A uPA peptide-imprinted conductive polymer, a biosensor, and its preparation method

Abstract

This invention relates to a uPA peptide-imprinted conductive polymer and its preparation method, particularly a peptide-imprinted conductive polymer comprising a monomer of a conductive polymer and a small portion of a uPA molecule peptide as a template. The uPA peptide-imprinted conductive polymer provided by this invention possesses advantages such as high sensitivity, high accuracy, fast analysis speed, small sample volume required, reusability, high chemical stability, simple preparation process, low cost, portability, and low detection limit. It can be effectively applied to the detection of uPA in human serum samples using extended gate field-effect transistors (EG-FETs) or electrochemical sensing.

Description

[Technical Field]

[0001] This invention relates to the technical field of epitope imprinting combined with electrochemical polymerization, and particularly to a peptide-imprinted conductive polymer and its use in detecting thrombotic diseases. The aforementioned peptide-imprinted conductive polymer comprises a conductive polymer monomer and a small portion of uPA molecules as a peptide as a template. [Previous Technology]

[0002] Atherosclerosis refers to the formation of atherosclerotic plaques on the arterial walls due to the accumulation of fat, cholesterol, and other substances and thickening of the intima. This leads to arteriosclerosis, narrowing, and even blockage of the arteries. Plaques may completely block blood vessels or rupture to form thrombi, causing acute vascular occlusion, such as acute myocardial infarction, angina pectoris, embolic stroke, and sudden death. Known risk factors include high cholesterol, high blood lipids, high blood pressure, smoking, obesity, diabetes, and family history. Furthermore, the involvement of inflammatory responses and the immune system is also considered an important influencing factor in the progression of atherosclerosis.

[0003] Treatment for atherosclerosis typically involves lifestyle modifications and medication. Lifestyle modifications include a healthy diet, increased physical activity, quitting smoking, and reducing alcohol consumption. Medications include cholesterol-lowering drugs (e.g., statins), blood pressure-lowering drugs (e.g., ACE inhibitors and beta-blockers), and antiplatelet drugs (e.g., aspirin) to prevent blood clots. In some cases, surgical treatment may be necessary, such as coronary artery bypass surgery or endarterectomy.

[0004] Urokinase-type plasminogen activator (uPA) is an enzyme that converts plasminogen into plasmin, thereby promoting thrombus dissolution. In thrombolytic therapy, uPA is used to treat thrombotic diseases such as acute myocardial infarction, pulmonary embolism, deep vein thrombosis, and atherosclerosis. uPA mainly dissolves existing thrombi by activating the plasmin system, thereby restoring blood flow. Compared to other plasminogen activators (e.g., tissue-type plasminogen activator (tPA)), uPA has a unique mechanism of action and efficacy.

[0005] However, the use of uPA still carries a risk of bleeding, especially in cases of overdose or in patients with a bleeding tendency. Therefore, close monitoring of the patient's bleeding and thrombolysis is necessary during uPA use. uPA is generally not recommended for patients with a bleeding tendency, recent surgery, active bleeding, or severe hypertension. To minimize the risk of bleeding, healthcare professionals typically calculate the required dosage and closely observe the patient's response.

[0006] Furthermore, the therapeutic effect of uPA can be influenced by a variety of factors, including the patient's health condition, the location and size of the thrombus, and the immediacy of treatment. With advancements in technology, new uPA variants and related treatments are being developed to improve efficacy and reduce side effects.

[0007] It is known that uPA protein is directly related to thrombotic diseases, and many studies have pointed out that uPA has important clinical significance in the diagnosis of thrombotic diseases. Among them, how to effectively, rapidly and accurately detect the concentration of uPA in the sample is an important issue to be solved. Therefore, there is an urgent need for a tool that can effectively detect uPA, a biomarker for thrombotic diseases.

[0008] Techniques for detecting uPA concentration in samples have been developed using antibodies or enzyme-linked immunosorbent assays (ELISA) that can identify uPA. However, these techniques are limited by the limit of detection (LOD) and may not be able to quantify the correct value of the target analyte. Furthermore, these techniques still have problems such as difficulty in antibody preservation, doubts about detection accuracy, high time consumption, and high labor costs.

[0009] In recent years, molecularly imprinted polymers (MIPs) have been rapidly developed to create new identification materials for the separation of optically active drugs or cells, biosensors, etc., due to their relative stability, simple operation process and low price. For example, the Republic of China Patent Publication No. TW201435344 "A method for preparing a coated polymer imprinted three-dimensional microstructure sensor that can detect biomarker molecules in urine" uses a biomolecular template to imprint in a polymer to form molecular pores that can complement the test sample, and then coats it on the surface of a nano-ceramic microstructure on a gold substrate to obtain a polymer imprinted three-dimensional microstructure sensor that can detect biomarker molecules in urine.

[0010] Proteins are an emerging field in molecular imprinting polymerization in recent years. Their large molecular weight and steric and thermodynamic effects hinder the formation of selective molecular imprinting pores. To address this, a new imprinting technique called epitope imprinting has been developed, using only a portion of the peptides in the protein molecule as a template. The aim is to create imprinting pores capable of recognizing the entire protein. Appropriate peptide length selection can improve sensing sensitivity; conversely, insufficient length will result in no recognition ability. Utilizing the hydrophobic amino acid properties within the peptide fragment, the imprinting template molecules can be directional within the polymer, producing consistent imprinting pores and thus endowing the imprinting pores with high selectivity. [Prior Art Literature] [Patent Literature]

[0011] [Patent Document 1]TW201435344 [Summary of the Invention]

[0012] [The technical problem that the invention is intended to solve]

[0013] uPA has been proven to be an indicator molecule for thrombotic diseases. Several methods for detecting thrombotic diseases by quantifying uPA in a sample have existed, such as antibody-specific binding and ELISA. However, due to limitations in the limit of detection (LOD), accuracy needs improvement. Furthermore, antibodies are difficult to preserve, have high manufacturing costs, require complex equipment, and face many restrictions in related experiments, hindering widespread adoption. For example, analytical instruments are expensive, or the procedures of immunoprecipitation and enzyme immunoassay are cumbersome and time-consuming, and errors caused by human experimental operation are difficult to avoid. In addition, the high cost of complete uPA protein prevents traditional measurement methods from achieving low-cost and portable applications, limiting the development of home healthcare systems.

[0014] In view of the above-mentioned problems, the present invention aims to provide a peptide-imprinted conductive polymer for detecting the intact uPA protein in human samples, which can be applied to a home biosensor for detecting uPA content. The present invention not only features simple experimental operation, short analysis time, reduced detection cost, high detection sensitivity, low detection limit, reusability, portability, and high specificity, but also achieves measurement efficacy with a wide current range, good stability, and high quality, whether using inexpensive extended gate field-effect transistors (EG-FETs) or expensive electrochemical detection. [Technical Means]

[0015] To achieve the above objectives, the present invention provides the following technical means.

[0016] In one embodiment, the present invention provides a uPA peptide-imprinted conductive polymer, characterized by comprising a conductive polymer monomer and a template molecule for identifying uPA. The template molecule for identifying uPA is selected from a peptide fragment of the complete uPA protein peptide sequence that has the following conditions: (1) the peptide fragment has a length of 5 to 30 amino acids; (2) the ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids contained in the peptide fragment is 0 to 75%; and (3) the peptide fragment contains 0 to 15 aromatic amino acids.

