An electrochemiluminescence biosensor based on functionalized nuclear pore membrane and its application in dopamine detection

Abstract

The present invention discloses an electrochemiluminescent biosensor based on a functionalized nucleopore membrane. The electrochemiluminescent biosensor uses a nucleopore membrane as a substrate, and the nanopores of the nucleopore membrane are modified with terpyridine ruthenium functionalized silver nanoparticles as ECL luminescent bodies; the outer surface of the nucleopore membrane is loaded with a gold nanolayer by spin coating, and a tribasic body composed of a dopamine aptamer and an auxiliary chain containing a hydrophobic group is fixed. When dopamine binds to the aptamer main chain, the structure of the tribasic body unwinds to release the auxiliary chain, and the hydrophobic barrier is eliminated, causing the nanopore to switch from a "closed state" to an "open state". The material in the pore contacts the co-reactant tripropylamine (TPrA) in the electrolyte, generating a significantly enhanced ECL signal. The dopamine concentration in the range of 0.1nM~10μM shows a good linear relationship with the ECL intensity (R 2 =0.995). The sensor achieves highly selective detection of dopamine by enhancing interface stability through a gold nanolayer, improving recognition accuracy through an aptamer, and dynamically regulating pore permeability through hydrophobic groups.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical analysis, in particular to an electrochemiluminescence biosensor based on a functionalized nuclear pore membrane and its application in dopamine detection. Background Art

[0002] Dopamine, a crucial neurotransmitter in the central nervous system, participates in numerous physiological processes, including motor control and mood regulation. Abnormal dopamine concentrations are closely associated with a variety of neurological disorders, including Parkinson's disease and schizophrenia. Accurately detecting dopamine concentrations in biological samples is crucial for the diagnosis and treatment of disease. Currently, methods for detecting dopamine primarily include high-performance liquid chromatography, mass spectrometry, and electrochemical methods. However, these methods suffer from complex procedures, high testing costs, and the need for specialized operators, making them difficult to meet the demands of rapid on-site testing. Therefore, accurate detection of dopamine in biological fluids (such as cerebrospinal fluid, blood, and urine) is crucial for the early diagnosis, efficacy monitoring, and pathological mechanism research of neurological diseases.

[0003] Electrochemiluminescence (ECL) biosensors combine the advantages of electrochemical analysis and luminescence detection, offering high sensitivity, good selectivity, and ease of operation. They hold great promise for biomolecule detection. However, existing ECL biosensors for dopamine detection still suffer from high detection limits and poor selectivity, making them difficult to meet practical detection requirements. Therefore, the development of a highly sensitive and selective dopamine biosensor is of great research significance and application value. Summary of the Invention

[0004] The present invention aims to solve the problems of high detection limit, poor selectivity, and complex operation in existing dopamine detection methods, and provides an electrochemiluminescence biosensor based on nuclear pore membrane to achieve ultrasensitive detection of dopamine. An electrochemiluminescence biosensor based on a functionalized nucleopore membrane comprises: a nucleopore membrane substrate, an outer surface of the nucleopore membrane being spin-coated with a gold nanolayer, the gold nanolayer being connected to a tribasic body, the tribasic body consisting of a main chain and an auxiliary chain, the main chain being a dopamine aptamer sequence, the two ends of the dopamine aptamer each containing eight thymines, the auxiliary chain comprising 11 sequences complementary to the 3' end of the dopamine aptamer sequence, and the 5' end of the auxiliary chain being modified with a C18 hydrophobic group. When dopamine binds to the aptamer main chain, the tribasic body unwinds and releases the auxiliary chain with the hydrophobic group, thereby eliminating the hydrophobic barrier on the outer surface of the nucleopore membrane and causing the nucleopore membrane nanopore to open. This allows the functionalized silver nanoparticles in the nanopore to contact with a coreactant TPrA in an electrolyte solution, further amplifying the ECL signal of the system. As a result, the ECL signal of the electrochemiluminescence biosensor increases significantly, enabling the detection of dopamine. After adding other interferents, the sensor showed no obvious signal change, demonstrating its good specificity.

