Integrated organic electrochemical transistor-based malaria-related biomarker detection chip as well as preparation method and application thereof

Abstract

The invention discloses a malaria-related biomarker detection chip based on an integrated organic electrochemical transistor as well as a preparation method and application of the malaria-related biomarker detection chip. According to the malaria-related biomarker detection chip based on the integrated organic electrochemical transistor, the integrated organic electrochemical transistor comprises an insulating substrate, a source electrode, a drain electrode and a gate electrode, the source electrode, the drain electrode and the gate electrode are arranged on the top of the insulating substrate, and an organic conductive channel is arranged between the source electrode and the drain electrode; the gate electrode and the organic conductive channel are located on the same plane, the gate electrode is a three-dimensional gold electrode obtained on the surface of a two-dimensional gold electrode by adopting an electrochemical deposition method, and the surface of the three-dimensional gold electrode is modified with a single-stranded DNA aptamer for identifying malaria-related biomarkers. The invention can realize high-sensitivity detection of malaria-related biomarkers, and has important significance for early diagnosis of diseases.

Description

Technical fields:

[0001] This invention relates to the field of biochemical sensor technology, specifically to a malaria-related biomarker detection chip based on an integrated organic electrochemical transistor, its preparation method, and its application. Background technology:

[0002] Early diagnosis of diseases is crucial for the prevention and treatment of many common and serious illnesses, such as cancer, diabetes, and Alzheimer's disease. With advancements in immunology, molecular biology, and genomics, researchers have discovered numerous biomarkers associated with early disease diagnosis. Currently, detecting the levels of these disease-related biomarkers has become an important method for early disease diagnosis.

[0003] Malaria is a deadly tropical disease with high morbidity and mortality rates in tropical countries, particularly in Africa and Southeast Asia. Among the antigens that trigger malaria, Plasmodium falciparum lactate dehydrogenase (PfLDH) plays a dominant role, accounting for 99.7% of all malaria cases. Therefore, highly sensitive detection of this disease biomarker is crucial. Currently, the gold standard for detecting this disease in most developing countries remains microscopic examination, which suffers from drawbacks such as being time-consuming, unreliable results at low hemoglobin concentrations, and requiring specialized operation and expert interpretation. These drawbacks, combined with the ambiguous symptoms of the infectious disease, lead to a large number of unidentified infections, especially in remote areas. Therefore, there is an urgent need to develop diagnostic tests with advantages such as high sensitivity and ease of processing.

[0004] Electrochemical sensors hold significant promise for the detection of disease-related biomarkers due to their low cost, simple structure, high sensitivity, and good stability. However, conventional electrochemical sensors typically exhibit low recognition element density and high background signal, limiting the detection of low-concentration targets. Organic electrochemical transistors (OECTs) possess excellent signal amplification capabilities, with their amplification capacity primarily dependent on the transconductance of the device. Therefore, enhancing recognition element density and improving transconductance are crucial for the detection of low-concentration targets. Summary of the Invention:

[0005] This invention solves the problems existing in the prior art. This invention provides a malaria-related biomarker detection chip based on integrated organic electrochemical transistors, its preparation method and application. The malaria-related biomarker detection chip proposed in this invention has high detection sensitivity for extremely low concentrations of the malaria-related biomarker PfLDH.

[0006] The first objective of this invention is to provide a malaria-related biomarker detection chip based on an integrated organic electrochemical transistor. The integrated organic electrochemical transistor includes an insulating substrate and a source electrode, a drain electrode, and a gate electrode disposed on top of the insulating substrate. An organic conductive channel is disposed between the source electrode and the drain electrode. The gate electrode and the organic conductive channel are on the same plane. The gate electrode is a three-dimensional gold electrode obtained on the surface of a two-dimensional gold electrode by an electrochemical deposition method. The surface of the three-dimensional gold electrode is modified with a single-stranded DNA aptamer for recognizing malaria-related biomarkers.

[0007] This invention prepares a three-dimensional gold electrode by electrochemical deposition, which improves the modification density of aptamer molecules on the electrode surface and the transconductance of the organic electrochemical transistor device. By using the organic electrochemical transistor device as a signal amplification element, it is possible to effectively detect extremely small gate electrode potential changes, thereby achieving high-sensitivity detection of analytes at extremely low concentrations.

