An electrochemical method for prostate specific antigen detection

Abstract

An electrochemical method for prostate specific antigen detection, comprising the following steps: S1: preparation of a molecular imprinting sensing electrode; S2: incubating the imprinting sensing electrode with PSA of a given concentration for 30 minutes, and washing the electrode clean after the PSA is captured by the imprinting sensing electrode; S3: taking the imprinting sensing electrode as a working electrode, a platinum wire as an auxiliary electrode, and a silver / silver chloride electrode as a reference electrode, and placing the three electrodes in a 5 mM [Fe(CN)6] (1:1) solution containing 0.1 M KCl for electrochemical impedance (EIS) measurement. 3− / 4− The imprinting sensing electrode is first incubated in a PSA solution of a given concentration for 30 min in step S2, and signal amplification detection is performed after the PSA is captured by the imprinting sensing electrode, and the imprinting sensing electrode is washed clean. The present application does not require expensive instruments.

Description

Technical Field

[0001] This invention relates to antigen detection, and more particularly to an electrochemical method for detecting prostate-specific antigen (PSA), belonging to the field of chemical technology. Background Technology

[0002] Prostate-specific antigen (PSA) is a biomarker used to diagnose prostate diseases. Electrochemical sensors, as point-of-care detection devices, can convert biorecognition events into electrochemical signals, showing great potential for sensitive detection of biomarkers. However, traditional electrochemical sensors often require antibodies or nucleic acid aptamers as recognition elements. Molecularly imprinted polymeric membranes (MIPs), as a class of artificially synthesized receptors, hold promise as alternatives to biological receptors. Compared to nucleic acids and antibodies, MIPs offer advantages such as long-term durability, low cost, and good robustness in the detection of low molecular weight targets. However, currently reported MIPs have many limitations in the recognition and detection of biomacromolecules such as proteins due to their large size, complex and variable structure, and poor environmental stability. For example, proteins undergo irreversible conformational changes during polymerization, the large size of MIPs makes it difficult to remove template proteins from the imprint cavity, and the rebinding ability of native proteins to the imprint cavity is weak. Epitope groups on the surface of biomacromolecules help proteins fixate on the surface of a substrate with specific molecular functionalization, thereby hindering the conformational changes of proteins during the imprinting process to some extent. Summary of the Invention

[0003] The purpose of this invention is to provide an electrochemical method for detecting prostate-specific antigen.

[0004] To achieve the objective of this invention, the following technical solution is adopted: an electrochemical method for detecting prostate-specific antigen, comprising the following steps: S1: Preparation of molecularly imprinted sensing electrode: The gold electrode was incubated for 12 hours with 50 μL of 2-morpholinoethanesulfonic acid buffer solution (50 mM, pH = 6.5) containing 0.1 mM mercaptotriacetic acid (NTA) and 1 mM tris(2-carboxyethyl)phosphine. Then the electrode was incubated with 10 μM Ni 2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complex; after rinsing the modified electrode with water, the electrode was incubated with 10 μg / mL of His6-tagged recombinant PSA protein for 30 minutes. The His6-tagged recombinant PSA protein, abbreviated as rPSA, forms a ternary NTA-Ni complex on the electrode surface. 2+ -rPSA complex, and then electropolymerize hyoscyamine on the electrode surface by electrodeposition, in NTA-Ni 2+A polyhyoscyamine-imprinted film is formed around the -rPSA complex. The specific polymerization steps and conditions are as follows: NTA-Ni 2+ The rPSA complex-modified electrode was subjected to multi-step amperometric scanning for 15–20 cycles in a 0.1 M NaCl solution containing 0.5 mM hyoscyamine, with applied potentials of 0 V for 5 s and 0.7 V for 1 s. The electrode was then rinsed multiple times with 20% ethanol and subsequently cleaned with 0.1 M HCl under ultrasonication to remove the template proteins rPSA and Ni. 2+ Thus, molecularly imprinted sensing electrodes were obtained; S2: Incubate the imprinted sensing electrode with a given concentration of PSA for 30 minutes. After the PSA is captured by the imprinted sensing electrode, rinse the electrode clean. S3: Using an imprinted sensing electrode as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode, the three electrodes are placed in a 5 mM solution containing 0.1 M KCl. 3− / 4− Electrochemical impedance spectroscopy (EIS) was performed in a 1:1 solution.

[0005] Further, in step S2, the imprinted sensing electrode is first incubated in a PSA solution of a given concentration for 30 min. After the PSA is captured by the imprinted sensing electrode, the signal is amplified and detected. Specifically, the imprinted sensing electrode that has captured PSA is incubated in 10 μL of 0.1 mg / mL ConA solution for 10 min, and then 10 μL of 0.5 mg / mL Avidin is added to the solution for further incubation for 30 min. The imprinted sensing electrode is then rinsed clean.

