Flexible field effect transistor biosensor for organophosphorus pesticide residue detection

Abstract

The invention relates to the field of flexible biosensing and electrochemical analysis, and provides a flexible biosensor based on an electrolyte grid-control field effect transistor (FET), which is used for in-situ detection of organophosphorus pesticide residues. The sensor is configured to be PDMS / PI / In2O3 / electrode / package / PEI adhesion layer, the sensor is provided with four FETs, an FET array is formed, the four FETs share one side grid, and channel regions of the FETs are covalently fixed by DNA hydrogel aptamers prepared through rolling circle amplification (RCA). Porous network collapse occurs after the hydrogel identifies the organophosphorus pesticide, so that more negative charges enter a Debye shielding region, thereby causing device current drop or threshold voltage positive shift, and realizing sensitive determination of pesticide residues. The device may be at lt; the device works under the low voltage of 1V, the detection range covers pg / mL to ng / mL, and the device has the advantages of flexible attachment, portable operation, quick response and the like, and can meet the on-site and nondestructive detection requirements of agricultural product circulation links on pesticide residues.

Description

I. Technical Field

[0001] This invention belongs to the field of flexible biosensing and electrochemical analysis technology, specifically relating to a flexible sensor based on electrolyte gate-controlled FET and DNA hydrogel aptamer recognition, which is suitable for in-situ and rapid detection of organophosphorus pesticides on plant leaves and other surfaces. II. Background Technology

[0002] Organophosphorus pesticides (OPs) are a class of small-molecule phosphate compounds widely used in fruit and vegetable cultivation due to their high efficiency, broad spectrum, and low cost. However, excessive or improper use often leads to excessive residue levels, threatening human health and damaging the ecological environment. Existing studies have shown that long-term exposure to OPs can cause neurotoxicity, endocrine disorders, and various chronic health problems. Therefore, developing rapid and reliable pesticide residue detection technologies is of great significance.

[0003] Current pesticide detection methods primarily rely on large, precision instruments, which are complex, time-consuming, and unsuitable for on-site testing. Aptamer sensors have attracted attention due to their specific recognition capabilities, offering advantages such as rapid response, high sensitivity, and strong specificity, significantly compensating for the shortcomings of traditional detection methods in terms of flexibility and portability. In recent years, DNA reactive hydrogels have been introduced into aptamer sensors. Upon recognizing target molecules, they can undergo structural collapse, volume changes, or release signal molecules, thereby enabling colorimetric or electrochemical detection. Hydrogels not only inherit the high specificity and excellent biocompatibility of aptamers but also possess strong water absorption and stability due to their porous network structure, providing a new approach for rapid on-site detection.

[0004] Meanwhile, the rapid development of flexible electronics technology has driven research into surface-mount biosensors. These sensors can be directly attached to irregular surfaces such as plant leaves, enabling non-destructive on-site sampling and detection. Field-effect transistors (FETs), in particular, have shown great potential in wearable sensors due to their miniaturization and the ability to fabricate thin-film transistors (TFTs) on flexible substrates. Electrolyte-gate FETs (EGFETs), relying on the high specific capacitance of nanoscale electrical double layers, can operate at voltages below 1V, making them suitable for flexible and low-power detection applications.

[0005] Unlike medical detection, which primarily relies on bodily fluids as liquid grid media, pesticide residues are mostly concentrated on the surface of vegetables and fruits, and can be collected easily through wiping, elution, or hydrogel adsorption. This provides feasibility and a new direction for developing novel sensors that combine aptamer hydrogels with flexible FETs to achieve in-situ, rapid, and non-destructive detection of pesticide residues. III. Summary of the Invention

[0006] This invention provides a layered, easily field-attached flexible FET biosensor: PDMS / PI / In2O3 / electrode / DNA hydrogel, for on-site detection of organophosphorus pesticides. The sensor mainly consists of an array of electrodes and a DNA hydrogel for OPs identification. Specifically, the bottom of the electrodes is a flexible PDMS support layer, above which is a PI electrode layer. An In2O3 semiconductor layer is deposited on the electrode layer. The semiconductor layer is covered with FET electrodes and wires with channels obtained through photolithography and magnetron sputtering. Above the wire area is a PDMS encapsulation layer, and the electrode surface is a PEI flexible encapsulation layer. The PEI-covered electrode area exposes the source, drain, and side gate electrodes with a square window cut by laser cutting. Four FETs share a single side gate. The PEI also provides double-sided adhesive adhesion. A responsive DNA hydrogel is constructed using RCA and fixed to the channel area. After OPs are identified, the mesh is disrupted / collapsed, allowing more negatively charged backbone to enter the Debye length, resulting in a decrease in IDS or a positive shift in the threshold, achieving highly sensitive readout. This invention enables low-voltage detection of organophosphorus pesticides (OPs) at the pg / mL level, providing a new method for rapid on-site screening of organophosphorus pesticide residues.