[0008] Preferably, the electrochemical deposition method is a chronoamperometry method, the electrochemical deposition solution is a 1-100 mM aqueous solution of HAuCl4·3H2O, the deposition potential is 0-0.7 V, and the deposition time is 5-60 min. The diameter of the two-dimensional gold electrode is 20-1000 μm. More preferably, the electrochemical deposition solution is a 10 mM aqueous solution of HAuCl4·3H2O, the deposition potential is 0.55 V, and the deposition time is 30 min.

[0009] Preferably, the active material of the organic conductive channel, by mass fraction, comprises 89% poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS), 10% dimethyl sulfoxide, and 1% (3-glycidoxypropyl)trimethoxysilane (GOPS). The thickness of the organic conductive channel is 115-500 nm, the width of the organic conductive channel is 10-1000 μm, and the length is 10-100 μm.

[0010] Preferably, the sequence of the single-stranded DNA aptamer molecule is: 5-Thiol-(CH2)6-CTG GGC GGT AGAACCATAGTGACC CAG CCG TCTAC-3.

[0011] The second objective of this invention is to protect the method for preparing the malaria-related biomarker detection chip based on integrated organic electrochemical transistors, comprising the following steps:

[0012] (1) Fabrication of integrated organic electrochemical transistor: The source electrode, drain electrode and gate electrode are separately fabricated on the upper surface of an insulating substrate. The active material of the organic conductive channel is disposed between the source electrode and the drain electrode to form an organic conductive channel. The organic conductive channel and the gate electrode are on the same plane.

[0013] (2) Preparation of three-dimensional gold electrode structure: The organic conductive channel part of the above integrated organic electrochemical transistor is isolated by polydimethylsiloxane, and only the gate electrode is placed in the electrochemical deposition solution. The two-dimensional gold structure gate electrode is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode. Chemical deposition is performed on the surface of the two-dimensional gold electrode by chronoamperometry to obtain the three-dimensional gold electrode.

[0014] (3) Preparation of the amperometric aptamer sensor: The three-dimensional gold electrode was placed in a single-stranded DNA aptamer / PBS aqueous solution and activated for 1-24 hours. Then, 6-mercapto-1-hexanol alcohol solution was used as the blocking solution and activated for 0.5-1.5 hours to obtain the amperometric aptamer sensor based on the gate electrode.

[0015] (4) Preparation of malaria-related biomarker detection chip: Remove polydimethylsiloxane from the surface of the above-mentioned integrated organic electrochemical transistor, and cover the source electrode, drain electrode and gate electrode with PBS electrolyte to obtain the malaria-related biomarker detection chip.

[0016] This invention employs an organic electrochemical transistor with high transconductance as the sensor device. Its gate electrode is a three-dimensional gold electrode obtained through electrochemical deposition, the channel is an organic conductive polymer material, and the molecular recognition element is a single-stranded DNA aptamer molecule. During the fabrication of the current-voltage sensor, the three-dimensional gold electrode can significantly increase the modification density of the aptamer molecule and improve the transconductance of the coplanar organic electrochemical transistor, thereby obtaining a high-performance miniaturized transistor device and achieving the goal of detecting target molecules at extremely low concentrations.

[0017] The fabrication process of organic electrochemical transistors includes metallization to form source, drain, and gate electrodes, passivation to determine the size of source, drain, and gate electrodes, and structuring to confine organic conductive materials within organic conductive channels (channels).

[0018] Preferably, the electrochemical deposition solution in step (1) is an aqueous solution of HAuCl4·3H2O with a concentration of 1–100 mM, a deposition potential of 0–0.7 V, and a deposition time of 5–60 min. More preferably, the electrochemical deposition solution is an aqueous solution of HAuCl4·3H2O with a concentration of 10 mM, a deposition potential of 0.55 V, and a deposition time of 30 min.

[0019] Preferably, the concentration of single-stranded DNA aptamer in the single-stranded DNA aptamer / PBS aqueous solution in step (2) is 0.1-1.0 μM, and the concentration of 6-mercapto-1-hexanol is 0.9-1.1 μM. More preferably, the concentration of single-stranded DNA aptamer in the single-stranded DNA aptamer / PBS aqueous solution in step (2) is 0.5 μM, and the concentration of 6-mercapto-1-hexanol is 1 μM.

