Preparation method of D-penicillamine molecular imprinting sensor based on HSA-assisted recognition

Abstract

The invention discloses a preparation method of a D-penicillamine molecular imprinting sensor based on HSA-assisted recognition, which relates to the technical field of molecular imprinting sensors. The preparation method comprises the following steps: S1. polishing a bare gold electrode on a suede surface with aluminum oxide polishing powder, placing the polished gold electrode in a nitric acid solution, anhydrous ethanol, and secondary water in sequence for ultrasonication, using a potassium ferrocyanide solution as a probe molecule, and measuring by cyclic voltammetry until the voltage response value of the gold electrode is less than 120 mV to obtain a cleaned gold electrode; S2. modifying the cleaned gold electrode with a cysteine ​​solution, then with a glutaraldehyde solution, immersing the washed gold electrode in a human serum albumin solution for carbonyl and amino group binding, and immersing the electrode in a D-penicillamine solution for binding and recognition to obtain a gold electrode modified with an HSA-D-Pen complex; S3. immersing the electrode in an o-phenylenediamine solution for pre-assembly and electropolymerization to obtain an imprinted polymer-modified gold electrode; and S4. placing the imprinted polymer-modified gold electrode in a PBS buffer solution for elution.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular imprinted sensors, and in particular to a method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition. Background Art

[0002] Chiral molecules are used in many fields, especially in medicine. Drugs containing chiral centers have very different properties of their enantiomers. In most chiral drugs, one enantiomer is medicinal, while the other is not, or even has toxic side effects. Therefore, the determination and separation of chiral drug enantiomers is crucial.

[0003] Conventional chiral separation and identification methods include high-performance liquid chromatography chiral stationary phase (CSPs) method, chromatographic separation method, high-speed countercurrent chromatography, capillary electrophoresis separation method, crystallization method, chemical resolution method, and biofilm resolution method. These methods have poor chiral separation effect, low separation efficiency, and environmental pollution. Therefore, it is necessary to introduce new technologies to develop an efficient chiral separation method.

[0004] Molecular imprinting sensors (MIPs) fabricated using molecular imprinting technology (MIT) possess unique characteristics such as structural predictability, recognition specificity, and wide applicability. They are able to selectively identify and detect specific target compounds, and have advantages such as simple design, high sensitivity, low cost, portability, and ease of miniaturization and automation. Due to their uniquely "specific" high chiral selectivity, they are widely used in fields such as chiral resolution and pharmaceuticals. However, conventional molecular imprinting sensors have weak anti-interference capabilities when identifying target molecules, can recognize substances and enantiomers with similar structures to the target molecule, and the resulting MIPs are structurally unstable after elution.

[0005] Human serum albumin (HSA), the primary soluble protein in the circulatory system, is characterized by its small molecular weight, high solubility, excellent stability, strong affinity, and the ability to bind to a variety of substances (such as metal ions, fatty acids, amino acids, hormones, and drugs). Proteins are natural chiral selectors, possessing numerous chiral centers. Most proteins are capable of chirally specific reverse bonding to molecules. HSA chiral recognition primarily occurs through Sites I and II, which are non-covalent bonds that interact between atoms in the amino acid residues surrounding the receptor's active site and atoms in the ligand corresponding to that active site.

[0006] Penicillamine (3,3-dimethylcysteine, Pen) is a low-molecular-weight amino acid produced by the hydrolysis of penicillin. It has right-hand and left-hand optical isomers with distinct chemical properties. For example, the chiral enantiomer of penicillamine, D-penicillamine (D-Pen), exhibits strong chelation with heavy metal ions such as copper, mercury, and lead. It forms a non-dissociable, low-toxic complex with mercury that is excreted in urine or feces. It is stable and highly soluble. Clinically, D-penicillamine is widely used to treat numerous diseases, such as rheumatoid arthritis and Wilson's disease, and as a chelating agent for the treatment of lead poisoning. L-penicillamine, on the other hand, is an intermediate in the synthesis of a highly effective protease inhibitor with strong antiviral properties and significant preventive and therapeutic effects against AIDS. However, L-penicillamine is toxic and can cause neurological disorders in humans, posing a threat to both human health and the environment. Currently, there are few methods published for the chiral separation of D- and L-penicillamine in pharmaceutical analysis, such as high performance liquid chromatography and capillary electrophoresis. However, these separation methods are relatively cumbersome.