[0017] In some embodiments, the aforementioned conductive polymer monomer is selected from at least one of the following groups: m-aminobenzenesulfonic acid (MSAN), 2-aminobenzenesulfonic acid (MSDS), 4-aminobenzenesulfonic acid (SDS), aniline (AN), 3,4-ethylenedioxythiophene (EDOT), hydroxymethyl 3,4-ethylenedioxy-thiophene (EDOT-OH), acetylene, pyrrole, thiophene, phenylene vinylene, isothianaphthene, 3-alkyl thiophene, 2,5-dialkoxy paraphenylene. Vinylene, paraphenylene, paraphenylene sulphide, and heptadiyne.

[0018] In some embodiments, the concentration of the conductive polymer monomer is between 0.001 and 50% by weight (wt%).

[0019] In some embodiments, the conductive polymer monomer is aniline, and at least one selected from the group consisting of m-aminobenzenesulfonic acid, 2-aminobenzenesulfonic acid, or 4-aminobenzenesulfonic acid. The molar ratio of aniline to m-aminobenzenesulfonic acid, 2-aminobenzenesulfonic acid, or 4-aminobenzenesulfonic acid can be from 10:1 to 1:10, for example, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0020] In some embodiments, the mole ratio of aniline is 80% of the total mole number of all conductive polymer monomers, that is, the mole ratio of aniline to other conductive polymer monomers is 4:1.

[0021] In some embodiments, the conductive polymer monomers are aniline and 2-aminobenzenesulfonic acid, and the mole ratio of aniline is 80% of the total mole number of all conductive polymer monomers.

[0022] In some embodiments, the peptide sequence of the template molecule for identifying uPA is selected from SEQ ID NO: 1 to SEQ ID NO: 11.

[0023] In some embodiments, the peptide sequence of the template molecule for identifying uPA is selected from SEQ ID NO: 1 or SEQ ID NO: 2.

[0024] In some embodiments, the concentration of the template molecule for identifying uPA is between 0.001 and 1000 picograms per milliliter.

[0025] In some embodiments, two-dimensional materials may be further added to the peptide-imprinted conductive polymer of the present invention. The aforementioned two-dimensional materials are selected from at least one of the structural formulas Mn+1Xn or MY2, where M represents a transition metal element, X represents carbon or nitrogen, Y represents a chalcogenide such as S, Se, or Te, and n = 1~3. Examples include: vanadium carbide (V4C3Tx), niobium carbide (Nb4C3Tx), tantalum carbide (Ta4C3Tx), chromium carbide (Cr2C), manganese carbide (Mn2C), molybdenum carbide (Mo2C), trimolybdenum dicarbide (Mo3C2), niobium carbide (Nb2C), hafnium carbide (Hf2C), scandium carbide (Sc2C), tantalum carbide (Ta2C), titanium carbide (Ti2C), zirconium carbide (Zr2C), CrC, MoN, WC, NbC, MoYC, chromium nitride (Cr2N), and hafnium nitride (Hf2C). 2N), molybdenum nitride (Mo 2N), tantalum nitride (Ta 2N), titanium nitride (Ti 2N), zirconium nitride (Zr 2N), tungsten nitride (W 2N), vanadium nitride (V 2N), niobium tricarbide (Nb 4C 3), tantalum tricarbide (Ta 4C 3), trititanium dicarbide (Ti 3C 2), trititanium dinitride (Ti 3N 2), trititanium trinitride (Ti 4N 3), trititanium carbonitride (Ti 3(CN)), zirconium dicarbide (Zr 3C 2), trititanium dicarbide ((Ti,V) 3C 2), trititanium dicarbide ((Ti 2,Nb)C 2), trititanium dicarbide ((Ti 2,Ta)C 2), trititanium dicarbide ((Ti 2,Mn)C 2) Tetracarbide (Ti,Nb)₄C₃, Tetracarbide (Niobium,Zr)₄C₃, Trihafnium dicarbide (Hf₃C₂), Vanadium dicarbide (V₂C), Tetracarbide (V₄C₃), Dihafnium dicarbide (Hf₂V)C₂, Dihafnium dimanganese dicarbide (Hf₂Mn)C₂, Divanadium dicarbide (V₂Ti)C₂, Dichromium dicarbide (Cr₂V)C₂, Dichromium dicarbide (Cr₂Nb)C₂, Dichromium dicarbide (Cr₂Ta)C₂, Dichromium dicarbide (Cr₂Ti)C₂ 2) Molybdenum dicarbide ((Mo₂Hf)C₂), Molybdenum dicarbide ((Mo₂Zr)C₂), Molybdenum dicarbide ((Mo₂Sc)C₂), Molybdenum dicarbide ((Mo₂Ta)C₂), Molybdenum dicarbide ((Mo₂Ti)C₂), Molybdenum dicarbide ((Mo₂Nb)C₂)2) Dicarbide (Mo₂V)C₂, Dicarbide (W₂Ti)C₂, Dicarbide (W₂Zr)C₂, Dicarbide (W₂Hf)C₂, Dicarbide (W₂Ti)C₃, Dicarbide (W₂Zr)C₃, Dicarbide (W₂Hf)C₃, Dicarbide (W₂Ti)C₃, Dicarbide (W₂Zr)C₃, Dicarbide (W₂Hf)C₃, Dicarbide (W₂Ti)C₃, Dicarbide (W₂Hf)C₃, Dicarbide (Mo₂Hf)C₃, Dicarbide (Mo₂V)C₃, Dicarbide (Mo₂Zr)C₃, Dicarbide (Mo₂Ti ... (2Ti ​​2)C 3), Tricarbide (dimolybdenum ditantalumide) ((Mo 2Ta 2)C 3), Tricarbide (dimolybdenum diniobide) ((Mo 2Nb 2)C 3), Tricarbide (dichromium dititanide) ((Cr 2Ti 2)C 3), Tricarbide (dichromium ditantalumide) ((Cr 2Ta 2)C 3), Tricarbide (dichromium diniobide) ((Cr 2Nb 2)C 3), Tricarbide (dichromium divanadate) ((Cr 2V 2)C 3), Tricarbide (diniobium ditantalumide) ((Nb 2Ta 2)C 3), Tricarbide (divanadium ditantalumide) ((V 2Ta 2)C 3), Tricarbide (divanadium diniobide) ((V 2Nb 2)C 3), Tricarbide (divanadium dititanide) ((V 2Ti 2)C 3), 3) Titanium tricarbide (Ti₂Ta₂)C₃, Titanium tricarbide (Ti₂Nb₂)C₃, Molybdenum disulfide (MoS₂), Tungsten disulfide (WS₂), Titanium disulfide (TiS₂), Vanadium disulfide (VS₂), Cobalt disulfide (CoS₂), Nickel disulfide (NiS₂), Zirconium disulfide (ZrS₂), Hormone disulfide (TcS₂), Rhodium disulfide (RhS₂), Palladium disulfide (PdS₂), Hafnium disulfide (HfS₂), Tantalum disulfide (TaS₂), Rhenium disulfide (ReS₂), Iridium disulfide (IrS₂), Indium disulfide (InS₂), Tin disulfide (SnS₂), Platinum disulfide (PtS₂), Molybdenum diselenide (MoSe₂) 2) Tungsten diselenide (WSe 2), Titanium diselenide (TiSe 2), Vanadium diselenide (VSe 2), Cobalt diselenide (CoSe 2), Nickel diselenide (NiSe 2), Zirconium diselenide (ZrSe 2), Hormone diselenide (TcSe 2), Rhodium diselenide (RhSe 2), Palladium diselenide (PdSe 2), Hafnium diselenide (HfSe 2), Tantalum diselenide (TaSe 2), Rhenium diselenide (ReSe 2), Iridium diselenide (IrSe 2), Indium diselenide (InSe 2), Tin diselenide (SnSe 2), Platinum diselenide (PtSe 2)2) Molybdenum ditelluride (MoTe 2), tungsten ditelluride (WTe 2), titanium ditelluride (TiTe 2), vanadium ditelluride (VTe 2), cobalt ditelluride (CoTe 2), nickel ditelluride (NiTe 2), zirconium ditelluride (ZrTe 2), uranium ditelluride (TcTe 2), rhodium ditelluride (RhTe 2), palladium ditelluride (PdTe 2), hafnium ditelluride (HfTe 2), tantalum ditelluride (TaTe 2), rhenium ditelluride (ReTe 2), iridium ditelluride (IrTe 2), indium ditelluride (InTe 2), tin ditelluride (SnTe 2), and platinum ditelluride (PtTe 2), etc.

