differences are crucial to the behavior of the entire organism. Analyzing cells using single-cell techniques helps reveal subtle and distinct characteristics that exist between cells of the same type, such as morphology, metabolism, or response to drugs, and provides insights into the causes and effects of cellular heterogeneity. It can also provide accurate information for pathological examination and biomedical treatments, reflecting the specific relationship between cell function and chemical composition. Therefore, the use of technologies and equipment capable of resolving the characteristics of individual biological cells is essential. Furthermore, due to the low molecular content within cells, developing single-cell analysis and detection technologies with high sensitivity and high resolution presents a significant challenge.

[0003] Adenosine triphosphate (ATP) is the primary energy source in organisms, participating in many biological processes such as biosynthesis and cellular metabolic regulation. Abnormal ATP levels are commonly associated with various diseases, such as Alzheimer's disease, hypoglycemia, cardiovascular disease, and some malignant tumors. Some studies have shown that cellular ATP content can serve as an indicator of whether certain small molecule drugs and biologics cause cell damage or proliferation. Therefore, in situ detection of dynamic changes in single-cell ATP can provide a more reliable scientific basis for understanding single-cell-related biochemical reactions and drug screening.

[0004] To date, several strategies, including colorimetry, chemiluminescence, fluorescence, and electrochemical methods, have been proposed for detecting ATP within single cells. For example, fluorescent probes for intracellular ATP imaging have been developed, some of which can be extended to subcellular organelles. The advantages of chemiluminescence and fluorescence are that they can be placed directly inside the cell to detect intracellular analytes and can selectively process a virtually unlimited range of macromolecules (proteins) and some small analytes. Their disadvantage is that the luminescent agents used in the detection process can interact with cellular components and are toxic, affecting cell viability and thus reducing the reliability of the detection, making long-term stable analysis impossible. Unlike the methods mentioned above, colorimetry itself does not leak or significantly affect cell viability. Colorimetric absorption characteristics vary with analyte concentration. They are typically attached to the tip of an optical fiber cable using chemical or physical methods. These fiber optic probes then measure extracellular and intracellular analyte concentrations using tapered fiber tips (less than 20 nm in diameter), but controlling and placing probes at this size is the most significant obstacle to intracellular monitoring. Electrochemical biosensors, due to their excellent selectivity and good controllability, can be used to detect ATP in cell lysates. However, due to their low sensitivity and slow response, they are difficult to measure intracellular ATP content.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a probe-type organic electrochemical transistor sensor for detecting ATP in single cells, as well as its preparation method and detection method, in order to solve the problem that the prior art cannot efficiently and sensitively detect the ATP content in single cells.

[0007] The technical solution of the present invention is as follows:

[0008] A probe-type organic electrochemical transistor sensor for detecting ATP in single cells includes a probe-type transistor inserted into a single cell and a probe-type gate electrode. The probe-type transistor is a micron-sized dual-pore capillary structure, with a conductive layer disposed within each pore of the micron-sized dual-pore capillary structure. Each conductive layer is connected to a metal wire and extends outward from the pore. An organic semiconductor thin film layer in contact with the conductive layer is disposed at the bottom end of the micron-sized dual-pore capillary structure. The probe-type gate electrode is a nano-sized single-pore capillary structure, with a functional layer modified on the inner wall of the tip of the nano-sized single-pore capillary structure. The functional layer includes 3-aminopropyltriethoxysilane, a first modifier bound to the 3-aminopropyltriethoxysilane, and an ATP aptamer bound to the first modifier. The binding strength of the ATP aptamer to ATP is greater than the binding strength of the ATP aptamer to the first modifier.

[0009] The probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, wherein the ATP aptamer is 5'-ACC TGG GGG AGT ATT GCG GAG GAA GGT-3', and the first modifier is 5'-CHO-ACCTTC CTC CGC AAT ACT-3' or glutaraldehyde.