[0007] To achieve the above functions, the electrode fabrication method designed in this invention is as follows: A PI precursor is spin-coated onto a polished silicon wafer and cured by programmed temperature rise to obtain a PI film with a thickness of approximately 5–20 μm; an indium salt solution containing alcohol is spin-coated and annealed at 150–350 °C to form an In₂O₃ channel layer with a thickness of approximately 3–10 nm. After impregnation with APTES ethanol solution, the surface is cleaned and dried to obtain a -NH₂ surface. After photolithography, Ni / Au (or Ni / Pt) is sputtered, preferably forming a comb-finger structure with a spacing and finger width of 10–30 μm. Subsequently, PDMS is brush-coated onto the electrode wire area to obtain an encapsulation layer with a thickness of 20–50 μm. Then, a window with double-sided adhesive function is laser-etched onto a PEI laser. The window is surrounded by three sides, with a window width of 0.5–10 mm, a wire lead width of 0.5–1 mm, a center-to-center spacing of 1.5 mm, and a lead centerline distance from the side edge of 0.75–1.25 mm.

[0008] The identification element for OPs pesticide residue detection is derived from the rolling circle amplification of broad-spectrum aptamers by OPs and immobilized in the channel of the electrode. Specifically, primers and linear DNA are mixed at a 1:1 molar ratio, with both reaching a final concentration of 20 μM. The mixture is heated at 95 °C for 5 min to fully denature the double strands, followed by rapid cooling on ice to maintain the single-stranded state. T4 DNA ligase (100 U / μL) is added to the cooled mixture, and the reaction is carried out at 22-25 °C for 12-24 h to achieve end-to-end ligation of the linear DNA, forming a circular DNA template. After the ligation reaction is complete, the ligase is inactivated by heating at 65 °C for 10 min; the resulting circular template is stored at 4 °C for later use. 20 μL of the obtained circular template is taken, and 2.5 μL of dNTPs, 6.5 μL of phi29 DNA polymerase (50 U / μL), and 21 μL of deionized water are added sequentially to prepare a reaction system with a total volume of 50 μL. The reaction system was subjected to rolling circle amplification at 22-32℃ for 12-24 h to generate a DNA hydrogel. The obtained DNA hydrogel was activated with -COOH in EDC / NHS (5 mg / mL) solution. After activation, it was thoroughly washed with deionized water; 0.5-3 μL of the activated DNA hydrogel was added dropwise to the aminated surface of the electrode channel region for covalent coupling. The hydrogel was allowed to dry, completing the sensor construction, and then stored at 4℃. IV. Description of the attached drawings

[0009] Figure 1 The following is a side view of the sensor structure of the present invention: 101 is a PEI double-sided adhesive film layer with a window, 102 is a circuit encapsulation layer, 103 is a PI electrode layer, and 104 is a PDMS support layer.

[0010] Figure 2 This is a top view of the electrodes before packaging. 201 is the electrode intercalation area, 202 is the source wire, 203 is the drain wire, and 203 is the common gate.

[0011] Figure 3 This is a top view of the packaged electrode, with 301 representing the detection area.

[0012] Figure 4 This is a standard curve for the detection of chlorpyrifos, dimethoate, methamidophos, and phorate. V. Detailed Implementation Methods

[0013] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0014] This sensor is characterized by: an array of electrodes and a DNA hydrogel for OPs recognition; a flexible PDMS support layer at the bottom of the electrodes; a PI electrode layer above the support layer; an In2O3 semiconductor layer deposited on the electrode layer; a FET electrode and wires with channels obtained by photolithography and magnetron sputtering on the semiconductor layer; a PDMS encapsulation layer above the wire area; a PEI flexible encapsulation layer on the electrode surface; and a square window exposed source, drain electrode, and side gate in the PEI-covered electrode area by laser cutting; four FETs sharing a single side gate; and a DNA hydrogel obtained after RCA organophosphorus pesticide broad-spectrum aptamer, which is fixed to the FET surface by covalent bonding.

[0015] Before detection, the electrode needs to be calibrated using blank PBS buffer, with VDS set to 0.4V and VGS to 0-1V. The transfer characteristic curve is then obtained, and the current value I corresponding to 0.7V is recorded. DSK As a blank value, the surface was then simply rinsed with ultrapure water, left to dry, and awaited testing.