[0026] In another embodiment, the present invention provides a biosensor comprising the above-mentioned uPA peptide imprinted conductive polymer.

[0027] In some embodiments, the aforementioned biosensor includes an electrode substrate for coating the peptide-imprinted conductive polymer. The aforementioned electrode substrate can be a substrate composed of any conductive material as understood by those skilled in the art to which this invention pertains, such as: indium tin oxide (ITO) glass substrate, screen-printed electrode, glassy carbon electrode, PET flexible conductive glass substrate, aluminum-doped zinc oxide (AZO) conductive substrate, fluorine-doped tin oxide (FTO) conductive substrate, and silicon dioxide conductive substrate, etc.

[0028] In some embodiments, the biosensor further includes an electrochemical analyzer or an extended gate field-effect transistor. The electrochemical analyzer may be, for example, a potentiostat electrochemical analyzer. The field-effect transistor may be, for example, a metal-oxide-semiconductor field-effect transistor.

[0029] In another embodiment, the present invention provides a method for preparing a biosensor, comprising: step (1) preparing a monomer solution of a conductive polymer monomer with a concentration between 0.001 and 50% by weight, wherein the conductive polymer monomer is selected from at least one of the following groups: m-aminobenzenesulfonic acid (MSAN), 2-aminobenzenesulfonic acid (MSDS), 4-aminobenzenesulfonic acid (SDS), aniline (AN), 3,4-ethylenedioxythiophene (EDOT), hydroxymethyl 3,4-ethylenedioxy-thiophene (EDOT-OH), acetylene, pyrrole, thiophene, phenylene Vinylene, isothianaphthene, 3-alkyl thiophene, 2,5-dialkoxy paraphenylene vinylene, paraphenylene, paraphenylene sulphide, and heptadiyne; Step (2) Add a template molecule for recognizing uPA to the monomer solution; Step (3) By electrochemical polymerization, imprint the molecular template onto the conductive polymer monomer, and coat the imprinted conductive polymer onto an electrode substrate to obtain a biosensor; The template molecule for recognizing uPA is selected from peptide fragments in the peptide sequence of the complete uPA protein that have the following conditions: (1) The length of the peptide fragment is 5 to 30 amino acids; (2) The ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids in the peptide fragment is 0 to 75%; and (3) This peptide fragment contains 0 to 15 aromatic amino acids.

[0030] In some embodiments, in the method for preparing the biosensor, the peptide sequence of the template molecule for identifying uPA is selected from SEQ ID NO: 1 to SEQ ID NO: 11.

[0031] In some embodiments, in the method for preparing the biosensor, the peptide sequence of the template molecule for identifying uPA is selected from SEQ ID NO: 1 or SEQ ID NO: 2.

[0032] In some embodiments, in the method for preparing the biosensor, before step (3), a two-dimensional material is further added to the conductive polymer monomer solution.

[0033] In some embodiments, in the method for preparing the biosensor, after step (3), the electrode substrate is further connected to an electrochemical analyzer or a field-effect transistor.

[0034] In another embodiment, the present invention provides a use of the above-mentioned uPA peptide imprinted conductive polymer, characterized in that the uPA peptide imprinted conductive polymer can be used to detect thrombotic diseases. Thrombotic diseases include acute myocardial infarction, pulmonary embolism, deep vein thrombosis, or atherosclerosis, etc.

[0035] In another embodiment, the present invention provides a method for detecting the concentration of uPA in a sample by electrochemical sensing, characterized by comprising the following steps: Step (1) Standard solutions containing different concentrations of uPA are respectively dropped onto a biosensor containing the above-mentioned peptide-imprinted conductive polymer, and then the response current values ​​of the cyclic voltammograms containing standard solutions of different concentrations of uPA are measured by a potentiostat, and the measurement is repeated at least twice; Step (2) The response current values ​​of the cyclic voltammograms obtained in Step (1) are integrated to obtain the uPA calibration curve; Step (3) The sample solution to be tested is dropped onto the biosensor, and the response current value of its cyclic voltammogram is measured, and the measurement is repeated at least twice; Step (4) The response current value of the sample is compared with the uPA calibration curve to calculate the uPA concentration of the sample.

[0036] In another embodiment, a method for detecting uPA concentration in a sample using an extended gate field-effect transistor (EGFET) is characterized by the following steps: Step (1) Connecting the biosensor of the present invention to an extended gate field-effect transistor (EGFET), setting the scan voltage to the drain terminal, setting different bias voltages to the gate terminal, and taking 60 data points from 0V to 6V; Step (2) Summarizing the characteristic graph (IDVD graph) of drain current versus drain voltage for different gate voltages (VG) to obtain the optimal VG; Step (3) Dropping standard solutions containing different uPA concentrations onto the biosensor, measuring them, and obtaining the adsorption drain current calibration curve with concentration on the x-axis and drain current on the y-axis; Step (4) Dropping the sample solution to be tested onto the biosensor, measuring the response current value, and repeating the measurement at least twice; Step (5) compares the response current value of the sample with the calibrator of the uPA to calculate the uPA concentration of the sample. [Effects of the Invention]

[0037] The length of the peptide and the hydrophobic amino acids contained within the fragment will affect whether the formed imprinted pores have specific recognition. The present invention provides uPA peptides of specific length with specific amino acid composition as template molecules for recognizing uPA. The peptide imprinted pores on the conductive polymer have high specific recognition for uPA.

[0038] This invention utilizes the characteristic that the specific binding of the sample and the polymer changes the electrical properties of the sensor. The biochemical signal of the sample is converted into an electrical signal that is easy to observe via a transducer. The concentration of uPA in the sample is deduced by quantitatively analyzing the response current signal, which serves as reference data for detecting thrombotic diseases.

[0039] This invention utilizes peptide-imprinted conductive polymer technology combining epitope imprinting and electrochemical polymerization to achieve a responsively sensitive deposition electrode surface. This provides a highly sensitive extended-gate field-effect transistor (EPFET) biosensor or electrochemical biosensor made from an EPFET conductive polymer used to detect uPA in human samples. Its circuit design allows for miniaturization, ease of manufacturing, and significantly reduced costs. Furthermore, the extended-gate field-effect transistor or electrochemical detection method exhibits high sensitivity and a low detection limit. Therefore, this novel sensor combines numerous advantages such as accurate detection, high sensitivity, rapid response, low detection limit, high chemical stability, simple fabrication process, easy operation, low cost, portability, reusability, and high specificity. It also achieves a wide current range, excellent stability, and high-quality measurement performance.

[0040] By using extended gate field-effect transistor (EG-FET) sensing or electrochemical sensing to detect uPA in human serum samples, the time and cost required to detect thrombotic diseases such as acute myocardial infarction, pulmonary embolism, deep vein thrombosis, and atherosclerosis can be shortened, and treatment can be initiated immediately. This can delay the progression of the disease or improve the effectiveness of treatment, thus solving the current dilemma in the medical diagnosis of thrombotic diseases.

[0041] The peptide-imprinted conductive polymer of the present invention has superior detection sensitivity and a low detection limit. Furthermore, when two-dimensional materials are added to the peptide-imprinted conductive polymer of the present invention, the linear region of the sensing and quantification lines for uPA can be further increased, thereby improving the response current range and imprinting efficiency.

