An electrochemical anti-fouling aptamer sensor based on y-shaped peptide modification and application research thereof

Abstract

The application discloses an electrochemical anti-fouling aptamer sensor based on Y-type peptide modification and application research thereof, and belongs to the technical field of anti-fouling biosensors. The electrochemical anti-fouling aptamer sensor is composed of a glassy carbon electrode, a sensitization material, an anti-fouling layer and a recognition part. The anti-fouling performance of the Y-type structure peptide is more excellent than that of straight-chain peptides and cyclic peptides with the same anti-fouling sequence. When detecting tetracycline residues in an actual sample, the sensor can effectively reduce the non-specific adsorption of non-detection substances in a food matrix, and can also realize detection of various target objects by changing the type of aptamer.

Description

Technical Field

[0001] This invention belongs to the field of antifouling biosensor technology, specifically a Y-peptide-modified electrochemical antifouling aptamer sensor and its application research. Background Technology

[0002] Tetracycline is a broad-spectrum antibiotic widely used to treat bacterial infections in animals. However, its overuse has led to tetracycline residues in animal-derived foods, posing a threat to human health. Therefore, an increasing number of methods are being used for tetracycline residue detection, with electrochemical sensors standing out due to their ease of operation, low cost, and high selectivity and sensitivity. However, in practical applications, non-detectable substances such as proteins and cells in the matrix can non-specifically adsorb onto the sensing surface, reducing the sensor's selectivity and sensitivity. Modifying the sensor surface with antifouling materials to enhance its antifouling ability is an effective strategy to address this problem. Currently recognized antifouling mechanisms include steric hindrance, hydration layers, and electrostatic repulsion. Based on these mechanisms, many antifouling materials have been designed, such as polyethylene glycol, zwitterionic polymers, and zwitterionic peptides. Zwitterionic peptides have attracted increasing attention due to their advantages of flexible design, strong biocompatibility, and industrial synthesis capabilities.

[0003] Zwitterionic peptides are polypeptide sequences composed mostly of positively and negatively charged amino acids, exhibiting excellent hydrophilicity. By adjusting the types and arrangement of amino acids, they can be made electrically neutral. While maintaining hydrophilicity and electrical neutrality, the modification density of zwitterionic peptides on the sensing surface can be increased through structural design and adjustment, thereby enhancing the steric hindrance effect and improving the sensor's antifouling ability. Compared to basic linear structures, cyclic and branched zwitterionic peptides show better antifouling performance. Currently, no antifouling material can completely resist interference from contaminants during sensor detection. Therefore, developing sensors with excellent antifouling properties remains a challenge and of significant importance. Summary of the Invention

[0004] The purpose of this invention is to construct an electrochemical antifouling aptamer sensor based on a branched-chain peptide. This branched-chain peptide contains two antifouling branches and macroscopically exhibits a Y-shape. Furthermore, a tetracycline aptamer is modified onto the sensor surface to enable sensitive detection of tetracycline in food. To achieve the above objective, the technical solution of this invention is as follows:

[0005] The first objective of this invention is to provide an antifouling Y-type peptide, comprising an anchoring end, a linking end, and two antifouling sequences, wherein the anchoring end is cysteine, the linking end comprises polyproline and two amino acid residues, and the antifouling sequences comprise zwitterionic peptides.

[0006] In one embodiment, the anchoring end includes one or more cysteine ​​residues.

[0007] In one embodiment, the polyproline comprises at least three prolines.

[0008] In one embodiment, the polyproline comprises PPPP.

[0009] In one embodiment, the two amino acid residues include glutamic acid and lysine.

[0010] In one embodiment, the zwitterionic peptide comprises alternating arrangements of arginine, serine, and glutamic acid.

[0011] In one embodiment, the zwitterionic peptide includes RSERSERSE.

[0012] In one embodiment, the sequence of the Y-type peptide is CPPPPEK-(RSERSERSE)2.

[0013] In one embodiment, the anchoring end and the connecting end form the main chain of the Y-type peptide, and the two antifouling sequences are the two branches of the Y-type peptide.