[0005] To achieve the above objectives, the present invention employs the following technical solution: Ruthenium terpyridine-functionalized silver nanoparticles are modified within the nanopores of a nuclear pore membrane. Since the etched nuclear pore membrane pores contain carboxyl groups, the pores are negatively charged, while the prepared Ruthenium terpyridine-functionalized silver nanoparticles are positively charged. The nanomaterial and the nuclear pore membrane nanopores are connected through electrostatic interactions. The nuclear pore membrane is then affixed to a working electrode, which is then mounted on a spin coater. Gold nanoparticles are spin-coated to form an Au nanolayer on the outer surface of the nuclear pore membrane nanopores. An aptamer and an auxiliary chain are then affixed to form a tribasic matrix. Nonspecific active sites are then blocked with 6-mercaptohexanol (MCH) to prevent nonspecific adsorption, resulting in an electrochemiluminescent biosensor based on the functionalized nuclear pore membrane.

[0006] The main chain is 5'-SH-C6-TTTTTTTTGTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCCTTTTTTTT-3'; and the auxiliary chain is 5'-C18-AAAAAAAAGGG-3'.

[0007] Furthermore, the nuclear pore membrane is a polyethylene terephthalate (PET) membrane, the pore size of the nanopores is 10-800 nm, and the distribution density of the nanopores in the nuclear pore membrane is 8×10 5 ~2×10 7 / cm 2 .

[0008] The present invention also provides a method for preparing the electrochemiluminescent biosensor based on the functionalized nuclear pore membrane, comprising the following steps: (1) ultrasonically cleaning the PET nuclear pore membrane with deionized water and ethanol in sequence to remove surface impurities, and drying the membrane for later use; (2) mixing a 0.1-1.5 mM terpyridine ruthenium solution with a 1-5 mM silver nitrate solution, adding a sodium citrate solution, placing the mixed solution in a magnetic stirrer for stirring, centrifuging the suspension at 2000-4000 rpm for 15-60 min, and rinsing the product with deionized water multiple times to obtain terpyridine ruthenium functionalized silver nanoparticles; (3) modifying the terpyridine ruthenium functionalized silver nanoparticles in the nanopores of the nuclear pore membrane. , fix the functionalized nuclear pore membrane on the working electrode; then fix the modified working electrode on the spin coater, and spin-coat the Au nanolayer on the outer surface of the nuclear pore membrane at a speed of 1000~10000rpm for 30~60s; (4) soak the functionalized working electrode in 20~100μM aptamer at 37~45℃ and incubate for 30~120min, then rinse it with deionized water several times to remove unbound aptamer, then soak it in the auxiliary chain and incubate it for 30~120min, dry it and clean it, then block it with 6-mercaptohexanol (MCH) to prevent nonspecific adsorption, then rinse it with ethanol and deionized water several times, dry it and set it aside.

[0009] Furthermore, the volume ratio of the terpyridine ruthenium solution to the silver nitrate solution in step (2) is 1:0.5 to 1:1.5; and the concentration ratio of the aptamer chain to the auxiliary chain in step (4) is 1:0.8 to 1:1.5. This concentration ratio facilitates the construction of an electrochemiluminescent biosensor with higher luminescence intensity.

[0010] The present invention also provides an application of the above-mentioned electrochemiluminescence biosensor based on the functionalized nuclear pore membrane in dopamine detection. The working electrode with the blocked active site is placed in a dopamine standard solution and incubated for 15 to 120 minutes, so that the tribasic body specifically captures dopamine, thereby releasing the auxiliary chain with the hydrophobic group, causing the nanopore to open. The working electrode incubated with the target object, an Ag / AgCl reference electrode, and a platinum wire counter electrode are placed together in an electrolytic cell to form a three-electrode system, and ECL detection is performed in a PBS electrolyte solution containing 50 to 100 mM tripropylamine (TPrA).

[0011] Furthermore, the electrolyte solution has a pH of 6.6-8.2.

[0012] Furthermore, an electrochemical method is used to excite the system to generate a luminescent signal.

[0013] Furthermore, the electrochemical method is cyclic voltammetry, which is performed in a potential range of -0.2~0.6V, with a potential increment of 10~50mV, a scan rate of 50~120mV / s, a photomultiplier tube voltage of 400~800V, and a luminescence intensity-time curve is recorded. A standard curve is drawn with dopamine concentration as the horizontal axis and the corresponding ECL signal as the vertical axis.