[0010] The probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, wherein the micron-sized dual-pore capillary structure and the nano-sized single-pore capillary structure are independently selected from borosilicate capillaries and quartz capillaries.

[0011] The probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, wherein the conductive layer is made of one of C, Pt, Au, Ag, and Cu.

[0012] The probe-type organic electrochemical transistor sensor for detecting ATP in single cells, wherein the material of the organic semiconductor thin film layer is at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polypyrrole, polythiophene, polyaniline, polycarbazole and their copolymers.

[0013] A method for preparing a probe-type organic electrochemical transistor sensor for detecting ATP in single cells, comprising the following steps:

[0014] By setting five parameters of the needle pulling device, namely heating temperature, filament mode, needle pulling speed, delay time, and pulling force, capillary tubes are pulled to produce micron-sized double-pore capillary tubes and nano-sized single-pore capillary tubes with a preset needle tip aperture.

[0015] Butane is introduced into a micron-sized double-pore capillary, and the capillary is heated to cause the butane to decompose into carbon black and deposit it in the pores of the capillary, forming a conductive layer.

[0016] Metal wires connected to the conductive layer are inserted into both pores of the micron-sized dual-pore capillary. An organic semiconductor thin film layer in contact with the conductive layer is generated at the bottom of the micron-sized dual-pore capillary using a three-electrode system and cyclic voltammetry, thus obtaining the micron-sized dual-pore capillary structure.

[0017] A solution of 3-aminopropyltriethoxysilane was poured into the inner wall of the tip of a nanoporous capillary. The capillary was placed vertically to remove air bubbles and incubated in the dark to obtain a nanoporous capillary tip functionalized with 3-aminopropyltriethoxysilane.

[0018] The first modifier solution was poured into the 3-aminopropyltriethoxysilane-functionalized nanoporous capillary tip, and after incubation, the first modifier was combined with the 3-aminopropyltriethoxysilane of the nanoporous capillary tip to obtain the first modifier-functionalized nanoporous capillary tip.

[0019] An ATP aptamer is infused into the tip of a nanoporous capillary that is functionalized with the first modifier. After incubation, the ATP aptamer is combined with the first modifier to obtain a nanoporous capillary structure with a functional layer formed on the inner wall of the tip.

[0020] Using the micron-sized dual-pore capillary structure as a probe-type transistor and the nano-sized single-pore capillary structure as a probe-type gate electrode, a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell is constructed.

[0021] A detection method using a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, comprising the steps of:

[0022] A probe-type transistor and a probe-type gate electrode were inserted into the electrolyte and assembled into a probe-type organic electrochemical transistor sensor. Different concentrations of ATP were then added dropwise to the electrolyte. A voltage was applied, and the Ig of the organic electrochemical transistor sensor was measured using a semiconductor parameter analyzer. DS -V G curve;

[0023] A probe-type transistor and a probe-type gate electrode were mounted on a 3D translation stage. Under microscopic observation, the probe-type transistor and probe-type gate electrode were slowly inserted into the single cell to be tested. After applying a voltage and waiting for the current to stabilize, the single cell was stimulated with a drug to induce ATP secretion, and then the current signal was collected. Based on the collected current signal and I... DS -V G The curve yields the ATP concentration of the single cell being tested.

[0024] The detection method of the probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, wherein the applied voltage parameter is V G =0-0.45V, V DS = -0.05V.