[0014] The second objective of this invention is to provide an electrochemically antifouling sensing surface, wherein the electrochemically antifouling sensing surface is an electrode with the above-mentioned Y-type peptide modified on its surface.

[0015] In one embodiment, the electrode comprises a glassy carbon electrode.

[0016] A third objective of this invention is to provide an electrochemical antifouling aptamer sensor, wherein the electrochemical antifouling sensor is modified with a Y-type peptide and an aptamer corresponding to the target to be detected.

[0017] In one embodiment, the target to be detected includes a small molecule compound.

[0018] In one embodiment, the small molecule compound includes tetracycline.

[0019] In one embodiment, the 5' end of the aptamer is modified with a thiol and a methylene group.

[0020] In one embodiment, the thiol-modifying group is -SH-(CH2)6-.

[0021] The fourth objective of this invention is to provide an electrochemical antifouling aptamer sensor, the specific steps of which are as follows:

[0022] 1) Pretreatment of glassy carbon electrode (GCE): Polish the glassy carbon electrode and ultrasonically clean it to obtain a GCE with a mirror-like surface;

[0023] 2) Preparation of gold nanoparticle (AuNPs) modified electrode (AuNPs / GCE): The pretreated GCE was immersed in chloroauric acid solution and AuNPs / GCE was obtained by constant potential deposition;

[0024] 3) Preparation of aptamer-Y peptide solution: Dissolve the aptamer and the above-mentioned Y peptide in phosphate buffer;

[0025] 4) Aptamer-Y peptide modification of AuNPs / GCE: AuNPs / GCE are immersed in aptamer-Y peptide solution at 2-6℃ for more than 8 hours to obtain electrochemical antifouling aptamer sensor (Apt-Pep / AuNPs / GCE).

[0026] In one embodiment, the concentration of chloroauric acid is 4–6 mmol / L. -1 .

[0027] In one embodiment, the electrodeposition voltage is -0.3 to -0.5V, and the time is 60s.

[0028] In one embodiment, the concentration of the Y-peptide is 0.05 mg / mL. -1 ~0.6mg mL -1 The concentration of the aptamer is 1 μmol L. -1 ~5μmol L -1 .

[0029] In one embodiment, the aptamer is an aptamer corresponding to the target to be tested, designed according to conventional techniques in the art.

[0030] In one embodiment, the 5' end of the aptamer is modified with a thiol and a methylene group.

[0031] In one embodiment, the thiol-modifying group is -SH-(CH2)6-.

[0032] In one embodiment, the aptamer sequence is 5'-SH-(CH2)6-CGTACG GAA TTC GCTAGCCCC CCG GCA GGC CAC GGC TTG GGT TGG TCC CAC TGC GCG TGG ATC CGA GCT CCACGT G-3'.

[0033] The fifth objective of this invention is to provide a method for detecting tetracycline, wherein the method utilizes the aforementioned electrochemical antifouling aptamer sensor (Apt-Pep / AuNPs / GCE) for detection, and the specific steps of the method are as follows:

[0034] (1) Apt-Pep / AuNPs / GCE was soaked in tetracycline standard solutions of different concentrations and incubated for more than 60 min. The current response value was recorded by differential pulse voltammetry and a standard curve was plotted.

[0035] (2) Soak Apt-Pep / AuNPs / GCE in a milk sample solution containing a specific concentration of tetracycline for more than 60 minutes, and determine the current response value by differential pulse voltammetry. Calculate the spiked recovery rate based on the standard curve drawn in step (1).

[0036] In one embodiment, the differential pulse voltammetry has a potential range of -0.2V to 0.6V, an amplitude of 50mV, and a scan rate of 0.1VS. -1 .

[0037] The present invention also provides the application of the above-mentioned antifouling Y-type peptide, the above-mentioned electrochemical antifouling sensing surface, or the above-mentioned electrochemical antifouling aptamer sensor in the field of biological detection.

[0038] In one implementation, it is applied in the fields of food safety testing or environmental monitoring.

[0039] The present invention also provides the application of the above-mentioned antifouling Y-type peptide, the above-mentioned electrochemical antifouling sensing surface, or the above-mentioned electrochemical antifouling aptamer sensor in the detection of small molecule compounds.