[0006] Furthermore, in step S2, the electrode is thoroughly rinsed with 10 mM phosphate buffer.

[0007] Further, the imprinted sensing electrode was rinsed clean with ultrapure water.

[0008] The electrochemical method provided by this invention does not require expensive equipment and offers significant advantages over traditional imprinted polymer membranes: it avoids the use of organic solvents for polymerization, provides mild template removal conditions, and exhibits excellent mass transfer performance of PSA both inside and outside the imprint cavity. Furthermore, ConA can simultaneously serve as a recognition element for PSA and a crosslinking agent for Avidin, triggering in-situ self-assembly of ConA / Avidin on the electrode surface to achieve signal amplification and detection. This simple self-assembly signal amplification strategy improves sensitivity by approximately 1000 times, providing crucial support for the preparation of imprinted materials and the development of protein detection platforms. Attached Figure Description

[0009] Figure 1 This is a schematic diagram illustrating the detection principle of this method.

[0010] Figure 2 The EIS curves of different concentrations of PSA were directly measured by the molecularly imprinted sensing electrode (without self-assembly signal amplification). The PSA concentrations were 0, 0.1, 1, 3.3, 6.7, and 10 ng / mL, respectively.

[0011] Figure 3 It is the impedance value R et Linear curves of PSA concentrations (without self-assembly signal amplification) are shown for PSA concentrations of 0.1, 1, 3.3, 6.7, and 10 ng / mL, respectively.

[0012] Figure 4 The EIS curves are of the molecularly imprinted sensing electrode (curve 1) after incubation in PSA (curve 2), PSA + ConA (curve 3), PSA + ConA + Avidin (curve 4), and PSA + ConA / Avidin (curve 5) solutions, respectively. The concentration of PSA was 3.3 ng / mL.

[0013] Figure 5 The image shows EIS curves of PSA at different concentrations determined by self-assembly signal amplification. The PSA concentrations were 0.1, 0.7, 3.3, 6.7, 10, 20, 50, and 100 pg / mL, respectively.

[0014] Figure 6 It is R et The graph shows the relationship between PSA concentration and PSA concentration (after amplification of the self-assembled signal). The inset is a linear curve (PSA concentrations are 0.1, 0.7, 3.3, 6.7, and 10 pg / mL, respectively).

[0015] Figure 7 This is a selective comparison chart of PSA detection using the self-assembly signal amplification method. Columns 1-7 represent, in order: blank control, CEA, AFP, CA125, 10% fetal bovine serum, and CEA + AFP + CA125 + 10% fetal bovine serum + PSA. The concentration of PSA is 10 pg / mL, and the concentration of other proteins is 1 ng / mL. Detailed Implementation

[0016] To more fully explain the implementation of the present invention, implementation examples are provided. These implementation examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[0017] Commercially available reagent kits contain PSA, a recombinant protein with a tag consisting of six histidine residues (His6). The multiple imidazole groups in this tag facilitate coordination between the protein and certain metal complexes. Based on the structural characteristics and physicochemical properties of commercially available PSA, this invention utilizes commercially available recombinant PSA containing the His6 tag (rPSA) and nickel hyponitrotriacetate (NTA-Ni) on an electrode surface. 2+ The complexes bind to form a ternary complex, enabling controlled immobilization of the template protein; then, electropolymerization is used to immobilize the NTA-Ni... 2+ A polyscopolamine-imprinted membrane forms around the -rPSA ternary complex. The template proteins rPSA and Ni are removed by washing the electrode with ethanol and dilute hydrochloric acid. 2+ Subsequently, an imprinted cavity capable of specifically recognizing natural PSA can be formed, thereby preparing a molecularly imprinted sensing electrode for recognizing PSA, and combining it with protein in situ self-assembly technology to realize signal amplification and detection of PSA.