[0025] Beneficial Effects: This invention provides a probe-type organic electrochemical transistor sensor for detecting ATP in single cells. Based on the probe-type organic electrochemical transistor, it amplifies weak current signal changes on the gate electrode by utilizing the OECT signal amplification function, achieving highly sensitive detection of ATP in single cells for the first time using a probe-type organic electrochemical transistor. A probe-type gate electrode is obtained by modifying a functional layer within a nanoporous capillary. The ATP aptamers contained in the inner functional layer of the probe-type nanogate electrode specifically bind to ATP and dissociate from cDNA within the functional layer, changing the surface charge of the probe tip. Therefore, the ATP concentration in single cells can be detected by detecting changes in the surface charge of the probe-type nanogate electrode. Furthermore, the cDNA immobilized on the tip surface can be re-incubated with fresh ATP aptamers, achieving cyclic regeneration. This invention uses ATP aptamers as recognition elements, which has low requirements for the detection environment and does not require consideration of the activity of the recognition element. Moreover, the use of probe-type nanoelectrodes as gate electrodes, due to the small size of nanoelectrodes, is less likely to affect cell activity and will not interfere with the behavior of single cells, allowing for continuous and stable detection. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the operational state of a probe-type organic electrochemical transistor sensor used to detect ATP in a single cell.

[0027] Figure 2 This is a schematic cross-sectional view of the micron-sized dual-pore capillary structure in the probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, as described in this invention.

[0028] Figure 3 This is a schematic cross-sectional view of the nanoporous capillary structure in the probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, as described in this invention.

[0029] Figure 4 This diagram illustrates the modification steps for a micro / nano probe-type organic electrochemical transistor sensor used for highly sensitive detection of ATP concentration in single cells.

[0030] Figure 5 This is a flowchart illustrating the preparation method of a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell according to the present invention.

[0031] Figure 6 The ΔI obtained by testing the micro / nano probe-type OECT device of this invention in PBS solution. DS Graph showing the relationship between ATP concentration and other factors.

[0032] Figure 7 The image shows the IV curve of a micro / nano probe-type OECT device in PBS solution.

[0033] Figure 8 The CV curves of a traditional nano-carbon electrode in ferrocene methanol solution are shown. Detailed Implementation

[0034] This invention provides a probe-type organic electrochemical transistor sensor for detecting ATP in single cells, along with its preparation and detection methods. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] Please see Figures 1-3 This invention provides a probe-type organic electrochemical transistor sensor for detecting ATP in single cells, as shown in the figure. It includes a probe-type transistor 1 inserted into a single cell and a probe-type gate electrode 2. The probe-type transistor 1 is a micron-sized dual-pore capillary structure. Each pore in the micron-sized dual-pore capillary structure has a conductive layer 3. Each conductive layer 3 is connected to a metal wire 4, which extends outward from the pore. An organic semiconductor thin film layer 5 is disposed at the bottom end of the micron-sized dual-pore capillary structure, in contact with the conductive layer 3. The probe-type gate electrode 2 is a nano-sized single-pore capillary structure. The inner wall of the tip of the nano-sized single-pore capillary structure is modified with a functional layer 6. The functional layer 6 includes 3-aminopropyltriethoxysilane, a first modifier bound to the 3-aminopropyltriethoxysilane, and an ATP aptamer bound to the first modifier. The binding strength between the ATP aptamer and ATP is greater than the binding strength between the ATP aptamer and the first modifier.

[0036] The probe-type organic electrochemical transistor sensor provided by this invention is a novel type of highly sensitive biosensor. This invention is the first to achieve ATP detection in single cells using a probe-type organic electrochemical transistor (OECT) sensor. Utilizing the signal amplification effect of OECT, the minute current generated by the probe-type gate electrode 2 can be amplified through the channel current of the probe-type transistor 1, resulting in higher detection sensitivity than traditional electrochemical nanoelectrode sensing technology.