[0014] The present invention is based on an ECL signal amplification strategy based on the coordinated regulation of the tribasic matrix and the nanopore. Its core working mechanism is as follows: in the absence of dopamine, the tribasic matrix on the outer surface of the nuclear pore membrane maintains a stable conformation before the target enters the system, wherein the hydrophobic group forms a barrier outside the nanopore. At this time, the electrode surface is blocked by the hydrophobic group because the nanopore is blocked by the hydrophobic group, and is isolated from the co-reactant in the electrolyte solution due to steric hindrance, preventing the nanomaterial in the nanopore from contacting the co-reactant in the solution, and the ECL signal is weak; when the target molecule dopamine is present in the system, dopamine is specifically captured by the aptamer, the tribasic matrix structure dissociates and releases a single chain containing the hydrophobic group, causing the nanopore to switch from a "closed" state to an "open" state. After the barrier is removed, TPrA can freely pass through the pore to react with the nanomaterial, significantly enhancing the ECL signal and achieving highly sensitive detection of the target.

[0015] In addition, the technical principle of the present invention is that a dopamine aptamer containing two symmetrical sequences and an auxiliary chain containing a hydrophobic group with a partially complementary sequence constitute a tribasic body. First, the aptamer is fixed to the outer surface of the nuclear pore membrane modified electrode through an Au-S bond, and then the auxiliary chain is introduced and allowed to hybridize, forming a tribasic body outside the nanopore. At this time, the nanopore is in a "closed" state. When dopamine is introduced into the system, dopamine is specifically captured by the aptamer, the tribasic body structure unwinds, and the auxiliary chain containing the hydrophobic structure is displaced. At this time, the nanopore is in an "open" state and the ECL signal is enhanced. The sensor is regulated by the tribasic body, and by measuring the signal change before and after incubation with the target substance dopamine, highly sensitive and rapid detection of the target molecule is achieved.

[0016] Compared with existing technologies, the present invention offers at least the following advantages: 1. Ruthenium terpyridine-functionalized silver nanoparticles, as ECL active materials, combine the efficient luminescence properties of ruthenium terpyridine with the localized surface plasmon resonance effect of silver nanoparticles. This not only produces a stronger electrochemiluminescence (ECL) signal but also significantly improves the sensor's sensitivity to dopamine by increasing the active area of ​​the working electrode. 2. The ECL signal is regulated through a synergistic regulation strategy between the tribasic structure and the nanopore. In the absence of dopamine, the hydrophobic groups form a barrier outside the nanopore, isolating the nanomaterial from the co-reactant, resulting in a weak ECL signal. In the presence of dopamine, the tribasic structure unwinds, releasing the auxiliary chain, opening the nanopore and allowing the co-reactant to contact the nanomaterial. The ECL signal is significantly enhanced, greatly improving detection sensitivity and specificity. 3. The hydrophobic groups form a lipid barrier outside the pore, dynamically regulating the pore permeability. 4. The detection device has a simple structure, is easy to prepare and operate, and enables rapid, real-time detection of dopamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0018] Figure 1 This is a detection principle diagram of a preparation method of an electrochemiluminescence biosensor based on functionalized nuclear pore membrane.

[0019] Figure 2 Transmission electron microscopy image of ruthenium terpyridine functionalized silver nanoparticles.

[0020] Figure 3 Optimization of experimental conditions (A is optimization of electrolyte solution pH, B is optimization of dopamine incubation time).

[0021] Figure 4 The ECL curves of responses to different concentrations of dopamine.

[0022] Figure 5 The standard curve of ECL intensity corresponding to different concentrations of dopamine.

[0023] Figure 6 To evaluate the stability of electrochemical biosensors for dopamine detection.

[0024] Figure 7 Evaluation of the specificity of electrochemical biosensors for dopamine detection. DETAILED DESCRIPTION

[0025] The present invention is further described below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0026] Example 1