[0040] Beneficial effects:

[0041] 1. Based on linear zwitterionic peptides, this invention modifies the structure of the peptides to design zwitterionic peptides with a macroscopic Y-shaped shape, giving them good antifouling properties, as well as good specificity, repeatability (RSD of 3.20%) and stability (RSD of 2.58%). The spiked recoveries are 91.0% to 103.1%, with RSDs all less than 3.9%.

[0042] 2. The preparation method of the present invention is simple, easy to miniaturize, and has a certain degree of universality. Electrochemical antifouling sensors that can identify different target analytes can be obtained by changing the type of aptamer. The aptamer can be obtained according to conventional technical means in the field. Attached Figure Description

[0043] Figure 1 (A) Zeta potential diagrams of linear peptide CPPPPRSERSERSE, (B) cyclic peptide CRSERSERSEC, and (C) Y-type peptide CPPPPEK-(RSERSERSE)2;

[0044] Figure 2An electrode modified with (A) the linear peptide CPPPPRSERSERSE, (B) the cyclic peptide CRSERSERSEC, and (C) the Y-type peptide CPPPPEK-(RSERSERSE)2 was used in 1 mg mL -1 DPV diagrams before and after incubation with bovine serum albumin solution;

[0045] Figure 3 Signal responses of GCE, AuNPs / GCE, and Pep / AuNPs / GCE in different protein solutions. Error bars represent the standard deviation of three repeated measurements;

[0046] Figure 4 Schematic diagram of contact angles for different modified electrodes: (A) GCE, (B) AuNPs / GCE, and (C) Pep / AuNPs / GCE;

[0047] Figure 5 Schematic diagram of the construction of an electrochemical antifouling aptamer sensor based on Y-peptide;

[0048] Figure 6 (A) Optimization of Y-peptide concentration, (B) Aptamer concentration, and (C) Tetracycline incubation time;

[0049] Figure 7 Electrochemical responses of different modified electrodes (GCE, AuNPs / GCE, Pep / AuNPs / GCE, Apt-Pep / AuNPs / GCE);

[0050] Figure 8 Scanning electron microscope image of AuNPs / GCE, magnification 50,000x;

[0051] Figure 9 A, B, C, and D represent GCE, AuNPs / GCE, Pep / AuNPs / GCE, and Apt-Pep / AuNPs / GCE, respectively, at concentrations of 100 ng / mL. -1 Electrochemical response before and after incubation with tetracycline;

[0052] Figure 10 (A): Electrochemical response of the sensor to different concentrations of tetracycline; (B): Curve showing the relationship between tetracycline concentration and current change. The inset shows the corresponding standard curve.

[0053] Figure 11 The sensor is effective at 100 ng / mL -1 The specificity of (a) tetracycline, (b) chlortetracycline, (c) doxycycline, (d) oxytetracycline, and (e) a mixture of the first four antibiotics was investigated.

[0054] Figure 12Sensor repeatability study: Tetracycline was detected using 7 batches of independently prepared sensors;

[0055] Figure 13 Sensor stability study: A single sensor was used to perform 7 consecutive tetracycline detections (A) and a 15-day long-term stability test (B). Detailed Implementation

[0056] The specific embodiments of the present invention will be further described below with reference to examples, and the technical solutions in the embodiments of the present invention will be clearly and completely described.

[0057] As described in the background section, zwitterionic peptide-modified surfaces can mitigate the non-specific adsorption of impurities in a matrix. Compared to basic linear peptides, surfaces modified with branched peptides exhibit greater steric hindrance, resulting in stronger antifouling properties and further improving sensor recognition performance. Therefore, this invention provides a method for constructing an electrochemical antifouling aptamer sensor based on Y-type peptides for the rapid and sensitive detection of tetracycline in food.

[0058] Differential pulse voltammetry (DPV) detection conditions: at 5 mmol / L -1 DPV was detected in a {K3[Fe(CN)6] / K4[Fe(CN)6]} solution, with a detection potential range of -0.2V to 0.6V, an amplitude of 50mV, and a scan rate of 0.1VS. -1 .