[0037] Specifically, this invention utilizes a micro / nanoscale probe combined with a novel probe-type OECT for the detection of ATP in single cells. By combining an organic electrochemical transistor (OECT) with a nanoscale probe single-cell analysis method, the detected signal is amplified, achieving higher sensitivity detection of single cells. The micron-sized dual-pore capillary structure serves as the source and drain electrode, while the nanoscale single-pore capillary structure acts as the gate electrode of the OECT. By modifying the inner wall of the nanoscale single-pore capillary tip, a positively charged 3-aminopropyltriethoxysilane (APTES) is applied, initially giving the tip a positive charge. Subsequently, a first modifier becomes negatively charged, converting the tip's ionic current to negative. The ATP aptamer, also negatively charged, connects to the first modifier, increasing the negative current at the tip. The ATP aptamer exhibits selective specificity for ATP; through binding with ATP, the ATP aptamer dissociates from the first modifier, altering the tip's surface charge and reducing the negative current. By modifying the probe tip with APTES, a first modifier, and an ATP aptamer, and interacting with ATP, the charge on the probe tip surface changes, resulting in a signal change and thus enabling the detection of ATP signals. The probe-type OECT sensor has been successfully applied to the detection of ATP in single cells, providing a new approach for highly sensitive detection in biosensing and single-cell analysis technologies, and has broad application prospects.

[0038] In some embodiments, the ATP aptamer is 5'-ACC TGG GGG AGT ATT GCG GAGGAAGGT-3', and the first modifier is 5'-CHO-ACC TTC CTC CGC AAT ACT-3' or glutaraldehyde. In this embodiment, taking 5'-CHO-ACC TTC CTC CGC AAT ACT-3' as the first modifier and 5'-ACCTGG GGG AGT ATT GCG GAG GAA GGT-3' as the ATP aptamer, for example... Figure 4As shown, the modification steps of the inner wall of the tip of the nanoporous capillary structure are as follows: After modifying 3-aminopropyltriethoxysilane (APTES) on the probe-type gate electrode, since the amino group in APTES is positively charged, it is modified on the inner wall of the probe-type gate electrode tip, and its ion current is positively rectified; then, aldehyde-functionalized cDNA (5'-CHO-ACC TTC CTC CGC AAT ACT-3') is modified on the inner wall of the probe-type gate electrode tip. The cDNA molecule is negatively charged, the tip begins to be negatively charged, and the ion current changes to negative; when the negatively charged ATP aptamer pairs complementaryly with the cDNA bases, the negative current of the tip further increases. After modification, the probe-type grid electrode introduces intracellular fluid into the needle tip via electroosmosis to detect the intracellular ATP concentration. Because the binding strength between the ATP aptamer and ATP is greater than that between the ATP aptamer and cDNA, the intracellular ATP specifically binds to the modified ATP aptamer within the needle tip. This causes the cDNA to separate from the ATP aptamer on the inner surface of the nanopore, resulting in a change in the charge on the needle tip surface. This reduces the negative charge on the inner surface of the glass nanopore, leading to a decrease in the negative charge density and a smaller negative current. The higher the ATP concentration, the more cDNA dissociates from the aptamer, and the more significant the decrease in the negative current at the needle tip.

[0039] In some embodiments, the micron-sized dual-pore capillary structure and the nano-sized single-pore capillary structure are independently selected from borosilicate capillaries and quartz capillaries, but are not limited thereto.

[0040] In some embodiments, the conductive layer is made of one of C, Pt, Au, Ag, and Cu, but is not limited thereto.

[0041] In some embodiments, the material of the organic semiconductor thin film layer is at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polypyrrole, polythiophene, polyaniline, polycarbazole and copolymers thereof, but is not limited thereto.

[0042] In some embodiments, a method for preparing a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell is also provided, such as... Figure 5 As shown, it includes the following steps:

[0043] S10. By setting five parameters of the needle pulling device, namely heating temperature, filament mode, needle pulling speed, delay time, and pulling force, the capillary is pulled to obtain micron double-pore capillary and nano single-pore capillary with a preset needle tip aperture size.

[0044] S20. Butane is introduced into the micron-sized double-pore capillary, and the micron-sized double-pore capillary is heated to cause the butane to decompose into carbon black and deposit in the pores of the micron-sized double-pore capillary to form a conductive layer.