[0020] Preferably, the three-dimensional gold electrode is placed in a single-stranded DNA aptamer / PBS aqueous solution and activated for 16 hours, followed by activation for 1 hour using 6-mercapto-1-hexanol alcohol solution as a blocking solution to obtain the gate electrode.

[0021] A third objective of this invention is to protect the application of the malaria-related biomarker detection chip based on integrated organic electrochemical transistors in the detection of malaria-related biomarkers.

[0022] This invention also protects a highly sensitive detection method for malaria, comprising the following steps: adding different concentrations of malaria-related biomarker molecule Plasmodium falciparum lactate dehydrogenase solution to the malaria-related biomarker detection chip, and testing the change of the gate electrode potential with the concentration of the target substance.

[0023] Preferably, the concentration of the Plasmodium falciparum lactate dehydrogenase solution is 10. -13 At -1 μM, the binding time of Plasmodium falciparum lactate dehydrogenase to single-stranded DNA aptamer molecules is 5-60 min.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The preparation of the organic channel active layer of organic electrochemical transistors does not require complex processing technology and can be obtained directly through solution processing.

[0026] 2. Organic electrochemical transistors are based on organic channel volume capacitance modulation, and their transconductance is significantly higher than that of traditional field-effect transistor devices.

[0027] 3. By optimizing the organic channel size and gate electrode deposition process conditions, the voltammetric sensor based on organic electrochemical transistors exhibits high detection sensitivity for extremely low concentrations of the malaria-related biomarker PfLDH.

[0028] 4. The operation method of the volt-ampere sensor based on organic electrochemical transistor is simple and easy to implement, and does not require professional training or complex analysis.

[0029] 5. This invention enables highly sensitive detection of malaria-related biomarkers, which is of great significance for early disease diagnosis. This method is versatile and also applicable to the detection of biomarkers for other types of diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancer. Attached image description:

[0030] Figure 1 Optical micrograph of an organic electrochemical transistor with coplanar gate electrodes;

[0031] Figure 2 A schematic diagram of an organic electrochemical transistor with coplanar gate electrodes;

[0032] Figure 3 This is a schematic diagram illustrating the process of gold electrode deposition, molecular modification recognition, and target analyte detection.

[0033] Figure 4 Optical micrographs of two different gold electrodes are shown, including: (a) an optical micrograph of the two-dimensional gold electrode in Example 4; and (b) an optical micrograph of the three-dimensional gold electrode in Example 5.

[0034] Figure 5 The image shows the cyclic voltammetry curves of the two-dimensional gold electrode (dashed line) and the three-dimensional gold electrode (solid line) formed by gold deposition (chronoamperometry, potential 0.5V, 30min) in Example 5. The solution is 50mM sulfuric acid solution, and the scan rate is 0.1V / s.

[0035] Figure 6 This example illustrates the signal changes of an electrochemical sensor based on a two-dimensional gold electrode and a three-dimensional gold electrode obtained through electrochemical deposition, as described in Example 5, under different concentrations of the analyte (Plasmodium falciparum lactate dehydrogenase, a malaria-related biomarker). The two-dimensional gold electrode has a size of 200 μm, and the three-dimensional gold electrode was obtained using a chronoamperometry method with a deposition potential of 0.55 V and a deposition time of 30 min.

[0036] Figure 7 The following are the conduction characteristic curves of organic electrochemical transistors with coplanar gate electrodes, wherein: (a) the output curve of the organic electrochemical transistor in Example 4; (b) the conduction curve of the organic electrochemical transistor in Example 4; (c) the output curve of the organic electrochemical transistor in Example 5; and (d) the conduction curve of the organic electrochemical transistor in Example 5.

[0037] Figure 8 The output and conduction curves of the organic electrochemical transistor sensor with coplanar gate electrodes in Example 5 are shown as changes in the concentration of the analyte; the gate electrode size is 200 μm, the deposition potential is 0.55 V, and the deposition time is 30 min.