Implementation Method

[0043] The following discloses embodiments of the present invention, which are not intended to limit the present invention to being implemented in the following manner, but are intended to illustrate the details of the present invention and the effects of its implementation.

[0044] The peptide-imprinted conductive polymer of the present invention comprises a conductive polymer monomer and a template molecule for recognizing uPA. The peptide-imprinted conductive polymer of the present invention may further comprise a two-dimensional material.

[0045] The conductive polymer monomer of the present invention is selected from at least one of the following groups: m-aminobenzenesulfonic acid (MSAN), 2-aminobenzenesulfonic acid (MSDS), aniline (AN), 4-aminobenzenesulfonic acid (SDS), 3,4-ethylenedioxythiophene (EDOT), hydroxymethyl 3,4-ethylenedioxy-thiophene (EDOT-OH), acetylene, pyrrole, thiophene, phenylene vinylene, isothianaphthene, 3-alkyl thiophene, 2,5-dialkoxy paraphenylene. Vinylene, paraphenylene, paraphenylene sulphide, and heptadiyne.

[0046] Preferably, the peptide-imprinted conductive polymer of the present invention may comprise two conductive polymer monomers, such as aniline (AN) and m-aminobenzenesulfonic acid (MSAN), aniline and 2-aminobenzenesulfonic acid (MSDS), or aniline and 4-aminobenzenesulfonic acid (SDS). The molar ratio of the two conductive polymer monomers may be controlled between 0.001 and 20, preferably between 0.25 and 5, more preferably between 0.5 and 4, and even more preferably 4. For example, the molar ratio of aniline to m-aminobenzenesulfonic acid, aniline to 2-aminobenzenesulfonic acid, or aniline to 4-aminobenzenesulfonic acid may be 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

[0047] The peptide-imprinted conductive polymer of the present invention preferably uses aniline and 2-aminobenzenesulfonic acid as the conductive polymers, and the mole ratio of aniline is 80% of the total mole number of all conductive polymer monomers, that is, the mole ratio of aniline to 2-aminobenzenesulfonic acid is 4:1. In subsequent embodiments, when using this specific ratio of conductive polymer monomers, it is also simply referred to as AN80%MSDS or AN80MSDS.

[0048] The concentration of the conductive polymer monomer of the present invention can be 0.001 to 50% by weight.

[0049] Exemplarily, the polymer monomers of the present invention are selected from three combinations of aniline and m-aminobenzenesulfonic acid, aniline and 2-aminobenzenesulfonic acid, and aniline and 4-aminobenzenesulfonic acid, and are used for electrochemical polymerization. The polymer monomers themselves have good conductivity and contain sulfonate groups, so there is no need to add additional organic acids. The monomers can be aniline-doped m-aminobenzenesulfonic acid, aniline-doped 2-aminobenzenesulfonic acid, or aniline-doped 4-aminobenzenesulfonic acid, both dissolved in deionized water, with concentrations ranging from 0.001 to 50% by weight, preferably 0.001 to 25% by weight, and more preferably 0.001 to 10% by weight.

[0050] The template molecule for identifying uPA in this invention utilizes a small portion of the uPA molecule's peptides as a template. Compared to the protein itself, short peptides are more stable and cheaper. However, the length of the peptide is closely related to the ease with which the molecule can enter and exit the pore. Among these factors, the hydrophobic properties of the amino acids contained in the peptide fragment have a more decisive influence on whether the formed imprinted pore has specific identification capability. Appropriate peptide selection can improve the reusability of the imprinted pore. Therefore, the template molecule of this invention is selected from peptide fragments in the peptide sequence of the complete uPA protein that have the following conditions: (1) The length of the peptide fragment is 5 to 30 amino acids; preferably, the length of the peptide fragment is 6 to 20 amino acids; more preferably, the length of the peptide fragment is 8 to 15 amino acids; even more preferably, the length of the peptide fragment is 9 to 12 amino acids; (2) To improve the solubility of the template molecule of the present invention in a conductive polymer solution, the ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids contained in the peptide fragment is 0-75%; preferably, the ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids contained in the peptide fragment is 2-60%; more preferably, the ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids contained in the peptide fragment is 20-55%, wherein the aforementioned hydrophobic amino acids may be selected from the group consisting of: glycine (Gly), alanine (Ala), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), methionine (Met), proline (Pro), valine (Val), and tryptophan (Trp); (3) The peptide fragment contains 0 to 15 aromatic amino acids; preferably, the peptide fragment contains 0 to 7 aromatic amino acids, which may be selected from the group consisting of: phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), histidine (His), and proline (Pro).

[0051] By way of example, the peptide sequence of the template molecule of the present invention may be selected from at least one of the groups of SEQ ID NO: 1 to SEQ ID NO: 11.

[0052] By way of example, the peptide sequence of the template molecule of the present invention may be selected from at least one of the following: peptide H (abbreviated as pH, SEQ ID NO: 1) and peptide D (abbreviated as pD, SEQ ID NO: 2). Peptide H is preferred.

[0053] The concentration of the aforementioned peptide template molecules is between 0.001 and 100 micrograms / mL, preferably 0.01 to 25 micrograms / mL, more preferably 0.1 to 10 micrograms / mL, and even more preferably 1 to 5 micrograms / mL.