[0007] Currently, there is no rapid chiral separation method with good separation effect, strong selectivity and high sensitivity. Summary of the Invention

[0008] In response to the deficiencies in the prior art, the present invention provides a method for preparing a molecularly imprinted sensor for D-penicillamine based on HSA-assisted recognition. The method provided by the present invention introduces human serum albumin into the preparation of molecularly imprinted materials, and uses a complex formed by HSA and the target molecule to fix the spatial conformation of the target molecule, thereby enhancing the specific recognition of D-penicillamine, while improving the fineness of the spatial structure of the imprinted pores and increasing the number of recognition sites. Cysteine ​​is combined with glutaraldehyde to modify the surface of a gold electrode, which is then bound to human serum albumin and finally to the target molecule D-penicillamine. O-phenylenediamine is used as a functional monomer, and an HSA-D-Pen complex imprinted polymer is constructed by electropolymerization. After eluting D-penicillamine alone, an imprinted cavity is obtained that matches and complements the spatial conformation, size, structure, and recognition site of D-penicillamine, thereby producing a chiral molecularly imprinted sensor with high-specificity conformational recognition of D-penicillamine.

[0009] The purpose of the present invention is to protect a method for preparing a D-penicillamine molecular imprinting sensor based on HSA-assisted recognition, which comprises the following steps:

[0010] S1. Polish a bare gold electrode on a suede surface with aluminum oxide polishing powder. Place the polished gold electrode in a nitric acid solution, anhydrous ethanol, and secondary water, sequentially, and ultrasonicate it. Cyclic voltammetry is performed using potassium ferrocyanide solution as a probe molecule until the voltage response of the gold electrode is less than 120 mV. This results in a cleaned gold electrode.

[0011] S2. The cleaned gold electrode is modified with a cysteine ​​solution, then with a glutaraldehyde solution, immersed in a human serum albumin solution for carbonyl and amino group binding, and then immersed in a D-penicillamine solution for binding and recognition, thereby obtaining a gold electrode modified with an HSA-D-Pen complex.

[0012] S3. Immerse the HSA-D-Pen complex-modified gold electrode in an o-phenylenediamine solution for pre-assembly and perform electropolymerization to obtain an imprinted polymer-modified gold electrode;

[0013] S4. The gold electrode modified with the imprinted polymer is placed in a PBS buffer solution for elution to obtain the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition.

[0014] Preferably, in step S1, polishing the suede surface with aluminum oxide polishing powder is polishing the suede surface with aluminum oxide polishing powder of 1.0, 0.3, and 0.05 μm in sequence.

[0015] Preferably, in step S1, the nitric acid solution is a 1:1 nitric acid solution; the ultrasonic time is 1 min; the concentration of the potassium ferricyanide solution is 5.0×10 -3 mol / L.

[0016] Preferably, in step S2, the concentration of the cysteine ​​solution is 0.1 mol / L, and the modification time by the cysteine ​​solution is 2 h;

[0017] The glutaraldehyde solution is a 5% glutaraldehyde solution.

[0018] Preferably, in step S2, the concentration of the human serum albumin solution is 4.0×10 -5 mol / L, and the time for the carbonyl and amino group to combine is 55 min.

[0019] Preferably, in step S2, the concentration of the D-penicillamine solution is 4.0×10 -5 mol / L; the immersion time for binding and recognition in D-penicillamine is 30 minutes.

[0020] Preferably, in step S3, the concentration of the o-phenylenediamine solution is 1.0×10 -4 mol / L.

[0021] Preferably, in step S3, the electropolymerization conditions are: the number of polymerization cycles is 15 cycles, the scanning range is -0.2V to +0.6V, and the scanning rate is 50mV / s.

[0022] Preferably, in step S4, the concentration of the PBS buffer solution is 0.20 mol / L, and the pH is 7.4.

[0023] Preferably, in step S4, the elution is performed using a time current potential of 1.05V for 50 minutes.