[0045] S30. Insert metal wires connected to the conductive layer into both holes of the micron-sized dual-pore capillary, and use a three-electrode system and cyclic voltammetry to electrochemically deposit an organic semiconductor thin film layer in contact with the conductive layer at the bottom of the micron-sized dual-pore capillary to obtain the micron-sized dual-pore capillary structure.

[0046] S40. Pour a 3-aminopropyltriethoxysilane solution into the inner wall of the tip of a nanoporous capillary, place it vertically to expel air bubbles, and incubate it in the dark to obtain a 3-aminopropyltriethoxysilane-functionalized nanoporous capillary tip.

[0047] S50. The first modifier solution is poured into the 3-aminopropyltriethoxysilane-functionalized nanoporous capillary tip, and after incubation, the first modifier is combined with the 3-aminopropyltriethoxysilane of the nanoporous capillary tip to obtain the first modifier-functionalized nanoporous capillary tip.

[0048] S60. The ATP aptamer is infused into the tip of the first modified nanoporous capillary, and after incubation, the ATP aptamer is combined with the first modified body to obtain a nanoporous capillary structure with a functional layer formed on the inner wall of the needle tip.

[0049] S70. Using the micron-sized dual-pore capillary structure as a probe-type transistor and the nano-sized single-pore capillary structure as a probe-type gate electrode, a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell is constructed.

[0050] The preparation method of the probe-type organic electrochemical transistor sensor of the present invention will be described below through specific embodiments:

[0051] (1) Fabrication of nanoporous glass electrodes and microporous glass electrodes:

[0052] Borosilicate capillaries with an outer diameter of 1.0 mm and an inner diameter of 0.59 mm were soaked in a freshly prepared piranha solution (98% H₂SO₄:30% H₂O₂ = 3:1 (v / v)) for 2 hours. The inside of the capillaries was rinsed repeatedly with deionized water and anhydrous ethanol to remove residual piranha solution. The cleaned glass capillaries were then dried with a nitrogen gun. Capillary electrodes were then fabricated using a P-2000 microelectrode drawing instrument. The drawing parameters of the P-2000 instrument for fabricating nanoporous glass electrodes were as follows:

[0053] HEAT=350, FIL=3, VEL=42, DEL=200, PUL=0;

[0054] HEAT=340, FIL=3, VEL=39, DEL=170, PUL=140;

[0055] The drawing parameters of the P-2000 drawing apparatus for preparing micron-sized quartz double-hole glass electrodes are as follows:

[0056] HEAT=660, FIL=3, VEL=30, DEL=140, PUL=120;

[0057] HEAT=680, FIL=3, VEL=30, DEL=140, PUL=130.

[0058] (2) Fabrication of source and drain electrodes for micro / nano probe-type organic electrochemical transistors:

[0059] The drawn capillary tube is placed inside a quartz glass tube, and argon gas is introduced into the quartz glass tube as a protective gas. Then, high-pressure butane gas is injected from the end of the capillary tube. At the same time, a heating nozzle is used to heat the tip of the capillary tube from the outer wall of the quartz glass tube. Under the high temperature of 800℃, the butane introduced into the capillary tube decomposes into carbon black, which is deposited on the inner wall of the capillary tube. Subsequent adjustments to parameters such as the inlet pressure of butane and argon gas, flame size, heating time, and heating temperature result in a quartz micron-sized dual-pore carbon electrode with a dense carbon layer and a smooth morphology.

[0060] (3) Deposition of PEDOT:PSS organic semiconductor thin film layer for micro / nano probe-type organic electrochemical transistors:

[0061] The prepared carbon electrode was placed in a solution containing 1 mM methanol-ferrocene and 0.1 M KCl for cyclic voltammetry scanning. A suitable source / drain electrode with an "S" shape was selected for subsequent electrodeposition experiments. The quartz micron-sized dual-pore electrode was cleaned and dried with ultrapure water and anhydrous ethanol, then treated in a plasma cleaner for 10 min to increase the hydrophilicity of the electrode surface. Using 15 mM EDOT and 20 mM PSS solution as the electrolyte, a three-electrode system was employed: an Ag / AgCl electrode as the reference electrode, a copper wire in the quartz micron-sized dual-pore electrode as the working electrode, and a platinum electrode as the counter electrode. The voltage was set to 0.9 V, and the deposition time was 150 s. A PEDOT:PSS thin film was deposited on the needle tip via polymerization to obtain micron-sized probe-type source and drain electrodes.