[0054] The two-dimensional material of the present invention may also be selected from at least one of the groups consisting of the general structural formula Mn+1Xn or MY2, which are transition metal carbides / nitrides (or referred to as MXene) or transition metal disulfides, respectively, wherein M represents a transition metal element, X represents a carbon element or a nitrogen element, Y represents a chalcogenide such as S, Se or Te, and n=1~3, for example: vanadium carbide (V4C3Tx), niobium carbide (Nb4C3Tx), tantalum carbide (Ta4C3Tx), chromium carbide (Cr2C), manganese carbide (Mn2C), molybdenum carbide (Mo2C), trimolybdenum dicarbide (Mo3C2), niobium carbide (Nb2C), hafnium carbide (Hf2C), scandium carbide (Sc2C), titanium carbide (Ti2C), tantalum carbide (Ta2C), zirconium carbide (Zr2C). 2C), CrC, MoN, WC, NbC, MoYC, chromium nitride (Cr2N), hafnium nitride (Hf2N), molybdenum nitride (Mo2N), tantalum nitride (Ta2N), titanium nitride (Ti2N), zirconium nitride (Zr2N), tungsten nitride (W2N), vanadium nitride (V2N), niobium tricarbide (Nb4C3), tantalum tricarbide (Ta4C3), trititanium dicarbide (Ti3C2), trititanium dinitride (Ti3N2), trititanium trinitride (Ti4N3), trititanium carbonitride (Ti3(CN)), zirconium dicarbide (Zr3C2), trititanium dicarbide ((Ti,V)3C2), trititanium dicarbide ((Ti2,Nb)C 2) Dicarbide (titanium, tantalum) ((Ti 2,Ta)C 2), Dicarbide (titanium, manganese) ((Ti 2,Mn)C 2), Tetracarbide (titanium, niobium) ((Ti,Nb) 4C 3), Tetracarbide (niobium, zirconium) ((Nb,Zr) 4C 3), Trihafnium dicarbide (Hf 3C 2), Vanadium dicarbide (V 2C), Vanadium tricarbide (V 4C 3), Dihafnium dicarbide (vanadium dicarbide) ((Hf 2V)C 2), Dihafnium dicarbide (manganese dihafnium) ((Hf 2Mn)C 2), Divanadium dicarbide (titanium divanadium) ((V 2Ti)C 2), Dichromium dicarbide (vanadium dichromium) ((Cr 2V)C 2), Dichromium dicarbide (niobium dichromium) ((Cr 2Nb)C 2) 2) Dicarbide (dichromium tantalum) ((Cr₂Ta)C₂), Dicarbide (dichromium titanate) ((Cr₂Ti)C₂), Dicarbide (dimolybdenum hafnium) ((Mo₂Hf)C₂), Dicarbide (dimolybdenum zirconate) ((Mo₂Zr)C₂), Dicarbide (dimolybdenum scandium) ((Mo₂Sc)C₂), Dicarbide (dimolybdenum tantalum) ((Mo₂Ta)C₂), Dicarbide (dimolybdenum titanate) ((Mo₂Ti)C₂), Dicarbide (dimolybdenum niobate) ((Mo₂Nb)C₂)2) Dicarbide (Mo₂V)C₂, Dicarbide (W₂Ti)C₂, Dicarbide (W₂Zr)C₂, Dicarbide (W₂Hf)C₂, Dicarbide (W₂Ti)C₃, Dicarbide (W₂Zr)C₃, Dicarbide (W₂Hf)C₃, Dicarbide (W₂Ti)C₃, Dicarbide (W₂Zr)C₃, Dicarbide (W₂Hf)C₃, Dicarbide (W₂Ti)C₃, Dicarbide (W₂Hf)C₃, Dicarbide (Mo₂Hf)C₃, Dicarbide (Mo₂V)C₃, Dicarbide (Mo₂Zr)C₃, Dicarbide (Mo₂Ti ... (2Ti ​​2)C 3), Tricarbide (dimolybdenum ditantalumide) ((Mo 2Ta 2)C 3), Tricarbide (dimolybdenum diniobide) ((Mo 2Nb 2)C 3), Tricarbide (dichromium dititanide) ((Cr 2Ti 2)C 3), Tricarbide (dichromium ditantalumide) ((Cr 2Ta 2)C 3), Tricarbide (dichromium diniobide) ((Cr 2Nb 2)C 3), Tricarbide (dichromium divanadate) ((Cr 2V 2)C 3), Tricarbide (diniobium ditantalumide) ((Nb 2Ta 2)C 3), Tricarbide (divanadium ditantalumide) ((V 2Ta 2)C 3), Tricarbide (divanadium diniobide) ((V 2Nb 2)C 3), Tricarbide (divanadium dititanide) ((V 2Ti 2)C 3), 3) Titanium tricarbide (Ti₂Ta₂)C₃, Titanium tricarbide (Ti₂Nb₂)C₃, Molybdenum disulfide (MoS₂), Tungsten disulfide (WS₂), Titanium disulfide (TiS₂), Vanadium disulfide (VS₂), Cobalt disulfide (CoS₂), Nickel disulfide (NiS₂), Zirconium disulfide (ZrS₂), Hormone disulfide (TcS₂), Rhodium disulfide (RhS₂), Palladium disulfide (PdS₂), Hafnium disulfide (HfS₂), Tantalum disulfide (TaS₂), Rhenium disulfide (ReS₂), Iridium disulfide (IrS₂), Indium disulfide (InS₂), Tin disulfide (SnS₂), Platinum disulfide (PtS₂), Molybdenum diselenide (MoSe₂) 2) Tungsten diselenide (WSe 2), Titanium diselenide (TiSe 2), Vanadium diselenide (VSe 2), Cobalt diselenide (CoSe 2), Nickel diselenide (NiSe 2), Zirconium diselenide (ZrSe 2), Hormone diselenide (TcSe 2), Rhodium diselenide (RhSe 2), Palladium diselenide (PdSe 2), Hafnium diselenide (HfSe 2), Tantalum diselenide (TaSe 2), Rhenium diselenide (ReSe 2), Iridium diselenide (IrSe 2), Indium diselenide (InSe 2), Tin diselenide (SnSe 2), Platinum diselenide (PtSe 2)2) Molybdenum ditelluride (MoTe 2), tungsten ditelluride (WTe 2), titanium ditelluride (TiTe 2), vanadium ditelluride (VTe 2), cobalt ditelluride (CoTe 2), nickel ditelluride (NiTe 2), zirconium ditelluride (ZrTe 2), tungsten ditelluride (TcTe 2), rhodium ditelluride (RhTe 2), palladium ditelluride (PdTe 2), hafnium ditelluride (HfTe 2), tantalum ditelluride (TaTe 2), rhenium ditelluride (ReTe 2), iridium ditelluride (IrTe 2), indium ditelluride (InTe 2), tin ditelluride (SnTe 2), and platinum ditelluride (PtTe 2). 2) Transition metal dichalcogenide two-dimensional materials have electrical conductivity close to that of semiconductors, while MXene two-dimensional materials have excellent electrical conductivity close to that of metals, excellent thermal stability and mechanical properties, and the particle size can be increased by 20 times.

[0055] The concentration of the aforementioned two-dimensional material may be 0.001~50 micrograms / mL, preferably 0.01~25 micrograms / mL, more preferably 0.05~10 micrograms / mL, even more preferably 1~5 micrograms / mL, and even more preferably 5 micrograms / mL.

[0056] For example, the present invention can add two-dimensional materials such as Ti2C, Ti2N, V2C, Ta2C or Nb2C as dopants to the peptide imprinting conductive polymer solution, thereby increasing the surface roughness and surface area of ​​the conductive polymer, thereby improving the overall conductivity and reducing the amount of organic solvent used in the process.

[0057] The present invention provides a biosensor comprising the above-mentioned peptide-imprinted conductive polymer. The biosensor may further comprise an electrochemical analyzer or a field-effect transistor (FET), enabling sensing to be either electrochemical sensing or extended gate field-effect transistor (EG-FET) sensing. The extended gate field-effect transistor may consist of a sensing electrode comprising the peptide-imprinted conductive polymer and a field-effect transistor (MOSFET).

[0058] The biosensor of the present invention may include an electrode substrate, which may be a substrate composed of any conductive material as understood by those skilled in the art to which this invention pertains, such as: indium tin oxide (ITO) glass substrate, screen-printed electrode, glassy carbon electrode, PET flexible conductive glass substrate, aluminum-doped zinc oxide (AZO) conductive substrate, fluorine-doped tin oxide (FTO) conductive substrate, and silicon dioxide conductive substrate, etc.

[0059] For example, as shown in FIG1, when the electrochemical biosensor of the uPA peptide imprinted conductive polymer of the present invention is further added with two-dimensional materials, the preparation steps may be as follows: Step (1) Prepare a monomer solution of conductive polymer monomer 11 with a concentration between 0.001 and 50% by weight, wherein the conductive polymer monomer is selected from at least one of the following groups: m-aminobenzenesulfonic acid (MSAN), 2-aminobenzenesulfonic acid (MSDS), 4-aminobenzenesulfonic acid (SDS), aniline (AN), 3,4-ethylenedioxythiophene (EDOT), hydroxymethyl 3,4-dioxyethylthiophene (EDOT-OH), acetylene, pyrrole, thiophene, phenylene vinylene, isothianaphthene, 3-alkyl thiophene, 2,5-dialkoxy paraphenylene Vinylene, paraphenylene, paraphenylene sulfide and heptadiyne; Step (2) Add a uPA peptide template molecule 10 with a concentration between 0.001 and 1000 μg / mL to the monomer solution and stir evenly; Step (3) Add a two-dimensional material 12 solution with a concentration between 0.001 and 50% by weight to the monomer solution; Step (4) Add the monomer solution to the electropolymerization cell, stir evenly with the magnet, then place the auxiliary electrode and the reference electrode, connect an electrode substrate 14 as the working electrode, and perform the electropolymerization step using a commercial potentiostat; Step (5) After the electropolymerization is completed, the electrode substrate 14 is coated with a conductive polymer 13 that combines the template molecule and is doped with two-dimensional material 12. Use a 5% volume molar concentration of alcohol aqueous solution and deionized water, and shake sequentially at a speed of 130 rpm for 10 minutes to remove the template molecule combined in the conductive polymer, leaving a distinctive imprint hole.