[0018] Preparation of the molecularly imprinted sensing electrode in this invention: A gold electrode was incubated for 12 hours with 50 μL of (2-morpholinoethanesulfonic acid) (MES) buffer solution containing 0.1 mM mercapto-NTA and 1 mM tris(2-carboxyethyl)phosphine (TCEP). Then the electrode was incubated with 10 μM Ni 2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complex. After rinsing the modified electrode with water, the electrode was incubated with 10 μg / mL of His6-tagged recombinant PSA protein (rPSA) for 30 minutes to form a ternary NTA-Ni complex on the electrode surface. 2+ -rPSA complex. Then, hyoscyamine is electropolymerized on the electrode surface via electrodeposition in NTA-Ni... 2+ A polyhyoscyamine-imprinted film is formed around the -rPSA complex. The specific polymerization steps and conditions are as follows: NTA-Ni 2+ The rPSA complex-modified electrode was subjected to multi-step amperometric scanning for 15–20 cycles in a 0.1 M NaCl solution containing 0.5 mM hyoscyamine, with applied potentials of 0 V (5 s) and 0.7 V (1 s). The electrode was then rinsed several times with 20% ethanol and subsequently cleaned with 0.1 M HCl under gentle sonication to remove the template proteins rPSA and Ni. 2+ .

[0019] PSA detection: To detect the target PSA, the sensing electrode was incubated with a given concentration of PSA or CEA for 30 minutes to allow the PSA to be captured by the blotted sensing electrode. After thoroughly rinsing the electrode with 10 mM phosphate buffer, the electrode was placed in a 5 mM solution of 0.1 M KCl [Fe(CN)6]. 3− / 4− Electrochemical impedance spectroscopy was performed in a (1:1) solution. The auxiliary electrode and the reference electrode were a platinum wire and an Ag / AgCl electrode, respectively.

[0020] PSA was detected using a self-assembled signal amplification method: First, the imprinted sensing electrode was incubated in a PSA solution of a predetermined concentration for 30 min to allow PSA capture by the electrode. Then, the electrode was incubated in 10 μL of 0.1 mg / mL ConA solution for 10 min, followed by the addition of 10 μL of 0.5 mg / mL Avidin for another 30 min. The electrode was then thoroughly rinsed with ultrapure water. Using the imprinted electrode as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode, all three electrodes were placed in a 5 mM [Fe(CN)6] solution containing 0.1 M KCl. 3− / 4− Electrochemical impedance spectroscopy was performed in a 1:1 solution.

[0021] To evaluate the sensitivity of the direct detection method (without self-assembled signal amplification), this invention measured the effect of PSA concentration on impedance changes. Figure 2 It can be seen that the impedance value R et The value gradually increases with increasing PSA concentration, indicating that this sensing electrode can be used for PSA detection. Figure 3 As shown, within the concentration range of 0.1 ~ 10 ng / mL, R et It has a linear relationship with PSA concentration, and the linear equation is R. et = 2106[PSA] (ng / mL) + 23309. The minimum detectable concentration is lower than the PSA concentration in the blood of prostate patients (4 ng / mL), indicating that this sensing electrode has great potential for practical sample detection.

[0022] While direct label-free detection of PSA can be achieved through impedance changes, its practical value is even greater if its sensitivity is improved. PSA is a glycoprotein capable of binding to lectins. Concanavalin A (ConA) is a tetrameric lectin protein that binds to mannose / glucose units and can trigger the assembly of dimer and tetrameric glycoproteins. Avidin is a common and inexpensive tetrameric glycoprotein that has been widely used in the construction of biosensors. This invention discovers that PSA captured by electrodes can be recognized by ConA through lectin-glucose interactions, thereby capturing the tetrameric glycoprotein Avidin. Figure 1 The exposed glycosidic chains on the Avidin surface can further capture ConA in solution through lectin-glycan interactions. The ConA adhering to the surface can then capture even more Avidin molecules through the same mechanism. This process repeats until ConA / Avidin assemblies are formed in situ on the electrode surface, effectively inhibiting [Fe(CN)6]. 3− / 4− Electron transfer on the electrode surface enables PSA signal amplification and detection.

[0023] like Figure 4 As shown, the impedance of the molecularly imprinted sensing electrode (curve 1) increases significantly after combining PSA (curve 2) or PSA+ConA (curve 3), indicating that the trapping effect of both PSA and ConA can hinder [Fe(CN)6]. 3− / 4− Electron transfer occurs on the electrode surface. When the electrode corresponding to curve 3 is co-incubated with Avidin again, its impedance further increases (curve 4), indicating that Avidin can also resist [Fe(CN)6]. 3− / 4− Electron transfer at the electrode surface. When the PSA-integrated sensing electrode is incubated with a ConA / Avidin mixture (curve 5), the impedance is significantly higher than that of a single ConA-Avidin conjugate (curve 4), indicating that the detection signal intensity can be improved through in-situ self-assembly of ConA and Avidin.