[0024] The beneficial effects of the present invention are embodied in:

[0025] (1) The method provided by the present invention introduces human serum albumin into the preparation of molecular imprinting materials, and uses the complex formed by HSA and the target molecule to fix the spatial conformation of the target molecule, thereby enhancing the specific recognition of D-penicillamine, while improving the fineness of the spatial structure of the imprinted pores and increasing the recognition sites. Cysteine ​​is combined with glutaraldehyde to modify it on the surface of a gold electrode, which is then combined with human serum albumin and finally with the target molecule D-penicillamine. O-phenylenediamine is used as a functional monomer, and an HSA-D-Pen complex imprinted polymer is constructed by electropolymerization. After eluting D-penicillamine alone, an imprinted cavity is obtained that matches and complements the spatial conformation, size, structure, and recognition site of D-penicillamine, thereby preparing a chiral molecular imprinting sensor that recognizes D-penicillamine with high specificity.

[0026] (2) The D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared by the method provided by the present invention can specifically recognize the chiral structure of D-penicillamine, has strong specificity, can effectively identify and separate chiral molecules, and has strong anti-interference ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0028] Figure 1 The voltammetric response diagram of the D-penicillamine molecular imprinted sensor based on HSA-assisted recognition provided in the embodiment;

[0029] Figure 2 is the voltammetric response diagram of the non-molecular imprinting sensor prepared in comparative example;

[0030] Figure 3 This is the impedance response diagram of the D-penicillamine molecular imprinted sensor based on HSA-assisted recognition prepared in Example;

[0031] Figure 4 The current response signals of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition provided in the embodiment at different concentrations of D-penicillamine;

[0032] Figure 5 This is the chiral recognition effect of conventional molecular imprinting sensor;

[0033] Figure 6 The chiral recognition effect of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in Example;

[0034] Figure 7 The anti-interference ability of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in the embodiment;

[0035] Attachment Figure 1 a-Gold electrode modified with cysteine, b-Gold electrode modified with glutaraldehyde, c-Gold electrode bound to human serum albumin, d-Gold electrode modified with HSA-D-Pen complex, e-Gold electrode modified with imprinted polymer, f-D-penicillamine molecular imprinting sensor based on HSA-assisted recognition, i-D-penicillamine molecular imprinting sensor based on HSA-assisted recognition re-adsorbs D-penicillamine;

[0036] Attachment Figure 2 a-bare gold electrode, b-gold electrode modified with glutaraldehyde, c-gold electrode bound to human serum albumin, d-non-molecularly imprinted sensor, e-non-molecularly imprinted sensor re-adsorbs D-penicillamine;

[0037] Attachment Figure 3 a-Gold electrode modified with cysteine, b-Gold electrode modified with glutaraldehyde, c-Gold electrode bound to human serum albumin, d-Gold electrode modified with HSA-D-Pen complex, e-Gold electrode modified with imprinted polymer, f-D-penicillamine molecular imprinting sensor based on HSA-assisted recognition, i-D-penicillamine molecular imprinting sensor based on HSA-assisted recognition re-adsorbs D-penicillamine;

[0038] Attachment Figure 4 The concentration of 1-D-penicillamine was 2.0×10 -14 mol / L, 2-D-penicillamine concentration was 4.0×10 -14 mol / L, 3-D-penicillamine concentration was 6.0×10 -14 mol / L, 4-D-penicillamine concentration was 2.0×10 -13 mol / L, 5-D-penicillamine concentration was 4.0×10 -13 mol / L, 6-D-penicillamine concentration was 6.0×10 -13 mol / L, 7-D-penicillamine concentration was 2.0×10 - 12 mol / L, 8-D-penicillamine concentration was 4.0×10 -12mol / L, 9-D-penicillamine concentration was 6.0×10 -12 mol / L, 10-D-penicillamine concentration was 2.0×10 -11 mol / L, 11-D-penicillamine concentration was 4.0×10 -11 mol / L, 12-D-penicillamine concentration was 6.0×10 -11 mol / L.

[0039] Attachment Figure 5 In the middle, a-blank, b-recognition of L-penicillamine, c-recognition of racemate, d-recognition of D-penicillamine;

[0040] Attachment Figure 6 In the figure, a-blank, b-recognition of L-penicillamine, c-recognition of racemate, d-recognition of D-penicillamine. DETAILED DESCRIPTION

[0041] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0042] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0043] Example

[0044] This embodiment provides a method for preparing a D-penicillamine molecular imprinting sensor based on HSA-assisted recognition, and the steps are as follows:

[0045] S1. Polish a bare gold electrode on a suede surface with aluminum oxide polishing powder. Place the polished gold electrode in a nitric acid solution, anhydrous ethanol, and secondary water, sequentially, and ultrasonicate it. Cyclic voltammetry is performed using potassium ferrocyanide solution as a probe molecule until the voltage response of the gold electrode is less than 120 mV. This results in a cleaned gold electrode.