[0062] (4) Modification of probe-type nanogrid electrode 3-aminopropyltriethoxysilane (APTES):

[0063] At room temperature, 5 μL of a 1% (v / v) solution of 3-aminopropyltriethoxysilane (APTES) was injected into the tip of a nanoporous glass electrode using a micro-syringe. The electrode was placed vertically to remove air bubbles and then incubated in the dark for 30 min. After the reaction, the nanoporous glass electrode was washed repeatedly with anhydrous ethanol, each time for 5 min, to prevent clogging of the nanopores. After washing, it was placed in a vacuum drying oven and dried at 60°C for 30 min.

[0064] (5) Functionalization process of the inner surface of probe-type nanogate electrode:

[0065] 20 μL of 10 μM aldehyde-functionalized cDNA (5'-CHO-ACC TTC CTC CGC AAT ACT-3') solution was injected into the tip of a 3-aminopropyltriethoxysilane (APTES)-functionalized nanoporous glass electrode using a micro-loading needle. The electrode was incubated at room temperature for 20 h, and then rinsed with 0.01 M PBS. Finally, the glass nanoelectrode containing the aldehyde-functionalized cDNA was injected with 20 μL of 10 μM ATP aptamer (5'-ACC TGG GGG AGT ATT GCGGAG GAA GGT-3') solution and incubated at room temperature for 24 h. The ATP aptamer partially paired with the cDNA to form a double-stranded structure, and the electrode was thoroughly rinsed with 0.01 M PBS.

[0066] (6) Constructing a probe-type OECT for detection in a simulated liquid:

[0067] A probe-type OECT is constructed by combining a probe-type source electrode, a drain electrode, and a probe-type gate.

[0068] This embodiment utilizes a combination of nanoprobes and organic electrochemical transistors (OECTs). Because OECTs combine sensing and signal amplification functions, they amplify the weak signal of the analyte on the gate electrode, enabling more sensitive detection of low concentrations of ATP within single cells. This embodiment is the first to employ micro / nanoprobe-type organic electrochemical transistors to detect ATP concentration within single cells, minimizing impact on cell activity and normal physiological processes, thus ensuring reliability. Furthermore, the modification of APTES, cDNA, and ATP aptamers, along with the change in surface charge caused by the ATP aptamer's dissociation from cDNA after binding with ATP, allows for the detection of ATP concentration. The specificity of ATP aptamer binding to ATP makes the detection more accurate and reliable. Moreover, after binding to ATP, the ATP aptamer linked to the needle tip dissociates from the cDNA and detaches from the needle tip surface. The cDNA immobilized on the needle tip surface can be re-incubated with fresh ATP aptamers to form a new double-stranded structure, allowing for further ATP testing and achieving a cycle of regeneration. Furthermore, the tip modifier can be replaced, enabling the sensor to be used more widely and to detect other corresponding biomolecules and chemical components.

[0069] In some embodiments, a detection method for a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell is also provided, comprising the steps of: inserting a probe-type transistor and a probe-type gate electrode into an electrolyte and assembling them into a probe-type organic electrochemical transistor sensor; sequentially adding different concentrations of ATP to the electrolyte; applying a voltage; and measuring the I of the organic electrochemical transistor sensor using a semiconductor parameter analyzer. DS -V G Curve; A probe-type transistor and probe-type gate electrode are mounted on a 3D translation stage, and under microscopic observation, the probe-type transistor and probe-type gate electrode are slowly inserted into the single cell to be tested. After applying a voltage and waiting for the current to stabilize, the single cell to be tested is stimulated with a drug to induce ATP secretion, and then the current signal is collected. Based on the collected current signal and I... DS -V G The curve yields the ATP concentration of the single cell being tested.