[0059] Cyclic voltammetry (CV) detection conditions: at 5 mmol / L -1 CV detection was performed in a {K3[Fe(CN)6] / K4[Fe(CN)6]} solution, with a detection potential range of -0.2V to 0.6V, an amplitude of 10mV, and a scan rate of 0.1VS. -1 .

[0060] Example 1: Design and Characterization of Zwitterionic Peptides

[0061] (1) Design of zwitterionic peptides

[0062] This invention systematically designed three zwitterionic peptides composed of alternating positively charged arginine (R), negatively charged glutamate (E), and uncharged serine (S) residues. Alternating positively charged arginine and negatively charged glutamate, followed by the insertion of uncharged hydrophilic serine, results in an overall electrically neutral and hydrophilic structure. The addition of polyproline facilitates the formation of an extended, rigid conformation of the peptide sequence. Cysteine ​​serves as an anchoring terminator, and gold-sulfur bonds are used to stably immobilize the zwitterionic peptide on an AuNPs-modified electrode. Based on this, a linear peptide, CPPPPRSERSERSE, was designed. Polyproline was removed from the linear peptide, and a cysteine ​​residue was added to the other end of the sequence, anchoring both ends to the sensing surface to form a cyclic peptide: CRSERSERSEC. Based on this, a Y-shaped branched peptide, CPPPPEK-(RSERSERSE)2, was designed. Based on this, the antifouling performance of the three zwitterionic peptides with the same antifouling sequence was compared.

[0063] (2) Characterization of zwitterionic peptides

[0064] The three peptides mentioned above (Y-type peptide, linear peptide, and cyclic peptide) were dissolved in 0.02 mol L⁻¹ water. -1 In PBS solution, solutions were prepared to a concentration of 0.3 mg / mL. -1 Three zwitterionic peptide solutions were analyzed. The charge in these solutions was determined using zeta potential. The results are as follows: Figure 1 As shown, the Zeta potentials of these three zwitterionic peptide solutions are all close to 0 mV, indicating that they are electrically neutral. This property can reduce the electrostatic interaction between the zwitterionic peptide-modified surface and the undetected substance, thereby avoiding non-specific adsorption to some extent.

[0065] Example 2: Preparation and Characterization of Antifouling Surface

[0066] (1) Electrode preparation

[0067] (a) Pretreatment of GCE: The bare GCE was polished in a circular motion on alumina powder slurry with particle sizes of 0.3 μm and 0.05 μm to remove surface impurities. Then, it was ultrasonicated in anhydrous ethanol and ultrapure water for 20 seconds to remove residual alumina powder and obtain a smooth surface.

[0068] (b) Preparation of AuNPs / GCE: A platinum electrode was used as the counter electrode, a saturated calomel electrode as the reference electrode, and a glassy carbon electrode pretreated in step (a) as the working electrode. All electrodes were immersed in a pre-prepared 5 mmol L solution. -1AuNPs / GCE were obtained by potentiostatic deposition in chloroauric acid solution. The deposition voltage was -0.5 V, the amplitude was 10 mV, and the deposition time was 60 s. This yielded a gold nanoparticle-modified electrode (AuNPs / GCE).

[0069] (c) Zwitterionic peptide modification of AuNPs / GCE: The Y-type peptide prepared in Example 1 was dissolved in 0.02 mol L... -1 0.3 mg mL of PBS was prepared. -1 A solution of Y-type peptides was prepared. The AuNPs / GCE prepared in step (b) was immersed in this solution and incubated at 4°C for 8 hours. During this time, the Y-type peptides self-assembled onto the AuNPs / GCE via gold-sulfur bonds, yielding the modified electrode (Pep / AuNPs / GCE). Modified electrodes with linear / cyclic peptides were prepared using the same method.