[0046] S2. The cleaned gold electrode is modified with a cysteine ​​solution, then with a glutaraldehyde solution, immersed in a human serum albumin solution for carbonyl and amino group binding, and then immersed in a D-penicillamine solution for binding and recognition, thereby obtaining a gold electrode modified with an HSA-D-Pen complex.

[0047] S3. Immerse the HSA-D-Pen complex-modified gold electrode in an o-phenylenediamine solution for pre-assembly and perform electropolymerization to obtain an imprinted polymer-modified gold electrode;

[0048] S4. The gold electrode modified with the imprinted polymer is placed in a PBS buffer solution for elution to obtain the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition.

[0049] In step S1 , the suede surface is polished with aluminum oxide polishing powder, which includes polishing the suede surface with aluminum oxide polishing powder of 1.0, 0.3, and 0.05 μm in sequence.

[0050] In step S1, the nitric acid solution is a 1:1 nitric acid solution; the ultrasonic time is 1 min; the concentration of the potassium ferricyanide solution is 5.0×10 -3 mol / L.

[0051] In step S2, the concentration of the cysteine ​​solution is 0.1 mol / L, and the modification time by the cysteine ​​solution is 2 hours; the glutaraldehyde solution is a 5% glutaraldehyde solution.

[0052] In step S2, the concentration of human serum albumin solution is 4.0×10 -5 mol / L, and the time for the carbonyl and amino group to combine is 55 min.

[0053] In step S2, the concentration of D-penicillamine solution is 4.0×10 -5 mol / L; the time for binding and recognition in D-penicillamine is 30 min.

[0054] In step S3, the concentration of o-phenylenediamine solution is 1.0×10 -4 mol / L.

[0055] In step S3 , the electropolymerization conditions are as follows: the number of polymerization cycles is 15, the scanning range is −0.2 V to +0.6 V, and the scanning rate is 50 mV / s.

[0056] In step S4, the concentration of the PBS buffer solution is 0.20 mol / L, and the pH is 7.4.

[0057] In step S4, elution is performed using a time current potential of 1.05 V for 50 minutes.

[0058] Comparative Example

[0059] This comparative example provides a preparation method of a non-molecular imprinting sensor, which is different from the embodiment in that the step of immersing in a D-penicillamine solution for binding and recognition in S2 is not included.

[0060] Test Example 1

[0061] Voltammetric response of the HSA-assisted recognition-based D-penicillamine molecular imprinted sensor prepared in the test example

[0062] Reagent preparation: Take three penicillamine tablets (mass 1.0698g) and grind them into a mortar. Weigh 0.0428g of the powder into a 50mL beaker, add 20mL of deionized water to dissolve it, and then put it into a 50mL centrifuge tube for centrifugation. Take the supernatant (wash the filter residue with deionized water several times - centrifuge - take the supernatant) and dilute it to a 1000mL volumetric flask. Use a pipette to remove 100μL of the supernatant and dilute it to a 100mL volumetric flask as the test solution.

[0063] Test method: The D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in the example was used to re-adsorb and identify D-penicillamine on the sample liquid, and the DPV response value was recorded.

[0064] Test results: see Figure 1 .

[0065] from Figure 1 It can be seen that the washed gold electrode was modified with cysteine ​​to obtain a cysteine-modified gold electrode (curve "a"), and then modified with 5% glutaraldehyde for 40 minutes to obtain a glutaraldehyde-modified gold electrode (curve "b"), and then immersed in 1.0×10 -5 mol / L human serum albumin was subjected to carbonyl and amino binding for 55 min to obtain a gold electrode bound to human serum albumin (curve "c"). The binding and recognition of 1.0×10 -12 mol / L target molecule D-penicillamine for 30 min to obtain a gold electrode modified with HSA-D-Pen complex (curve "d"). The above steps hindered the transfer of electrons to a certain extent. Compared with the bare gold electrode, the reduction peak current intensity of the probe molecule was significantly reduced.