[0070] Specifically, after constructing a probe-type OECT by combining a probe-type source electrode, a drain electrode, and a probe-type gate, 0.1M KCl solution was used as the electrolyte. Different ATP concentrations were prepared using 0.1M KCl solution for measurement. The results were obtained using a Keithley 4200 semiconductor parameter analyzer at V... G =0-0.45V, V DS Measuring the I of the device under the condition of -0.05V DS -V G curve; Figure 6ΔI obtained by testing micro / nano probe-type OECT devices in PBS solution DS The graph shows the relationship between ΔI and different ATP values. This graph illustrates that, in the simulated solution, the probe-type OECT device of this invention, as the ATP concentration increases, [ΔI / ... DS The size also increases accordingly, the device responds to the ATP concentration and the current signal is amplified by OECT, the effect is quite obvious.

[0071] Then, HeLa cells were cultured: HeLa cells were cultured at 37°C for 24 hours in DMEM medium containing 10% fetal bovine serum (FBS), 1% antibiotics, and 5% CO2. Prior to single-cell ATP concentration analysis, cells were incubated at a relatively low density in confocal cell culture dishes at 37°C for 24 hours. Calcein-AM and propidium iodide (PI) were added to the cell culture medium at a final concentration of 3 μg / mL to stain the cells and confirm cell viability. After incubation under cell culture conditions for 30 minutes, residual dye was washed away.

[0072] Finally, the probe-type OECT sensor was inserted into HeLa cells to detect ATP concentration: the cells were first washed and then recultured in a culture dish containing 1X PBS solution. With the assistance of an MP-225 micromanipulator (CA) equipped with an inverted microscope, two capillaries were slowly brought closer to the cells under microscopic observation by adjusting the three-dimensional micromanipulator, and the micro / nano probe-type OECT sensor was inserted into a single HeLa cell for detection. HeLa cells were incubated with 100 μM etoposide for 20 minutes or 3 μg / mL oligomycin for 3 hours. After the current stabilized, ATP was induced by etoposide stimulation and oligomycin was used to inhibit ATP consumption, and the current signal was collected, demonstrating the potential of the micro / nano probe-type OECT sensor to detect drug effects. The test values ​​are the average of three measurements of the same sample.

[0073] Figure 7 The I of the micro / nano probe type OECT device of this invention DS -V G The graph shows that the displayed current is around 10. -5 In comparison, this invention also measured the CV curves of conventional carbon nanoelectrodes in ferrocene methanol solution, as shown in section A. Figure 8 As shown, its display current level is 10. -9 A. By comparison, it can be found that the micro / nano probe type OECT device of the present invention has a current magnitude that is 4 orders of magnitude higher than that obtained by traditional electrochemical methods. The larger the current magnitude obtained, the more sensitive it is to changes in weak biological signals, so it can achieve a highly sensitive detection effect for low concentrations of biomolecules.

[0074] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A probe-type organic electrochemical transistor sensor for detecting ATP in a single cell, characterized in that, The device includes a probe-type transistor and a probe-type gate electrode inserted into a single cell. The probe-type transistor is a micron-sized dual-pore capillary structure, in which each pore contains a conductive layer, and each conductive layer is connected to a metal wire extending outward from the pore. An organic semiconductor thin film layer in contact with the conductive layer is disposed at the bottom end of the micron-sized dual-pore capillary structure. The probe-type gate electrode is a nano-sized single-pore capillary structure, in which the inner wall of the tip of the nano-sized single-pore capillary structure is modified with a functional layer. The functional layer includes 3-aminopropyltriethoxysilane, a first modifier bound to the 3-aminopropyltriethoxysilane, and an ATP aptamer bound to the first modifier. The binding strength of the ATP aptamer to ATP is greater than the binding strength of the ATP aptamer to the first modifier. The ATP aptamer is 5'-ACC TGG GGG AGT ATT GCG GAG GAA GGT-3', and the first modifier is 5'-CHO-ACC TTC CTC CGC AAT ACT-3'.