[0038] Figure labeling: 1. Source electrode; 2. Drain electrode; 3. Gate electrode; 4. Organic conductive channel; 5. Single-stranded DNA aptamer molecule; 6. PBS electrolyte. Detailed implementation method:

[0039] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0040] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental materials and reagents used herein are commercially available products commonly used in this field. In the examples below, PBS solution or PBS electrolyte refers to a 0.1M phosphate buffer solution. Single-stranded DNA aptamers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] A malaria-related biomarker detection chip based on an integrated organic electrochemical transistor includes an integrated organic electrochemical transistor and a single-stranded DNA aptamer for identification. The integrated organic electrochemical transistor includes an insulating substrate and a source electrode 1, a drain electrode 2, and a gate electrode 3 disposed on top of the insulating substrate. An organic conductive channel 4 is disposed between the source electrode 1 and the drain electrode 2. The gate electrode 3 and the organic conductive channel 4 are on the same plane. The gate electrode is a three-dimensional gold electrode obtained by electrochemical deposition on the surface of a two-dimensional gold electrode. The surface of the three-dimensional gold electrode is modified with a single-stranded DNA aptamer molecule 5.

[0042] In the following preferred embodiments, the electrochemical deposition method is chronoamperometry, the electrochemical deposition solution is a 1-100 mM aqueous solution of HAuCl4·3H2O, the deposition potential is 0-0.7 V, and the deposition time is 5-60 min. The diameter of the two-dimensional gold electrode is 20-1000 μm. More preferably, the electrochemical deposition solution is a 10 mM aqueous solution of HAuCl4·3H2O, the deposition potential is 0.55 V, and the deposition time is 30 min.

[0043] In the preferred embodiments described below, the active material of the organic conductive channel, by mass fraction, comprises 89% poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS), 10% dimethyl sulfoxide, and 1% (3-glycidoxypropyl)trimethoxysilane (GOPS). The thickness of the organic conductive channel is 115-500 nm, the width of the organic conductive channel is 10-1000 μm, and the length is 10-100 μm.

[0044] In the following preferred embodiments, the sequence of the single-stranded DNA aptamer molecule is: 5-Thiol-(CH2)6-CTG GGCGGTAGA ACCATAGTGACC CAG CCG TCTAC-3.

[0045] The preparation method of the above-mentioned malaria-related biomarker detection chip includes the following steps:

[0046] (1) Fabrication of integrated organic electrochemical transistor: Source electrode 1, drain electrode 2 and gate electrode 3 are separately fabricated on the upper surface of an insulating substrate. The active material of the organic conductive channel is disposed between the source electrode 1 and the drain electrode 2 to form an organic conductive channel 4. The organic conductive channel 4 and the gate electrode 3 are on the same plane.

[0047] (2) Preparation of three-dimensional gold electrode structure: The organic conductive channel part of the above integrated organic electrochemical transistor is isolated by polydimethylsiloxane, and only the gate electrode is placed in the electrochemical deposition solution. The two-dimensional gold structure gate electrode is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode. Chemical deposition is performed on the surface of the two-dimensional gold electrode by chronoamperometry to obtain the three-dimensional gold electrode.

[0048] (3) Preparation of amperometric aptamer sensor: The gate electrode (three-dimensional gold electrode) of the integrated organic electrochemical transistor is placed in single-stranded DNA aptamer / PBS aqueous solution and activated for 1-24 hours. Then, 6-mercapto-1-hexanol alcohol solution is used as blocking solution and activated for 0.5-1.5 hours to obtain amperometric aptamer sensor based on gate electrode.

[0049] (4) Preparation of malaria-related biomarker detection chip: Remove polydimethylsiloxane from the surface of the above integrated organic electrochemical transistor, and cover the source electrode 1, drain electrode 2 and gate electrode 3 with PBS electrolyte 6 to obtain malaria-related biomarker detection chip.

[0050] In the following preferred embodiment, the electrochemical deposition solution in step (2) is an aqueous solution of HAuCl4·3H2O with a concentration of 1-100mM, the deposition potential is 0-0.7V, and the deposition time is 5-60min.

[0051] In the following preferred embodiments, the concentration of single-stranded DNA aptamer in the single-stranded DNA aptamer / PBS aqueous solution in step (3) is 0.1-1.0 μM, and the concentration of 6-mercapto-1-hexanol is 0.9-1.1 μM. More preferably, the concentration of single-stranded DNA aptamer in the single-stranded DNA aptamer / PBS aqueous solution in step (3) is 0.5 μM, and the concentration of 6-mercapto-1-hexanol is 1 μM.

[0052] In the preferred embodiment described below, a three-dimensional gold electrode is placed in a single-stranded DNA aptamer / PBS aqueous solution and activated for 16 hours, followed by activation for 1 hour using a 6-mercapto-1-hexanol alcohol solution as a blocking solution, to obtain an amperometric aptamer sensor based on a gate electrode.