[0024] To evaluate the sensitivity of the amplified self-assembled signal, this patent measured different concentrations of PSA. For example... Figure 5 As shown, increased PSA concentration leads to increased impedance, indicating that higher PSA concentrations promote the formation of more ConA / Avidin assemblies. Based on the impedance value R... et Relationship with PSA concentration ( Figure 6 A good linear fit curve was obtained in the range of 0.1–10 pg / mL (inset), and the linear equation was R. et= 3154 [PSA] (ng / mL) + 25407. The lowest detectable concentration is lower than that of the previous direct impedance method (without self-assembly signal amplification), and this value is comparable to or even lower than that of other electrochemical biosensors that employ enzyme and nanomaterial signal amplification. Such high sensitivity can be attributed to the in-situ formed ConA / Avidin insulating assembly effectively blocking [Fe(CN)6]. 3− / 4− Electron transfer on the electrode surface. Furthermore, the self-assembly signal amplification method of the present invention is simple to operate and does not require the use of expensive proteins and special nanomaterials for signal amplification.

[0025] To evaluate the selectivity of this method, samples including carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), antigen 125 (CA125), and 10% fetal bovine serum were analyzed. Figure 7 As shown, none of these samples caused R et Significant changes occurred, with corresponding impedance values ​​comparable to the blank control experiment (columns 1-5), indicating that this method has good specificity for PSA detection. The presence of these test samples did not affect the PSA measurement (column 6), indicating that the method has good anti-interference ability. Furthermore, considering the high sensitivity of this method, multiple dilutions of the real sample can effectively reduce potential interference from other biological matrices.

[0026] The PSA detection involved in this invention does not involve human samples, and the reagents and samples used can be purchased.

[0027] After a detailed description of the embodiments of the present invention, those skilled in the art will clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent applications. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention, and the present invention is not limited to the embodiments of the examples given in the specification.

Claims

1. An electrochemical method for detecting prostate-specific antigen, characterized in that... Includes the following steps: S1: Preparation of molecularly imprinted sensing electrode: A gold electrode was incubated for 12 hours with 50 μL of 50 mM 2-morpholinoethanesulfonic acid buffer solution containing 0.1 mM mercapto-NTA and 1 mM tris(2-carboxyethyl)phosphine. The pH of the buffer solution was 6.

5. Then the electrode was incubated with 10 μM Ni 2+ Incubation with the solution resulted in the formation of NTA-Ni on the gold surface. 2+ Complex; after rinsing the modified electrode with water, the electrode was incubated with 10 μg / mL of His6-tagged recombinant PSA protein for 30 minutes. The His6-tagged recombinant PSA protein, abbreviated as rPSA, forms a ternary NTA-Ni complex on the electrode surface. 2+ -rPSA complex, and then electropolymerize hyoscyamine on the electrode surface by electrodeposition, in NTA-Ni 2+ A polyhyoscyamine-imprinted film is formed around the -rPSA complex. The specific polymerization steps and conditions are as follows: NTA-Ni 2+ The rPSA complex-modified electrode was subjected to multi-step amperometric scanning for 15–20 cycles in a 0.1 M NaCl solution containing 0.5 mM scopolamine, with applied potentials of 0 V for 5 s and 0.7 V for 1 s. The electrode was then rinsed multiple times with 20% ethanol, followed by ultrasonic cleaning with 0.1 M HCl to remove the template proteins rPSA and Ni. 2+； ; molecularly imprinted sensing electrodes were obtained; S2: Incubate the imprinted sensing electrode with a given concentration of PSA for 30 minutes to allow the PSA to be captured by the imprinted sensing electrode, and then rinse the imprinted sensing electrode clean. S3: Using an imprinted sensing electrode as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode, the three electrodes are placed in a 5 mM solution containing 0.1 M KCl. 3− / 4− Electrochemical impedance spectroscopy was performed in a 1:1 solution.

2. The electrochemical method for detecting prostate-specific antigen according to claim 1, characterized in that: In step S2, the imprinted sensing electrode is first placed in a PSA solution of a given concentration and incubated for 30 min to allow PSA to be captured by the imprinted sensing electrode; then the signal amplifier is assembled, specifically: the imprinted sensing electrode is placed in 10 μL of 0.1 mg / mL ConA solution and incubated for 10 min, then 10 μL of 0.5 mg / mL Avidin is added to the solution and incubated for another 30 min; the imprinted sensing electrode is then rinsed clean.

3. The electrochemical method for detecting prostate-specific antigen according to claim 1, characterized in that: In step S2, the electrode is thoroughly rinsed with 10 mM phosphate buffer.

4. The electrochemical method for detecting prostate-specific antigen according to claim 2, characterized in that: Rinse the imprinted sensing electrode with ultrapure water.

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