[0066] HSA-D-Pen imprinted polymer was prepared by electropolymerization using 10.00 mL of 1.0×10-4 mol / L o-phenylenediamine as a substrate, resulting in a polymer-modified gold electrode. A dense, poorly conductive imprinted film formed on the electrode surface after polymerization, severely hindering signal transmission efficiency and causing a sharp drop in probe signal intensity (curve "e"). Elution of D-penicillamine alone revealed electron-transferring imprinted pores, resulting in a D-penicillamine molecularly imprinted sensor based on HSA-assisted recognition. This significantly increased the probe's response signal current intensity (curve "f"). After 20 minutes of re-adsorption of the D-penicillamine target molecule, the molecule entered the imprinted pores, again hindering electron transfer, causing a sharp increase in the probe's response signal (curve "i").

[0067] Test Example 2

[0068] Test the voltammetric response of the non-molecular imprinting sensor prepared in the comparative example

[0069] Reagent preparation: Take three penicillamine tablets (mass 1.0698g) and grind them into a mortar. Weigh 0.0428g of the powder into a 50mL beaker, add 20mL of deionized water to dissolve it, and then put it into a 50mL centrifuge tube for centrifugation. Take the supernatant (wash the filter residue with deionized water several times - centrifuge - take the supernatant) and dilute it to a 1000mL volumetric flask. Use a pipette to remove 100μL of the supernatant and dilute it to a 100mL volumetric flask as the test solution.

[0070] Test method: The non-molecular imprinting sensor prepared in the comparative example was used to re-adsorb the sample liquid to identify D-penicillamine, and the DPV response value was recorded.

[0071] Test results: see Figure 2 .

[0072] from Figure 2 It can be seen that glutaraldehyde was modified with cysteine ​​on the bare gold electrode (curve "a") to obtain a gold electrode modified with glutaraldehyde (curve "b"), and then 4.0×10 -5 mol / L human serum albumin, and a gold electrode bound to human serum albumin was obtained (curve "c"). At this time, the response signal of the probe molecule dropped sharply. After elution, the response signal intensity of the non-molecular imprinted sensor (curve "d") was basically consistent with the response signal intensity of the electropolymerization. -12 The DPV response after addition of 1 mol / L D-penicillamine (curve "e") is essentially the same as after elution (curve "d"). Because the target molecule is not bound during the nMIPs sensor preparation process, molecularly imprinted pores are not formed after elution, resulting in a lack of electron transfer. Therefore, the molecularly imprinted sensor prepared using human serum albumin as the auxiliary target molecule for D-penicillamine specifically adsorbs D-penicillamine. This also demonstrates that the elution potential of 1.05 V has no effect on other materials modified on the electrode during the elution process.

[0073] Test Example 3

[0074] Impedance response of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in the test example

[0075] Test results: see Figure 3 shown.

[0076] from Figure 3 It can be seen that the bare gold electrode has the best conductivity without any modification material and its resistance value is the smallest (curve "a").

[0077] Gold electrode modified with glutaraldehyde: After glutaraldehyde was modified on the electrode surface using cysteine, the resistance increased and the current passing through it decreased slightly (curve "b"). The modification hindered the electron transfer on some electrodes.

[0078] Gold electrode bound to human serum albumin: When bound to human serum albumin, the resistance value increases rapidly (curve "c"); Gold electrode modified with HSA-D-Pen complex: After binding to the target molecule, the resistance value continues to increase (curve "d");

[0079] Imprinted polymer-modified gold electrode: After 15 cycles of electropolymerization with o-phenylenediamine, a dense polymer film with poor conductivity forms on the electrode surface, severely hindering electron transfer and causing a sharp increase in resistance (curve "e").

[0080] D-penicillamine molecularly imprinted sensor based on HSA-assisted recognition: After the target molecule is eluted, imprinted holes that can pass electrons are left behind, and the resistance value decreases (curve "f");

[0081] The HSA-assisted recognition D-penicillamine molecularly imprinted sensor re-adsorbed D-penicillamine. After re-adsorption of the target molecule, D-penicillamine entered the recognition pore, blocking the electron transfer path and causing the resistance to increase again (curve "g"). This indicates that the HSA-assisted recognition D-penicillamine molecularly imprinted sensor exhibited consistent electrochemical properties under all preparation conditions, and the conclusions obtained were consistent with the response results from differential pulse voltammetry.

[0082] Test Example 4

[0083] The current response signals of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition provided in the test example at different concentrations of D-penicillamine were measured.