[0053] Example 1:

[0054] A highly sensitive detection method for malaria includes the following steps:

[0055] S1. A two-dimensional gold electrode with a diameter of 24 μm is subjected to plasma treatment to remove surface contaminants;

[0056] S2. A 0.5 μM recognition molecular element (single-stranded DNA aptamer) was modified onto the surface of a two-dimensional gold electrode, reacted for 16 h, and then washed with PBS solution. Subsequently, the electrode was placed in 1 M 6-MCH ethanol solution, reacted for 1 h, washed with PBS solution, and dried with nitrogen to obtain an amperometric sensor.

[0057] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 100μm×10μm and a channel thickness of 115nm as the signal amplification element, a current-voltage sensor is obtained.

[0058] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0059] Example 2

[0060] A highly sensitive detection method for malaria includes the following steps:

[0061] S1. A two-dimensional gold electrode with a diameter of 24 μm was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 30 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0062] S2. The gold electrode was immersed in deionized water for 10 min and dried with nitrogen, followed by plasma treatment to remove surface contaminants; a recognition molecular element (single-stranded DNA aptamer) at a concentration of 0.5 μM was modified on the electrode surface, reacted for 16 h, and then washed with PBS solution; the electrode was then placed in 1 M 6-MCH ethanol solution, reacted for 1 h, washed with PBS solution, and dried with nitrogen to obtain the amperometric aptamer sensor.

[0063] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 100μm×10μm and a channel thickness of 115nm as the signal amplification element, a voltammetric sensor (malaria-related biomarker detection chip) is obtained.

[0064] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0065] Example 3

[0066] A highly sensitive detection method for malaria includes the following steps:

[0067] S1. A gold electrode with a diameter of 24 μm was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 30 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0068] S2. The gold electrode was immersed in deionized water for 10 min and dried with nitrogen, followed by plasma treatment to remove surface contaminants; a recognition molecular element (single-stranded DNA aptamer) at a concentration of 0.5 μM was modified on the electrode surface, reacted for 16 h, and then washed with PBS solution; the electrode was then placed in 1 M 6-MCH ethanol solution, reacted for 1 h, washed with PBS solution, and dried with nitrogen to obtain the amperometric aptamer sensor.

[0069] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 1000μm×100μm and a channel thickness of 115nm as the signal amplification element, a voltammetric sensor (malaria-related biomarker detection chip) is obtained.

[0070] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0071] Example 4

[0072] A highly sensitive detection method for malaria includes the following steps:

[0073] S1. A two-dimensional gold electrode with a diameter of 200 μm is subjected to plasma treatment to remove surface contaminants;

[0074] S2. The electrode surface was modified with a recognition molecular element (single-stranded DNA aptamer) at a concentration of 0.5 μM, reacted for 16 h, and then washed with PBS solution; the electrode was then placed in 1 M 6-MCH ethanol solution, reacted for 1 h, washed with PBS solution, and dried with nitrogen to obtain the amperometric aptamer sensor.

[0075] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 100μm×10μm and a channel thickness of 115nm as the signal amplification element, a voltammetric sensor (malaria-related biomarker detection chip) is obtained.

[0076] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10-2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0077] Example 5

[0078] A highly sensitive detection method for malaria includes the following steps:

[0079] S1. A gold electrode with a diameter of 200 μm was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 30 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0080] S2. Immerse the gold electrode in deionized water for 10 min to remove surface contaminants and dry it with nitrogen; modify the electrode surface with recognition molecular elements (single-stranded DNA aptamers) to obtain an amperometric aptamer sensor.

[0081] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 100μm×10μm and a channel thickness of 115nm as the signal amplification element, a voltammetric sensor (malaria-related biomarker detection chip) is obtained.

[0082] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0083] Example 6

[0084] A highly sensitive detection method for malaria includes the following steps:

[0085] S1. A gold electrode with a diameter of 200 μm was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 30 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0086] S2. Immerse the gold electrode in deionized water for 10 min to remove surface contaminants and dry it with nitrogen; modify the electrode surface with recognition molecular elements (single-stranded DNA aptamers) to obtain an amperometric aptamer sensor.

[0087] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 1000μm×100μm and a channel thickness of 115nm as the signal amplification element, a voltammetric sensor (malaria-related biomarker detection chip) is obtained.