[0027] A method for preparing an electrochemiluminescent biosensor based on a functionalized nuclear pore membrane: (1) ultrasonically cleaning the nuclear pore membrane with deionized water and ethanol in sequence to remove surface impurities; drying and setting aside; (2) mixing a 0.8 mM tert-pyridine ruthenium solution with a 2 mM silver nitrate solution in a volume ratio of 1:0.5, adding a sodium citrate solution, placing the mixed solution in a magnetic stirrer for stirring, centrifuging the suspension at 2000 rpm for 15 minutes, and rinsing the product with deionized water several times to obtain tert-pyridine ruthenium functionalized silver nanoparticles; (3) modifying the tert-pyridine ruthenium functionalized silver nanoparticles in the nanopores of the nuclear pore membrane, and fixing the nuclear pore membrane on a working electrode; then fixing the prepared functionalized working electrode on a spin coater, and spinning the spin coater on the outside of the nuclear pore membrane at a speed of 2000 rpm. The surface was spin-coated with an Au nanolayer for 30 seconds; (4) the functionalized working electrode was immersed in 80 μM aptamer (5'-SH-C6-TTTTTTTTGTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCCTTTTTTTT-3') at 40 °C for 90 minutes, and then rinsed with deionized water several times to remove unbound aptamer, and then immersed in auxiliary chain (5'-C18-AAAAAAAAGGG-3') for 90 minutes, dried and cleaned, and then blocked with 6-mercaptohexanol (MCH) to prevent nonspecific adsorption, and then rinsed with ethanol and deionized water several times, dried and set aside.

[0028] Example 2

[0029] The application of an electrochemiluminescence biosensor based on a functionalized nuclear pore membrane in dopamine detection includes the following steps: placing a working electrode with blocked active sites in a dopamine standard solution for incubation for 30 minutes, allowing the tribasic body to specifically capture dopamine, thereby releasing auxiliary chains containing hydrophobic groups, causing the nanopores to open; placing the working electrode incubated with the target, an Ag / AgCl reference electrode, and a platinum wire counter electrode in an electrolytic cell to form a three-electrode system; and performing ECL detection in a PBS electrolyte solution (pH 7.4) containing 50mM tripropylamine. The optimization parameters include electrolyte solution pH optimization ( Figure 3 A) and dopamine incubation time optimization ( Figure 3 B). Cyclic voltammetry was used to excite the system to generate luminescence signals. The potential was scanned in the range of -0.2 to 0.6 V with a potential increment of 20 mV, a scan rate of 100 mV / s, and a photomultiplier tube voltage of 600 V. The luminescence intensity-time curve was recorded ( Figure 4), with dopamine concentration as the horizontal axis and the corresponding ECL signal as the vertical axis, a standard curve was drawn. As can be seen from the figure, as the dopamine concentration increases in the range of 0.1nM~10μM, the corresponding ECL intensity also increases, and the two have a good nonlinear relationship (R 2 =0.995). The results showed that after adding 1nM dopamine standard solution, the ECL intensity increased by 6.45 times compared with the blank sample, and increased by 31.55 times at 1μM. The linear correlation coefficient R²=0.995, and the detection limit (3σ) reached 0.217nM ( Figure 5 ).

[0030] To evaluate the stability of the sensor, the ECL sensor was scanned continuously for 9 cycles. The results showed that the ECL signal intensity did not change significantly between cycles, and the relative standard deviation (RSD) was only 1.03%, indicating that the sensor has good detection stability ( Figure 6 ).

[0031] Example 3

[0032] During the dopamine detection process, some common interfering substances may compete with the target substance for deposition sites during the test incubation phase, thereby affecting the accuracy of the test results. Therefore, 100-fold concentrations of interfering substances: epinephrine (EP), glucose (Glu), and uric acid (UA) were added to a 1μM dopamine standard solution to evaluate the sensor's anti-interference ability ( Figure 7 The results showed that the standard deviation of the luminescence intensity between a mixed solution containing the interfering substances EP, Glu, and UA and a standard dopamine solution was 1.04%. Because the aptamer is highly specific for dopamine, the sensor does not capture other interfering substances, demonstrating its excellent selectivity for dopamine.

[0033] The above description is only the best embodiment of the present invention and is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent replacements, improvements, etc. made should be included in the scope of protection of the present invention.