[0070] (2) Comparison of antifouling performance of different antifouling surfaces

[0071] 1 mg mL was prepared using PBS. -1 Bovine serum albumin (BSA) solution was used to simulate pollutants. The three modified electrodes were incubated in BSA for 30 min, and the changes in current signals before and after incubation were recorded using DPV to examine the strength of protein adsorption on the surfaces of the different modified electrodes. Figure 2 Y-peptide-modified electrodes at 1 mg / mL -1 After incubation in BSA for 30 minutes, the peak current remained almost unchanged. In a single protein solution, this electrode showed better antifouling performance compared to electrodes modified with linear peptides / cyclic peptides, so it was used for subsequent sensor fabrication.

[0072] To further verify the antifouling performance of the Y-peptide-based antifouling surface, GCE, AuNPs / GCE, and Pep / AuNPs / GCE were mixed at different concentrations (1 mg / mL). -1 2mg mL -1 5mg mL -1 The samples were incubated for 30 min in negatively charged BSA, positively charged lysozyme solution (LYS), and uncharged hemoglobin (HB). The corresponding current changes before and after incubation were recorded using DPV. The electrical signal before incubation was I0, and the electrical signal after incubation was I. The signal inhibition rate was calculated. Results are as follows: Figure 3 As shown, after incubation in different concentrations and different single-protein solutions, the signal inhibition rates of Pep / AuNPs / GCE were significantly lower than those of the bare electrode and the electrode modified only with AuNPs. Compared to GCE at 1 mg / mL... -1 The signal inhibition rate after BSA incubation was 70.36%, and the Pep / AuNPs / GCE ratio was 1 mg / mL.-1 The signal inhibition rate incubated in BSA was only 3.5%; even at high concentrations (5 mg / mL), the inhibition rate was still low. -1 In the BSA of Pep / AuNPs / GCE, the signal suppression rate was only 7.56%, indicating that Pep / AuNPs / GCE has excellent antifouling performance.

[0073] Signal Suppression (%) = (Electrical signal I0 - Electrical signal I) / Electrical signal I0 * 100%;

[0074] Finally, the water contact angle of the Y-peptide-modified antifouling surface was measured using the static drop method to examine the hydrophilicity of the electrode surface modified with this peptide and to explore the antifouling mechanism of the Y-peptide-based antifouling surface. The results are as follows: Figure 4 As shown, the contact angle of the bare GCE surface is 60.6°; after modification with AuNPs, the contact angle increases to 69.2°, indicating that the electrodeposited AuNPs enhance the surface's hydrophobicity; after modification with a Y-peptide, the contact angle significantly decreases to 27.7°, indicating that the peptide has good hydrophilicity. This may be the reason why the antifouling surface based on the Y-peptide exhibits excellent antifouling performance.

[0075] Example 3: Preparation of an electrochemical antifouling aptamer sensor

[0076] (1) Sensor fabrication

[0077] (a) AuNPs / GCE were prepared according to Example 2.

[0078] (b) Aptamer and Y-peptide modification of AuNPs / GCE: AuNPs / GCE were immersed in a solution of Y-peptide and aptamer of a certain concentration and incubated at 4°C for 8 hours. The Y-peptide and aptamer self-assembled onto the electrode via gold-sulfur bonds, and then the surface was rinsed with ultrapure water to remove unbound components, completing the preparation of the electrochemical antifouling aptamer sensor (Apt-Pep / AuNPs / GCE). The construction process is as follows: Figure 5 aptamer sequence:

[0079] 5'-SH-(CH2)6-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTGCGCGTGGATCCGAGCTCCACGTG-3'.

[0080] (2) Concentration optimization

[0081] (a) Optimization of Y-peptide concentration

[0082] To ensure the constructed electrochemical antifouling aptamer sensor exhibits good antifouling performance, the effect of Y-peptide concentration on the antifouling properties of the electrode surface was investigated. The concentration of the Y-peptide was set to 0.1 mg / mL. -1 0.2 mg mL -1 0.3 mg mL -1 0.4 mg mL -1 0.5 mg mL -1 A series of electrochemical antifouling aptamer sensors were prepared using the method described above. These electrochemical antifouling aptamer sensors were then tested at 1 mg / mL. -1 Incubate in BSA for 30 min. The electrical signal before incubation is recorded as I0, and the electrical signal after incubation is recorded as I. The signal inhibition rate is calculated. The results are as follows: Figure 6 As shown in (A), with the increase of Y-type peptide concentration, at 0.3 mg / mL... -1 The suppression rate of the current signal was 4.6%. 0.4–0.5 mg / mL -1 The suppression rate of the current signal was related to 0.3 mg / mL. -1 It is quite effective at resisting staining.