[0088] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0089] Example 7

[0090] A highly sensitive detection method for malaria includes the following steps:

[0091] S1. A gold electrode with a diameter of 200 μm was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 30 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0092] S2. Immerse the gold electrode in deionized water for 10 min to remove surface contaminants and dry it with nitrogen; modify the electrode surface with recognition molecular elements (single-stranded DNA aptamers) to obtain an amperometric aptamer sensor.

[0093] S3. Using the ampere sensor as the gate electrode, and an organic electrochemical transistor with a channel size of 1000μm×100μm and a channel thickness of 500nm as the signal amplification element, a voltammetric sensor (malaria-related biomarker detection chip) is obtained.

[0094] S4. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -410 -3 10 -2 The conduction curve of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0095] Comparative Example 1

[0096] A highly sensitive detection method for malaria includes the following steps:

[0097] S1. A two-dimensional gold electrode with a diameter of 24 μm is subjected to plasma treatment to remove surface contaminants;

[0098] S2. The electrode surface was modified with a recognition molecular element (single-stranded DNA aptamer) at a concentration of 0.5 μM, reacted for 16 h, and then washed with PBS solution; the electrode was then placed in 1 M 6-MCH ethanol solution, reacted for 1 h, washed with PBS solution, and dried with nitrogen to obtain the amperometric sensor.

[0099] S3. Characterization at different concentrations (10) was performed using AC voltammetry with an electrochemical workstation. -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 Current-voltage curves of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0100] Comparative Example 2

[0101] A highly sensitive detection method for malaria includes the following steps:

[0102] S1. A 24 μm gold electrode was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 5 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0103] S2. The gold electrode was immersed in deionized water for 10 min to remove surface contaminants and dried with nitrogen. The electrode surface was modified with a recognition molecular element (single-stranded DNA aptamer) at a concentration of 0.5 μM and reacted for 16 h. The electrode was then washed with PBS solution. Subsequently, the electrode was placed in 1 M 6-MCH ethanol solution and reacted for 1 h. The electrode was then washed with PBS solution and dried with nitrogen to obtain the amperometric sensor.

[0104] S3. Characterization at different concentrations (10) was performed using AC voltammetry with an electrochemical workstation. -8 10 -7 10 -6 10 -5 10 -410 -3 10 -2 Current-voltage curves of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0105] Comparative Example 3

[0106] A highly sensitive detection method for malaria includes the following steps:

[0107] S1. A 24 μm gold electrode was placed in a tetrachloroauric acid solution (concentration of 10 mM) and deposited for 30 min at a bias voltage of 0.55 V using a chronoamperometry method to obtain a three-dimensional gold electrode.

[0108] S2. The gold electrode was immersed in deionized water for 10 min to remove surface contaminants and dried with nitrogen. The electrode surface was modified with a recognition molecular element (single-stranded DNA aptamer) at a concentration of 0.5 μM and reacted for 16 h. The electrode was then washed with PBS solution. Subsequently, the electrode was placed in 1 M 6-MCH ethanol solution and reacted for 1 h. The electrode was then washed with PBS solution and dried with nitrogen to obtain the amperometric sensor.

[0109] S3. Characterization at different concentrations (10) was performed using AC voltammetry with an electrochemical workstation. -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 Current-voltage curves of the device under the action of the target substance (malaria-associated biomarker Plasmodium falciparum lactate dehydrogenase) at a concentration of μM.

[0110] Comparative Example 4 (using graphene transistors)

[0111] A highly sensitive detection method for malaria includes the following steps:

[0112] S1. A reduced graphene oxide transistor was prepared by chemical vapor deposition. The transistor channel size was 1000μm×10μm, and the gate electrode of the transistor was an externally connected large-size Ag / AgCl electrode. The graphene sensor was used as a signal amplification element.

[0113] S2. DNA aptamer molecular chains (2008s) with pyrene functional groups at the ends were modified onto the graphene surface, reacted for 16 h, and washed with PBS solution to remove excess aptamer molecules, thus obtaining a current-voltage sensor based on graphene transistors.

[0114] S3. The Keithley 4200 semiconductor analyzer was used to characterize the results at different concentrations (10). -11 10-10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The conduction curve of the device under the action of a μM target material.