[0060] Different concentrations of the target peptide or uPA were prepared and dropped onto the electrochemical biosensor described above to detect the re-adsorption effect of the electrochemical biosensor on the target peptide and uPA. [Example]

[0061] Next, specific embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

[0062] Example 1: Test of different doping ratios of conductive polymer monomers (aniline-doped m-aminobenzenesulfonic acid, aniline-doped 2-aminobenzenesulfonic acid, and aniline-doped 4-aminobenzenesulfonic acid)

[0063] Aniline, m-aminobenzenesulfonic acid, 2-aminobenzenesulfonic acid, and 4-aminobenzenesulfonic acid are dissolved in 0.5 liters of deionized water to make the concentration of conductive monomer in the resulting solution 0.01~2 M, and then doped with different molar ratios: (1) The molar ratio of aniline to m-aminobenzenesulfonic acid is 4:1, 2:1, 1:1, 1:2 or 1:4; (2) The molar ratio of aniline to 2-aminobenzenesulfonic acid is 4:1, 2:1, 1:1, 1:2 or 1:4; (3) The molar ratio of aniline to 4-aminobenzenesulfonic acid is 4:1, 2:1, 1:1, 1:2 or 1:4.

[0064] To the conductive monomer solutions prepared in different molar ratios as described above, template molecule peptide H (SEQ ID NO: 1) or peptide D (SEQ ID NO: 2) was added to prepare MIP electropolymerization solutions with a total volume of 20 mL and template molecule peptide concentrations of 1 µg / mL, 2.5 µg / mL, and 5 µg / mL, respectively. Simultaneously, NIP electropolymerization solutions containing only the mixed conductive monomers and without the template molecule peptide were also prepared.

[0065] A platinum sheet (Pt) is placed in the electrolytic cell as an auxiliary electrode, and a silver / silver ion (Ag / Ag+) electrode is used as a reference electrode. An indium tin oxide (ITO) glass with a Ω of 5-50 mm (height) × 5-50 mm (width) × 0.5-5 mm (thickness) and 2-20 Ω is used as the working electrode and fixed with a working electrode clamp. Next, an electropolymerization reaction is performed using an electropolymerization solution to coat the working electrode with a non-imprinted polymer (NIPs) or a molecularly imprinted polymer (MIPs). The area of ​​the imprinted molecular film is 0.1-3 × 0.01-3 cm².

[0066] After polymerization, add a 5% (v / v) alcohol aqueous solution and agitate at 130 rpm for 15 minutes to clean and remove the template molecules bound to the conductive polymer, leaving identifiable imprinted holes in the polymer. Place in a 55°C constant temperature oven and dry for 10 minutes. The resulting sensing electrode can be stored in a dehumidifier until needed.

[0067] The completed peptide imprinting electrode and the measured ΔC current difference are shown in Figure 2. Among them, the peptide imprinting electrode difference is the largest when aniline and 2-aminobenzenesulfonic acid with a molar ratio of 4:1 are used as polymer monomers and peptide H with a molecular template of 5.0 μg / mL is used.

[0068] Example 2: Re-adsorption experiment of peptide-imprinted conductive polymers (aniline with m-aminobenzenesulfonic acid, aniline with 2-aminobenzenesulfonic acid, aniline with 4-aminobenzenesulfonic acid)

[0069] The target molecule sensing performance of peptide-imprinted electrodes (MIPs) and unimprinted electrodes (NIPs) was tested when the polymer monomers were selected in a molar ratio of 4:1 for aniline:aminobenzenesulfonic acid (m-aminobenzenesulfonic acid, 2-aminobenzenesulfonic acid, 4-aminobenzenesulfonic acid). The imprinted molecular template was also peptide H at 5.0 μg / mL.

[0070] The electrolytic cell is also equipped with the aforementioned auxiliary electrode, reference electrode, and working electrode. Potassium ferricyanide is added to deionized water to prepare a buffer solution, which is then added to the electrolytic cell. After standing for 10 minutes, the cyclic voltammogram of the sensing sample in the buffer solution is measured using a commercial potentiostat (Figure 3). Next, after the measured solution is adsorbed with filter paper, different concentrations of the target molecule (peptide H) sample are added, with the concentration increasing sequentially by powers of 10 from a minimum of 0.001 picograms / mL to a maximum of 1000 picograms / mL. After standing for 10 minutes, the measurement is performed again. The response current value is obtained by summing the data from each measurement. This is further plotted as a graph with the peptide concentration from 0.001 to 1000 picograms / mL on the x-axis and the current density on the y-axis, which is called the calibration curve (Figure 4).

[0071] The aforementioned target molecule samples were prepared by dissolving the target molecules in ultrapure water in a sterile cryotube, mixing them evenly with a shaker, and then mixing the target molecules with a buffer solution to prepare test samples of different concentrations.

[0072] The imprinting efficiency α (α = MIPs current / NIPs current) is obtained by using the difference in current density between the electrochemical biosensor of peptide-imprinted conductive polymers (MIPs) and the difference in current density between the electrochemical biosensor of unimprinted conductive polymers (NIPs).

[0073] The results are shown in Figure 4 and Table 1. Among the conductive polymers imprinted using peptide H, the largest current difference, 1.1 microamperes, was obtained when using a conductive monomer with a doping ratio of aniline to 2-aminobenzenesulfonic acid of 4:1. Furthermore, the imprinting efficiency was also better when using a conductive monomer with a doping ratio of aniline to 2-aminobenzenesulfonic acid of 4:1.

[0074] [Table 1] Sensing effect of conductive monomers in a mixture of aniline and three aminobenzenesulfonic acids (4:1) Monomers mixed with aniline MSDS MSAN SDS Difference 1.1 0.8 0.7 Printing efficiency 2.4 1.7 1.1

[0075] The results above show that the unprinted conductive polymer does not have identification pores, so it cannot significantly increase the adsorption current density as the concentration of target molecules in the test solution increases. When the conductive polymer with imprinted peptide H adsorbs peptide H and uPA intact protein, the adsorption current density gradually reaches saturation.

[0076] Example 3: Electrochemical sensing of the scan rate of a biosensor based on peptide-imprinted conductive polymer

[0077] First, a molar ratio of aniline to 2-aminobenzenesulfonic acid of 4:1 (AN80%MSDS) was selected as the conductive monomer for imprinting. The imprinting molecular template was also peptide H at 5.0 μg / mL. After preparing peptide H imprinted conductive polymers (AN80%MSDS pHIPs) and unimprinted conductive polymers (NIPs) coated on the electrode using the same steps as in Example 1, measurements were taken at three different scan rates of 0.1 V / s, 0.05 V / s, and 0.02 V / s, and the effective electrode area of ​​pHIPs and NIPs was determined using the Randles-Sevcik equation.

[0078] The electrolytic cell is also equipped with the aforementioned auxiliary electrode, reference electrode, and working electrode. Potassium ferricyanide is added to deionized water to prepare a buffer solution, which is then added to the electrolytic cell. After standing for 10 minutes, the buffer solution sample is sensed using the aforementioned pHIPs and NIPs sensors. Next, the measured solution is adsorbed using filter paper, and then 20 μl of peptide H (concentration of 1 pg / ml) is added as the target molecule sample. After standing for 10 minutes, the measurement is performed again.