Claims

1. An electrochemiluminescence biosensor based on a functionalized nuclear pore membrane, characterized in that: The electrochemiluminescence biosensor comprises: a nucleopore membrane substrate, wherein the nanopores of the nucleopore membrane are modified with terpyridine ruthenium functionalized silver nanoparticles, and the nucleopore membrane is fixed to a working electrode; a gold nanolayer is spin-coated on the outer surface of the nucleopore membrane, and a tribasic body is connected to the gold nanolayer. The tribasic body is composed of a main chain and an auxiliary chain. The main chain is a dopamine aptamer sequence, and its two ends contain the same pyrimidine sequence. The auxiliary chain contains a sequence complementary to the 3' end of the dopamine aptamer sequence, and the 5' end of the auxiliary chain is modified with a hydrophobic group. The main chain is 5'-SH-C6-TTTTTTTTGTCTCTGTGTGCGCCAGAGAACACTGGGG CAGATATGGGCCAGCACAGAATGAGGCCCTTTTTTTT-3'; and the auxiliary chain is 5'-C18-AAAAAAAAGGG-3'.

2. The electrochemiluminescence biosensor based on a functionalized nucleopore membrane according to claim 1, characterized in that: The nuclear pore membrane is a polyethylene terephthalate (PET) membrane, the pore size of the nanopores is 10 to 800 nm, and the distribution density of the nanopores in the nuclear pore membrane is 8×10 5 ~2×10 7 / cm 2 .

3. The method for preparing an electrochemiluminescence biosensor based on a functionalized nucleopore membrane according to claim 1, wherein: The following steps are involved: 1) The PET nucleopore membrane was ultrasonically cleaned with deionized water and ethanol to remove surface impurities, and then dried for later use; 2) mixing a 0.1-1.5 mM terpyridine ruthenium solution with a 1-5 mM silver nitrate solution, adding a sodium citrate solution, placing the mixed solution in a magnetic stirrer for stirring, centrifuging the suspension at 2000-4000 rpm for 15-60 minutes, and rinsing the product with deionized water multiple times to obtain terpyridine ruthenium functionalized silver nanoparticles; 3) functionalizing silver nanoparticles within the nanopores of the nuclear pore membrane and fixing the functionalized nuclear pore membrane on a working electrode; then fixing the functionalized working electrode on a spin coater, and spin-coating an Au nanolayer on the outer surface of the nuclear pore membrane at a speed of 1000 to 10000 rpm for 30 to 60 seconds; 4) The functionalized working electrode is immersed in 20-100 μM aptamer at 37-45° C. and incubated for 30-120 min. Subsequently, it is rinsed with deionized water multiple times to remove unbound aptamer, and then immersed in auxiliary chain and incubated for 30-120 min. After drying and cleaning, it is blocked with 6-mercaptohexanol (MCH) to prevent nonspecific adsorption, and then rinsed with ethanol and deionized water multiple times, dried and set aside.

4. The method for preparing an electrochemiluminescence biosensor based on a functionalized nucleopore membrane according to claim 3, characterized in that: In step (2), the volume ratio of the terpyridine ruthenium solution to the silver nitrate solution is 1:0.5 to 1:1.5; and in step (4), the concentration ratio of the aptamer chain to the auxiliary chain is 1:0.8 to 1:1.

5.

5. The use of the electrochemiluminescence biosensor based on the functionalized nucleopore membrane in dopamine detection according to claim 1, characterized in that: The working electrode with the blocked active site was placed in a standard dopamine solution of different concentrations and incubated for 15 to 120 minutes, allowing the tribasic body to specifically capture dopamine, thereby releasing the auxiliary chain with a hydrophobic group, causing the nanopore to open. The working electrode incubated with the target object was placed together with an Ag / AgCl reference electrode and a platinum wire counter electrode in an electrolytic cell to form a three-electrode system. The system was excited to generate a luminescent signal using an electrochemical method, and electrochemiluminescence detection was performed in a PBS electrolyte solution containing 50 to 100 mM tripropylamine (TPrA).

6. Use of the electrochemiluminescence biosensor based on functionalized nuclear pore membrane in dopamine detection according to claim 5, characterized in that: The electrolyte solution has a pH of 6.6 to 8.

2.

7. Use of the electrochemiluminescence biosensor based on functionalized nuclear pore membrane in dopamine detection according to claim 5, characterized in that: The electrochemical method is cyclic voltammetry, which is performed in a potential range of -0.2 to 0.6 V, with a potential increment of 10 to 50 mV, a scan rate of 50 to 120 mV / s, a photomultiplier tube voltage of 400 to 800 V, and a luminescence intensity-time curve is recorded. A standard curve is drawn with dopamine concentration as the horizontal axis and the corresponding electrochemiluminescence signal as the vertical axis.

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