[0084] Test method: D-penicillamine concentration 1→12 are: 2.0×10 -14 , 4.0×10 -14 , 6.0×10 -14 , 2.0×10 -13 , 4.0×10 -13 , 6.0×10 -13 , 2.0×10 -12 , 4.0×10 -12 , 6.0×10 -12 , 2.0×10 -11 , 4.0×10 -11 , 6.0×10 -11 mol / L.

[0085] Test results: see Figure 4 shown.

[0086] from Figure 4It can be seen that the intensity change of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition decreases with the increase of the logarithm of the D-penicillamine concentration (logC), and it can be detected at 2.0×10 -14 mol / L~6.0×10 -11 mol / L showed a good positive correlation linearity within the gradient concentration range, and the detection limit was 5.39×10 -15 mol / L. The linear equation is Δi(μA)=1.0737×10 - 6 lgC(mol / L)+3.1065×10 -6 (r=0.9937).

[0087] Test Example 5

[0088] Detection of the chiral recognition effect of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in Example

[0089] The conventional molecular imprinting sensor preparation method is as follows: a bare gold electrode is placed on a 1.0×10 -4 mol / L o-phenylenediamine and 4.0×10 -5 Cyclic voltammetry was performed for 15 cycles of electropolymerization in a solution of a 3:1 mol / LD-penicillamine mixture over a scan range of -0.2 V to +0.6 V at a scan rate of 50 mV / s. The polymerized electrode material was eluted at an IT potential of 1.05 V for 50 minutes to obtain a conventional molecularly imprinted sensor.

[0090] The conventional molecular imprinting sensor was used to measure the concentration of 1.0×10 -12 mol / L D-penicillamine, 1.0×10 - 12 mol / L L-penicillamine and the racemate of the same concentration were re-adsorbed and identified. The results are shown in the figure: blank voltammetric response value (curve "a"); identification of L-penicillamine molecules (curve "b"); identification of racemate (curve "c"); identification of D-penicillamine molecules (curve "d"); Figure 5 It can be seen that conventional molecular imprinting sensors can identify 1.0×10 -12 Voltammetric responses were observed for L-penicillamine, D-penicillamine, and its racemate at concentrations of 1 mol / L. While the D-penicillamine response was significant, the racemate response was more than half that of the D-penicillamine response, significantly impacting the accuracy of the test results. This is due to the inability of conventional molecularly imprinted sensors to form a fixed chiral conformational cavity, preventing chiral selection during the recognition process.

[0091] The HSA-assisted recognition-based D-penicillamine molecular imprinting sensor prepared in the embodiment has a concentration of 1.0×10 -12 mol / L D-penicillamine, L-penicillamine and racemates were identified, such as Figure 6 ,from Figure 6 It can be seen that the response signal intensity for recognizing L-penicillamine is only 2.87% of that for recognizing D-penicillamine, and the signal intensity for recognizing the racemate is 5.74% of that for D-penicillamine. That is, the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition provided by the present invention can specifically recognize D-penicillamine. This is because human serum albumin fixes the chiral structure of D-penicillamine after binding to the D-penicillamine complex, and forms a hole with the same chiral conformation after elution. When recognizing D-penicillamine, it can be quickly and accurately recognized, and the interference of the racemate and L-penicillamine is greatly reduced.

[0092] Test Example 6

[0093] Stability and reproducibility of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in the test example

[0094] The D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in the embodiment was used to measure D-penicillamine in parallel 5 times, and the relative standard deviation RSD of the experimental results was 3.89%. It can be seen that the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition provided by the present invention has good stability.

[0095] At the same time, 5 D-penicillamine molecular imprinting sensors based on HSA-assisted recognition prepared in the embodiment were prepared, and 1.0×10-12 mol / L of D-penicillamine was measured successively. The relative standard deviation RSD of the obtained results was 4.1%. It can be seen that the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition provided by the present invention has good reproducibility.

[0096] Test Example 7

[0097] Detection of the anti-interference ability of the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition prepared in Example

[0098] Substances with similar structures to the target molecule, including alanine (Ala), valine (Vla), cysteine ​​(Cys), and phenylalanine (Phe), were added to 1.0×10-12 mol / L D-penicillamine for re-adsorption identification. The results of the transfer assay were compared with those of the D-penicillamine assay. The results are shown in the table. Figure 7 .from Figure 7As can be seen from the figure, the response signal of cysteine ​​(Cys), which has the greatest interference with D-penicillamine, is only 4.93%. Therefore, the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition provided by the present invention has weak recognition ability for substances with similar structures and can specifically recognize D-penicillamine.