[0079] The results showed that the pHIPs sensor had a larger effective electrode area, and the increase in surface area indicated that the target molecules did indeed fill the pores of the peptide imprinted electrode.

[0080] Example 4: Interference test of biosensor based on peptide-imprinted conductive polymer for electrochemical sensing

[0081] To verify whether the pores left after imprinting peptide H with conductive polymer have specificity, thereby reducing the false positive rate and effectively applying them to real samples, conductive polymer AN80%MSDS pHIPs imprinted with 5.0 μg / mL peptide H were used to sense peptide H (SEQ ID NO:1), uPA molecules, peptide D (SEQ ID NO:2), and lysozyme, respectively.

[0082] Cyclic voltammetry of peptide D and lysozyme was obtained using a commercial potentiostat with AN80% MSDS pHIPs, and the results are shown in Figure 5. The detection curves (with the detection concentration increasing sequentially by powers of 10 from a minimum of 0.001 picograms / mL to a maximum of 1000 picograms / mL) obtained using AN80% MSDS pHIPs are shown in Figure 6. Figure 6 shows that the adsorption current difference for peptide H was the highest with AN80% MSDS pHIPs. The adsorption current difference for uPA molecules with AN80% MSDS pHIPs was also higher than that for peptide D and lysozyme. This result confirms that even in the presence of other interfering substances in the test sample, the peptide-imprinted conductive polymer of this invention still exhibits high specificity for the target molecule and can be effectively used for testing real samples.

[0083] Example 5: Impedance Analysis of Biosensors Based on Electrochemical Sensing of Peptide-Imprinted Conductive Polymers

[0084] The purpose of this embodiment is to obtain the charge transfer resistance value and electrochemical impedance spectroscopy of the peptide-imprinted conductive polymer.

[0085] Conductive polymer AN80%MSDS pHIPs were imprinted using peptide H at a concentration of 5.0 μg / mL. The coated electrodes (AN80%MSDS pHIPs) and unimprinted conductive polymers (NIPs) were prepared using the same steps as in Example 1. The aforementioned auxiliary electrode, reference electrode, and working electrode were also configured in the electrolytic cell. Potassium ferricyanide was added to deionized water to prepare a buffer solution, which was then added to the electrolytic cell. After standing for 10 minutes, the buffer solution sample was sensed using the aforementioned AN80%MSDS pHIPs and NIPs sensors. Next, after adsorbing the measured solution with filter paper, 20 μl of peptide H (concentration 1 pg / ml) was added as the target molecule sample, and the sample was again allowed to stand for 10 minutes before measurement. Measurements were performed using electrodes with different parameters as described below. By summarizing the electrochemical impedance spectroscopy data of various parameters and performing curve fitting using Zahner Aanalysis software, and setting the x-axis as the real impedance and the y-axis as the imaginary impedance, the resistance value of charge transfer and the electrochemical impedance spectroscopy can be obtained.

[0086] The electrochemical analysis parameters are set as follows: the mode is electrochemical impedance spectroscopy (EIS potentiostatic), the diagram type is Nyquist, the AC display is Z' and Z", the frequency scan strategy is 100mHz to 100KHz, the spectrum is single sine, the frequency step is set to 10 for high frequency and 5 for low frequency, and the measurement period is set to 20 for high frequency and 4Ω for low frequency.

[0087] The measurement results are as follows: the impedance of NIPs to the buffer solution is 6.49Ω, and the impedance to peptide H (concentration of 1 pg / ml) is 5.33Ω. The impedance of pHIPs to the buffer solution is 4.48Ω, and the impedance to peptide H (concentration of 1 pg / ml) is 3.82Ω.

[0088] Example 6: Reusability of a Biosensor Based on Electrochemical Sensing of Peptide-Imprinted Conductive Polymers

[0089] The purpose of this embodiment is to verify that the peptide-imprinted conductive polymer of the present invention can be reused for several cycles during testing.

[0090] An AN80%MSDS pHIPs sensor was prepared using the same steps as in Example 1, and then the peptide H (concentration of 1 pg / ml) added to the buffer solution was sensed using the same steps as in Example 5. After repeated measurements, the experimental data from each measurement were summarized to observe the degradation trend of the conductive polymer sensing performance.

[0091] The measurement results are shown in Figure 7. The x-axis in the figure represents the number of cycles, and the y-axis represents the relative current density. In the seven-cycle test, the AN80%MSDS pHIPs maintained more than 97% sensing performance in the second to fourth cycles compared to the first cycle. Although the sensing performance decreased slightly after the fifth cycle, it still maintained close to 90% sensing performance in the sixth cycle.

[0092] Example 7: Real Specimen Sensing Using a Biosensor Based on Electrochemically Sensing Peptide Imprinted Conductive Polymer

[0093] In order to test whether the peptide imprinted holes of the present invention are capable of recognizing the intact uPA protein present in human serum, a real sample sensing experiment was conducted.

[0094] An AN80% MSDS pHIPs sensor was prepared using the same steps as in Example 1, and samples were tested using the same steps as in Example 4. The samples tested were: human type AB serum, and uPA intact protein at known concentrations of 10 pg / ml and 30 pg / ml.

[0095] Summarize the data from multiple measurements to obtain the adsorption current value. Next, perform curve fitting and spiking to estimate the concentration of the actual sample and compare it with the theoretical concentration of the sample to obtain the recovery rate. The closer the recovery rate is to 100%, the higher the accuracy.

[0096] The formula for calculating accuracy is as follows:

[0097] Accuracy (%) = ×100

[0098] Measured value: The original concentration calculated using the calibration curve for the detection current.

[0099] Theoretical value: The concentration added plus the concentration of the detection solution.

[0100] The results are shown in Table 2. The concentration of human AB serum diluted 10⁶ times was 2.51 ng / mL. Using the spiking method, the highest recovery rate reached 94.5%, demonstrating high accuracy. The sensing limit was 0.032 fg / ml, and the sensing sensitivity was 120.19 (μA / fg)∙mL.

[0101] The sensing sensitivity is calculated as follows: Sensing sensitivity = (maximum current density in sensing area - minimum current density in sensing area) / maximum concentration in sensing area.

[0102] [Table 2]

[0103] Example 8: Testing of Extended Gate Field-Effect Transistor

[0104] The purpose of this embodiment is to verify that the present invention can also achieve excellent detection results when using extended gate field-effect transistor (EG-FET) sensing method.

[0105] First, following the same steps as in Example 1, electrodes coated with AN80% MSDS pHIPs and NIPs were prepared. 20 mL of PBS buffer solution was added to the electrolytic cell. The prepared working electrode and reference electrode (platinum sheet) were fixed and connected to a field-effect transistor. The current-voltage control system parameters were set as follows: the scan voltage was at the drain terminal, different bias voltages were set at the gate terminal, and 60 data points were collected from 0V to 6V. Next, the I-D-D plots of different VGs were summarized (Figure 8) to obtain the optimal VG.

[0106] The VGS of this extended gate field-effect transistor was set to 4.0V and the VDS was set to 3.0V. Peptide H sensing was performed using two working electrodes coated with AN80% MSDS pHIPs and NIPs, respectively, and a calibration curve was obtained with the peptide concentration (0.001~1000 picograms / mL) on the x-axis and the adsorption-drain current on the y-axis.

[0107] The I DV D diagram is shown in Figure 8, and the peptide H sensing detection lines for the two electrodes are shown in Figure 9. The peptide H sensing results show that the AN80% MSDS pHIPs have a sensing range of 0.001~1000 fg / mL for peptide H. The sensing limit is 0.013 fg / mL, and the sensing sensitivity is 477.39 (μA / fg)∙mL.

[0108] The same sensing steps were performed on real samples. The samples tested were: human AB serum, and uPA intact protein at known concentrations of 10 ag / mL and 40 ag / mL.