[0016] During the detection, first spray 80-100 μL of PBS buffer (0.1M concentration, with ≤10% acetone added) onto the vegetable leaves. Then, peel off the protective film from the PEI electrode and rub and stir it in the solution to facilitate rapid pesticide extraction. Next, firmly adhere the electrode to the surface. The semi-dry hydrogel has good water absorption properties; after absorbing and dissolving the OPs buffer, it stabilizes after 30-40 minutes. Measure the I at VGS = 0.7V according to the blank calibration parameters. DSS Through ΔIDS=I DSK -I DSS Record ΔIDS and calculate the possible residue levels of these four pesticides using a standard curve.

[0017] VI. Beneficial Effects

[0018] This invention proposes a method and sensor for rapid on-site screening of surface organophosphorus pesticides. The sensor can perform detection without damaging the sample, eliminating the need for complex sample pretreatment. The sensor is small and portable, which meets the needs of rapid detection in various circulation links of vegetables with short market cycles and is in line with the development trend of rapid food safety detection in my country.

Claims

1. A flexible field-effect transistor biosensor for pesticide residue detection, characterized in that, Including those stacked from bottom to top: (1) PDMS flexible support layer; (2) Flexible PI substrate; (3) A semiconductor channel layer disposed on the PI substrate and a source, drain and gate electrode paired therewith; (4) A DNA hydrogel recognition layer fixed on the surface of the semiconductor channel region; (5) The composite upper layer structure located at the top and coplanar with the electrode region includes: (5a) An encapsulation layer that covers the metal wires and leaves the electrode area empty; (5b) A PEI film with double-sided adhesive is disposed in the electrode area. The PEI film is laser-cut to form a square window, exposing the source / drain electrodes and forming a detection area. The PEI film is used to reversibly attach the sensor to the surface to be measured.

2. The sensor according to claim 1, wherein, The semiconductor channel layer is an indium oxide (In2O3) thin film with a thickness of 3–20 nm.

3. The sensor according to claim 1, wherein, The source / drain electrodes are metal stacks of Ni adhesive layer / Au conductive layer, preferably forming comb-finger type electrodes with a finger width and finger spacing of 10-30μm, a wire lead width of 0.5-1mm, a center-to-center spacing of 1.5mm, and a lead center line distance from the side edge of 0.75-1.25mm.

4. The sensor according to claim 1, wherein, The sensor includes four independent FET unit electrodes, and the four units share a single side gate.

5. The sensor according to claim 1, wherein, The DNA hydrogel is prepared by rolling circle amplification (RCA) and contains a broad-spectrum aptamer sequence targeting organophosphorus compounds (OPs). The hydrogel ends or side chains have carboxyl groups (-COOH), which are activated by EDC / NHS and then covalently coupled to the aminated layer (formed by APTES or other organosilane coupling agents) on the channel surface to fix it.

6. The sensor according to claim 5, wherein, The primers and templates used for RCA include the following sequences: Primer: 5'-COOH-GTTCACCGGATACGGGTCTGATCCGGCTGC-3'; Template: 5' -PO4-CCGTATCCGGTGAACCATTCTTGCATGGTCACACGTCGTTCTAGTACGCTTCTTGCAGCCGGATCAGAC-3'.

7. The sensor according to claim 1, wherein, The encapsulation layer is selected from polyimide, epoxy, SU-8, Parylene, PDMS or a combination thereof, and its thickness is preferably 2-50 μm. It is used to insulate and protect the metal wires and to remain coplanar with the PEI film.

8. The sensor according to claim 1, wherein, The PEI film is a polyetherimide film with a thickness of 10–200 μm; the side length of the laser-cut square window is 0.5–10 mm.

9. The sensor according to claim 1, in operation, employs an electrolyte gate-controlled field (EGFET) method, uses a buffer solution as the gate medium, and modulates the channel conductivity by screen printing the Ag / AgCl side gate voltage.

10. The application of the sensor according to any one of claims 1-9 in the in-situ detection of organophosphorus pesticide residues (including but not limited to chlorpyrifos, phosphamidon, methamidophos, phorate, etc.), characterized in that: Spray or apply a small amount of electrolyte solution to the surface to be tested, and use PEI double-sided tape to attach the electrode area to the surface to be tested. Under electrolyte grid control, collect the changes in drain-source current / migration curves to qualitatively or quantitatively determine the residual level. It is preferred to perform this under low voltage conditions of <1V.

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