2. The probe-type organic electrochemical transistor sensor for detecting ATP in a single cell according to claim 1, characterized in that, The micron-sized dual-pore capillary structure and the nano-sized single-pore capillary structure are independently selected from borosilicate capillaries and quartz capillaries.

3. The probe-type organic electrochemical transistor sensor for detecting ATP in a single cell according to claim 1, characterized in that, The conductive layer is made of one of the following materials: C, Pt, Au, Ag, and Cu.

4. The probe-type organic electrochemical transistor sensor for detecting ATP in a single cell according to claim 1, characterized in that, The material of the organic semiconductor thin film layer is at least one of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid, polypyrrole, polythiophene, polyaniline, polycarbazole and their copolymers.

5. A method for preparing a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell as described in any one of claims 1-4, characterized in that, Including the following steps: By setting five parameters of the needle pulling device, namely heating temperature, filament mode, needle pulling speed, delay time, and pulling force, capillary tubes are pulled to produce micron-sized double-pore capillary tubes and nano-sized single-pore capillary tubes with a preset needle tip aperture. Butane is introduced into a micron-sized double-pore capillary, and the capillary is heated to cause the butane to decompose into carbon black and deposit it in the pores of the capillary, forming a conductive layer. Metal wires connected to the conductive layer are inserted into both pores of the micron-sized dual-pore capillary. An organic semiconductor thin film layer in contact with the conductive layer is generated at the bottom of the micron-sized dual-pore capillary using a three-electrode system and cyclic voltammetry, thus obtaining the micron-sized dual-pore capillary structure. A solution of 3-aminopropyltriethoxysilane was poured into the inner wall of the tip of a nanoporous capillary. The capillary was placed vertically to remove air bubbles and incubated in the dark to obtain a nanoporous capillary tip functionalized with 3-aminopropyltriethoxysilane. The first modifier solution was poured into the 3-aminopropyltriethoxysilane-functionalized nanoporous capillary tip, and after incubation, the first modifier was combined with the 3-aminopropyltriethoxysilane of the nanoporous capillary tip to obtain the first modifier-functionalized nanoporous capillary tip. An ATP aptamer is infused into the tip of a nanoporous capillary that is functionalized with the first modifier. After incubation, the ATP aptamer is combined with the first modifier to obtain a nanoporous capillary structure with a functional layer formed on the inner wall of the tip. Using the micron-sized dual-pore capillary structure as a probe-type transistor and the nano-sized single-pore capillary structure as a probe-type gate electrode, a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell is constructed.

6. A detection method for a probe-type organic electrochemical transistor sensor for detecting ATP in a single cell as described in any one of claims 1-4, characterized in that, Including the following steps: A probe-type transistor and a probe-type gate electrode were inserted into the electrolyte and assembled into a probe-type organic electrochemical transistor sensor. Different concentrations of ATP were then added dropwise to the electrolyte. A voltage was applied, and the Ig of the organic electrochemical transistor sensor was measured using a semiconductor parameter analyzer. DS -V G curve; A probe-type transistor and a probe-type gate electrode were mounted on a 3D translation stage. Under microscopic observation, the probe-type transistor and probe-type gate electrode were slowly inserted into the single cell to be tested. After applying a voltage and waiting for the current to stabilize, the single cell was stimulated with a drug to induce ATP secretion, and then the current signal was collected. Based on the collected current signal and I... DS -V G The curve yields the ATP concentration of the single cell being tested.

7. The detection method of the probe-type organic electrochemical transistor sensor for detecting ATP in a single cell according to claim 6, characterized in that, The applied voltage parameter is V G =0-0.45 V, V DS =-0.05 V.

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