[0099] Test Example 8

[0100] Sample testing

[0101] Accurately pipette 100 μL of the sample solution into a 10 mL beaker and dilute with 10.00 mL of deionized water. Four HSA-assisted D-penicillamine molecularly imprinted sensors prepared using the method described in Example were used to identify the sample solution for 20 minutes, and the voltammetric response signals were measured. The sensor accuracy was verified using the spike recovery method.

[0102] The sample solution was prepared as follows: three penicillamine tablets (1.0698 g) were ground into a mortar, 0.0428 g of the powder was weighed into a 50 mL beaker, 20 mL of deionized water was added to dissolve the powder, and the mixture was placed into a 50 mL centrifuge tube for centrifugation. The supernatant was taken (the filter residue was washed with deionized water several times, centrifuged, and the supernatant was taken) and the volume was adjusted to a 1000 mL volumetric flask. 100 μL of the supernatant was removed with a pipette and the volume was adjusted to a 100 mL volumetric flask as the test solution.

[0103] As can be seen from Table 1, the recovery rate of D-penicillamine in the sample by the HSA-assisted recognition-based D-penicillamine molecular imprinting sensor provided by the present invention is 97.20% to 101.80%. That is, the HSA-assisted recognition-based D-penicillamine molecular imprinting sensor of the present invention can be applied to the detection of D-penicillamine in penicillamine tablets, and has good detectability, and can be actually used in detection.

[0104] Table 1

[0105]

[0106]

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for preparing a D-penicillamine molecular imprinting sensor based on HSA-assisted recognition, characterized in that: The preparation method comprises the following steps: S1. Polish a bare gold electrode on a suede surface with aluminum oxide polishing powder. Place the polished gold electrode in a nitric acid solution, anhydrous ethanol, and secondary water, sequentially, and ultrasonicate it. Cyclic voltammetry is performed using potassium ferrocyanide solution as a probe molecule until the voltage response of the gold electrode is less than 120 mV. This results in a cleaned gold electrode. S2. The cleaned gold electrode is modified with a cysteine ​​solution, then with a glutaraldehyde solution, immersed in a human serum albumin solution for carbonyl and amino group binding, and then immersed in a D-penicillamine solution for binding and recognition, thereby obtaining a gold electrode modified with an HSA-D-Pen complex. S3. Immerse the HSA-D-Pen complex-modified gold electrode in an o-phenylenediamine solution for pre-assembly and perform electropolymerization to obtain an imprinted polymer-modified gold electrode; S4. The gold electrode modified with the imprinted polymer is placed in a PBS buffer solution for elution to obtain the D-penicillamine molecular imprinting sensor based on HSA-assisted recognition.

2. The method for preparing a D-penicillamine molecular imprinting sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S1, the polishing of the suede surface with aluminum oxide polishing powder is performed by sequentially polishing the suede surface with aluminum oxide polishing powder of 1.0, 0.3, and 0.05 μm.

3. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S1, the nitric acid solution is a 1:1 nitric acid solution; the ultrasonic time is 1 min; the concentration of the potassium ferricyanide solution is 5.0×10 -3 mol / L.

4. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S2, the concentration of the cysteine ​​solution is 0.1 mol / L, the modification time by the cysteine ​​solution is 2 hours, and the glutaraldehyde solution is a 5% glutaraldehyde solution.

5. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S2, the concentration of the human serum albumin solution is 4.0×10 -5 mol / L, and the time for the carbonyl and amino group to combine is 55 min.

6. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S2, the concentration of the D-penicillamine solution is 4.0×10 -5 mol / L; the immersion time for binding and recognition in D-penicillamine is 30 minutes.

7. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S3, the concentration of the o-phenylenediamine solution is 1.0×10 -4 mol / L.

8. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S3, the electropolymerization conditions are as follows: the number of polymerization cycles is 15 cycles, the scanning range is -0.2V to +0.6V, and the scanning rate is 50mV / s.

9. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S4, the concentration of the PBS buffer solution is 0.20 mol / L, and the pH is 7.

4.

10. The method for preparing a D-penicillamine molecular imprinted sensor based on HSA-assisted recognition according to claim 1, characterized in that: In step S4, the elution is performed at a time current potential of 1.05 V for 50 minutes.

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