[0115] Comparative Example 5 Colorimetric Method

[0116] A highly sensitive detection method for malaria includes the following steps:

[0117] S1. Single-stranded DNA aptamer molecules (2008s) with the same sequence as in the example were modified on streptavidin-coated 96-well culture plates for 2 hours. The plates were then washed with PBS solution to remove unbound DNA molecular segments.

[0118] S2. Different concentrations of PfLDH / PBS solution were added to the aptamer-modified culture plates, reacted for 1 h, and then washed with PBS solution. Subsequently, L-lactic acid / nitrotetrazole chloride blue solution was added, and the reaction was carried out for 45 min.

[0119] S3. Recordings at different concentrations (10) were performed using a UV-Vis spectrometer. -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 The absorption intensity of the sample at 570 nm under the action of the target substance (μM).

[0120] The sensitivity of a device depends on its lowest detection limit (LOD). LOD = ks / m, where k is a constant of random error, usually taken as k = 3, s is the standard deviation, and m is the slope of the linear detection region.

[0121] The sensor devices of the embodiments and comparative examples were tested to obtain their lowest detection limits. The test results are shown in Table 1 below:

[0122] Table 1. Sensitivity results of the device for the malaria-related biomarker PfLDH (limit of detection)

[0123]

[0124] As shown in Table 1, Examples 1-7 utilize organic electrochemical transistors as current-voltage sensors (malaria-related biomarker detection chips). By amplifying the target analyte signal, only adjustments to the surface area of ​​the gate electrode and the volume parameters of the organic conductive channel are needed to achieve high-sensitivity detection of the analyte. Compared to the amperometric sensors used in Comparative Examples 1-3, the current-voltage sensors based on organic electrochemical transistors can detect lower concentrations of the target analyte, with the detection limit decreasing from 1.52 pM to 0.00002 pM, a reduction of nearly six orders of magnitude. Compared to the graphene sensor used in Comparative Example 4, both have the same detection mechanism, but the organic electrochemical transistor has higher transconductance. Therefore, the current-voltage sensors based on organic electrochemical transistors have higher detection sensitivity, with the detection limit reduced by a factor of three. Compared to the colorimetric sensor in Comparative Example 5, the electrochemical sensor is more advantageous in detecting low-concentration target analytes.

[0125] Figure 1-2 This is a schematic diagram of an organic electrochemical transistor with coplanar gate electrodes. Figure 3 This diagram illustrates the process of gold electrode deposition, molecular modification recognition, and target analyte detection. Figure 4 Optical micrographs of two gold electrodes with different structures. Figure 5 The cyclic voltammetry curves are shown for a two-dimensional gold electrode (dashed line) and a three-dimensional gold electrode (solid line) formed by gold deposition (chronoamperometry, potential 0.5V, 30min). Figure 6 This study examines the signal changes of an electrochemical sensor based on a two-dimensional gold electrode and a three-dimensional gold electrode obtained through electrochemical deposition under different concentrations of a detection substance (Plasmodium falciparum lactate dehydrogenase, a malaria-related biomarker). Figure 7 The graphs show the conduction characteristics of an organic electrochemical transistor with coplanar gate electrodes. Figure 8 The output and conduction curves of an organic electrochemical transistor sensor with coplanar gate electrodes vary with the concentration of the detectable. Figure 1-6 It can be seen that electrochemical deposition reduced the surface area of ​​the gold electrode from 0.031 mm². 2 Increased to 3.37mm 2 The increase is approximately 100 times. From Figure 7 It can be seen that the amperometric sensor based on three-dimensional gold electrodes exhibits higher detection sensitivity compared to the amperometric sensor based on two-dimensional gold electrodes, with the detection limit decreasing from 1.52 pM in the two-dimensional device to 0.17 pM, a reduction of approximately one order of magnitude. Figure 8 It can be seen that using organic electrochemical transistors as signal amplification elements can significantly amplify the detection signal of the ampere sensor, reducing the sensor's detection limit from 0.17 pM to 0.00003 pM, a reduction of nearly four orders of magnitude.

[0126] This invention enables highly sensitive detection of malaria-related biomarkers, which is of great significance for early disease diagnosis. This method is also versatile and applicable to the detection of biomarkers for other types of diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancer.