[0109] Summarize multiple measurement data to obtain the adsorption and drain current values. Next, perform curve fitting and slaking to estimate the actual concentration of the sample, and compare it with the theoretical concentration of the sample to obtain the recovery rate. The closer the recovery rate is to 100%, the higher the accuracy.

[0110] The results are shown in Figure 10 and Table 3. The sensing range of AN80%MSDS pHIPs for intact uPA protein in real samples was 0.001~1000 fg / mL. When detecting human AB serum diluted 10⁹ times, the conversion concentration was 2.82 ng / mL. Using the spiking method, the highest recovery rate in the experiment reached 96.1%, demonstrating high accuracy.

[0111] [Table 3]

[0112] It can be seen that even when using extended gate field-effect transistors for sensing, the peptide imprinted conductive polymer of the present invention still has high specificity and can be applied to the effective detection of real samples.

[0113] Example 9: Electrode Surface Analysis of Biosensors

[0114] The surface of the electrodes of sensors coated with peptide-imprinted conductive polymers was analyzed using four methods: cold field emission electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy, and chemical analytical electron spectroscopy. The analysis focused on the electrode surface conditions of both AN80% MSDS pHIPs and NIPs sensors before washing (BW), after washing (AW), or re-adsorption (RB) during electrochemical sensing. "Before washing (BW)" refers to the situation where the peptide-imprinted electrode was directly air-dried without being washed with ethanol. "After washing (AW)" refers to the situation where the peptide-imprinted electrode was further washed with 5% (v / v) ethanol for 15 minutes, and then air-dried again. "Re-adsorption (RB)" refers to the situation where the ethanol-washed electrode was further immersed in a target molecule test solution with a concentration of 1 pg / mL for 10 minutes. The results of the atomic force microscopy analysis are shown in Figures 11 and 12. The results showed that the roughness before washing was 11.9 nm, which was less than the roughness after washing (17.1 nm). In the electrode for "re-adsorption", the target molecules were adsorbed on the imprinted surface, so the roughness value was between that before and after washing, at 12.8 nm. [Simplified Explanation of the Diagram]

[0042] [Figure 1] Figure 1a is a schematic diagram of the preparation process of the electrode coated with the peptide-imprinted conductive polymer of the present invention; Figure 1b is a schematic diagram of the structure of the extended gate field-effect transistor biosensor connected to the metal oxide semiconductor field-effect transistor (MOSFET). [Figure 2] ΔC current difference values ​​of each conductive monomer combination measured in Example 1 of the present invention. [Figure 3] Cyclic voltammetry diagrams of MIP and NIP of each conductive monomer combination measured in Example 2 of the present invention. [Figure 4] Detection curves of each conductive monomer combination for peptide H measured in Example 2 of the present invention. [Figure 5] Cyclic voltammetry diagrams of peptide D and lysozyme sensed with AN80%MSDS pHIPs in Example 4 of the present invention. [Figure 6] Detection curves of peptide H, uPA molecule, peptide D and lysozyme sensed with AN80%MSDS pHIPs in Example 4 of the present invention. [Figure 7] Sensing performance change graph measured in Example 6 of the present invention. [Figure 8] I-DV-D plot obtained by extended gate field-effect transistor measurement in Example 8 of the present invention. [Figure 9] Detection curves of peptide H sensed by AN80%MSDS pHIPs and NIPs in Example 8 of the present invention. [Figure 10] Detection curves of peptide H and intact uPA protein of real sample by AN80%MSDS pHIPs in Example 8 of the present invention. [Figure 11] Atomic force microscopy analysis results of the electrode surface of 1×1μm pHIP in Example 9 of the present invention. [Figure 12] Atomic force microscopy analysis results of the electrode surface of 1×1μm NIP in Example 9 of the present invention.

Claims

1. A uPA peptide-imprinted conductive polymer, comprising: a conductive polymer monomer and a template molecule for identifying uPA; the template molecule for identifying uPA is selected from a peptide fragment of the complete uPA protein peptide sequence that has the following conditions: (1) the peptide fragment has a length of 5 to 30 amino acids; (2) the ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids contained in the peptide fragment is 0 to 75%; and (3) the peptide fragment has 0 to 15 aromatic amino acids.

2. The uPA peptide-imprinted conductive polymer as described in claim 1, wherein, The conductive polymer monomer is selected from at least one of the following groups: m-aminobenzenesulfonic acid (MSAN), 2-aminobenzenesulfonic acid (MSDS), 4-aminobenzenesulfonic acid (SDS), aniline (AN), 3,4-ethylenedioxythiophene (EDOT), hydroxymethyl 3,4-ethylenedioxy-thiophene (EDOT-OH), acetylene, pyrrole, thiophene, phenylene vinylene, isothianaphthene, 3-alkyl thiophene, 2,5-dialkoxy paraphenylene vinylene, paraphenylene, and paraphenylene sulfide. sulphide and heptadiyne.

3. The uPA peptide-imprinted conductive polymer as described in claim 2, wherein, The concentration of the conductive polymer monomer is between 0.001 and 50% by weight; the conductive polymer monomer is aniline, and at least one selected from the group consisting of m-aminobenzenesulfonic acid, 2-aminobenzenesulfonic acid or 4-aminobenzenesulfonic acid; the mole ratio of the aniline is 80% of the total mole number of all conductive polymer monomers.

4. The uPA peptide-imprinted conductive polymer as described in claim 3, wherein, The conductive polymer monomers are aniline and 2-aminobenzenesulfonic acid.

5. The uPA peptide-imprinted conductive polymer as described in claim 1, wherein, The peptide sequence of the template molecule for identifying uPA is selected from SEQ ID NO: 1 or SEQ ID NO:

2.

6. The uPA peptide-imprinted conductive polymer as described in any one of claims 1 to 5, wherein, It further includes two-dimensional materials.

7. A biosensor comprising the uPA peptide-imprinted conductive polymer as described in any one of claims 1 to 6.

8. A biosensor as described in claim 6, wherein, The biosensor system further includes an electrochemical analyzer or an extended gate field-effect transistor.

9. A method for preparing a biosensor, comprising: step (1) preparing a monomer solution of a conductive polymer monomer with a concentration between 0.001 and 50% by weight, wherein, The conductive polymer monomer is selected from at least one of the following groups: m-aminobenzenesulfonic acid (MSAN), 2-aminobenzenesulfonic acid (MSDS), 4-aminobenzenesulfonic acid (SDS), aniline (AN), 3,4-ethylenedioxythiophene (EDOT), hydroxymethyl 3,4-ethylenedioxy-thiophene (EDOT-OH), acetylene, pyrrole, thiophene, phenylene vinylene, isothianaphthene, 3-alkyl thiophene, 2,5-dialkoxy paraphenylene vinylene, paraphenylene, and paraphenylene sulfide. sulphide and heptadiyne; Step (2) Add a template molecule for recognizing uPA to the monomer solution; Step (3) By electrochemical polymerization, the molecular template is imprinted on the conductive polymer monomer, and the imprinted conductive polymer is coated on an electrode substrate to obtain a biosensor; The template molecule for recognizing uPA is selected from peptide fragments in the peptide sequence of the complete uPA protein that have the following conditions: (1) The length of the peptide fragment is 5 to 30 amino acids; (2) The ratio of the number of hydrophobic amino acids in the peptide fragment to the total number of amino acids contained in the peptide fragment is 0 to 75%; and (3) The peptide fragment has 0 to 15 aromatic amino acids.

10. A method for fabricating a biosensor as described in claim 9, wherein, The peptide sequence of the template molecule for identifying uPA is selected from SEQ ID NO: 1 or SEQ ID NO: 2.