[0127] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A malaria-related biomarker detection chip based on an integrated organic electrochemical transistor, characterized in that: The integrated organic electrochemical transistor includes an insulating substrate and a source electrode, a drain electrode and a gate electrode arranged on the top of the insulating substrate. An organic conductive channel is arranged between the source electrode and the drain electrode. The gate electrode and the organic conductive channel are in the same plane. The gate electrode is a three-dimensional gold electrode obtained by an electrochemical deposition method on the surface of a two-dimensional gold electrode. The surface of the three-dimensional gold electrode is modified with a single-stranded DNA aptamer for identifying malaria-related biomarkers.

2. The malaria-related biomarker detection chip based on integrated organic electrochemical transistor according to claim 1, characterized in that: The electrochemical deposition method is a chronoamperometry method, the electrochemical deposition solution is a HAuCl4·3H2O aqueous solution with a concentration of 1-100mM, the deposition potential is 0-0.7V, and the deposition time is 5-60min.

3. The malaria-related biomarker detection chip according to claim 1 or 2, characterized in that: The active material of the organic conductive channel includes, by mass fraction, 89% of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), 10% of dimethyl sulfoxide and 1% of (3-glycidyloxypropyl)trimethoxysilane. The thickness of the organic conductive channel is 115-500nm, the width of the organic conductive channel is 10-1000μm, and the length is 10-100μm.

4. The malaria-related biomarker detection chip according to claim 1 or 2, characterized in that: The sequence of the single-stranded DNA aptamer molecule is: 5-Thiol-(CH2)6-CTG GGC GGTAGAACCATAGTGACC CAG CCG TCTAC-3.

5. The method for preparing the malaria-related biomarker detection chip according to claim 1, characterized in that: The method comprises the following steps: (1) preparing an integrated organic electrochemical transistor: separately preparing a source electrode, a drain electrode and a gate electrode on the upper surface of an insulating substrate, wherein an active material of an organic conductive channel is arranged between the source electrode and the drain electrode to form an organic conductive channel, wherein the organic conductive channel and the gate electrode are in the same plane; (2) Preparation of a three-dimensional gold electrode structure: The organic conductive channel portion of the above-mentioned integrated organic electrochemical transistor is isolated by polydimethylsiloxane, and only the gate electrode is placed in an electrochemical deposition solution. The gate electrode of the two-dimensional gold structure is used as a working electrode, the Ag / AgCl electrode is used as a reference electrode, and the platinum wire electrode is used as a counter electrode. Chemical deposition is performed on the surface of the two-dimensional gold electrode by chronoamperometry to obtain a three-dimensional gold electrode; (3) Preparation of amperometric aptamer sensor: placing the three-dimensional gold electrode in a single-stranded DNA aptamer / PBS aqueous solution and activating it for 1-24 hours, and then using 6-mercapto-1-hexanol alcohol solution as a blocking solution and activating it for 0.5-1.5 hours to obtain an amperometric aptamer sensor based on a gate electrode; (4) Preparation of malaria-related biomarker detection chip: The polydimethylsiloxane on the surface of the above-mentioned integrated organic electrochemical transistor is removed, and PBS electrolyte is covered on the surface of the source electrode, the drain electrode, and the gate electrode to obtain the malaria-related biomarker detection chip.

6. The preparation method according to claim 5, characterized in that: The electrochemical deposition solution in step (1) is a HAuCl4·3H2O aqueous solution with a concentration of 1 to 100 mM, a deposition potential of 0 to 0.7 V, and a deposition time of 5 to 60 min.

7. The preparation method according to claim 5, characterized in that: The concentration of the single-stranded DNA aptamer in the single-stranded DNA aptamer / PBS aqueous solution in step (2) is 0.1-1.0 μM, and the concentration of 6-mercapto-1-hexanol is 0.9-1.1 μM.

8. Use of the malaria-related biomarker detection chip according to claim 1 in detecting malaria-related biomarkers.

9. A highly sensitive method for detecting malaria, characterized in that: The method comprises the following steps: adding different concentrations of malaria-related biomarker molecule Plasmodium falciparum lactate dehydrogenase solution to the malaria-related biomarker detection chip according to claim 1, and testing the change of gate electrode potential with the concentration of the target substance.

10. The detection method according to claim 9, characterized in that: The concentration of the Plasmodium falciparum lactate dehydrogenase solution is 10 -13 -1μM, the binding time of Plasmodium falciparum lactate dehydrogenase and single-stranded DNA aptamer molecules is 5-60min.

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