[0083] (b) Optimization of aptamer concentration

[0084] To ensure good detection performance of the constructed electrochemical antifouling aptamer sensor, the effect of aptamer concentration on tetracycline detection was investigated. The aptamer concentration was set to 1 μmol / L. -1 2 μmol L -1 3 μmol L -1 4 μmol L -1 5 μmol L -1 A series of electrochemical antifouling aptamer sensors were prepared using the method described above. These electrochemical antifouling aptamer sensors were then tested in 100 ng / mL... -1 The sample was incubated in a tetracycline standard solution for 30 minutes, and the signal inhibition rate was calculated. The results are as follows: Figure 6 As shown in (B), when the aptamer concentration is 3 μmol L... -1 At that time, it can be clearly seen that the signal inhibition rate of DPV tends to stabilize before and after incubation.

[0085] Based on the above, AuNPs / GCE were soaked in 0.3 mg / mL solution at 4°C. -1 Y-type peptide and 3 μmol L -1 The aptamer was incubated in a mixed solution for 8 hours, and then the surface was gently rinsed with ultrapure water to complete the preparation of the entire electrochemical antifouling aptamer sensor (Apt-Pep / AuNPs / GCE).

[0086] (3) Characterization of the sensing surface

[0087] (a) DPV characterization

[0088] To demonstrate the successful construction of this electrochemical antifouling aptamer sensor, the change in current signal during the stepwise modification of the DPV detection electrode was used.

[0089] like Figure 7 As shown, the peak current significantly increases when AuNPs are modified onto the GCE surface. This change is attributed to the excellent conductivity of AuNPs, proving their successful modification onto the GCE surface. After the Y-peptide and aptamer are self-assembled onto AuNPs / GCE, the peak current decreases significantly. This is because the poor conductivity of the Y-peptide and aptamer hinders electron transfer. When the aptamer specifically binds to tetracycline, the conductivity of the electrode surface further decreases, thus further reducing the peak current. The changes in the DPV response current values ​​of the above-mentioned modified electrodes demonstrate the successful construction of this electrochemical antifouling aptamer sensor.

[0090] (b) Scanning electron microscopy (SEM) characterization

[0091] The surface morphology of AuNPs / GCE was characterized using SEM. Figure 8 As shown, AuNPs can be observed to be uniformly distributed on the surface of GCE under a 50,000x microscope, proving that AuNPs were successfully modified on the surface of GCE.

[0092] Example 4: Detection of Tetracycline

[0093] (1) Feasibility test of tetracycline detection

[0094] Several electrodes without aptamers (GCE, AuNPs / GCE, Pep / AuNPs / GCE) and an electrode modified with aptamers (Apt-Pep / AuNPs / GCE) were respectively tested at 100 ng / mL. -1 The electrodes were incubated in tetracycline for 60 min, thoroughly rinsed with ultrapure water, and the current changes of each electrode before and after incubation were recorded using DPV. The results are as follows: Figure 9 As shown, the peak current of the DPV curves for several electrodes without aptamers showed almost no change before and after tetracycline incubation, indicating that tetracycline was not captured. Only the electrode modified with the aptamer showed a significant change in peak current after incubation. This is because tetracycline binds to the aptamer, hindering electron transfer. This result demonstrates that the aptamer can sensitively recognize tetracycline.

[0095] (2) Optimization of tetracycline incubation time

[0096] The electrochemical antifouling aptamer sensor prepared in Example 3 was immersed in 100 ng / mL water. -1In tetracycline, the cells were incubated for 20 min, 40 min, 60 min, 80 min, and 100 min, respectively. The changes in DPV current before and after incubation were compared, and the signal inhibition rate was calculated. The results are as follows: Figure 6 As shown in (C), when the incubation time is less than 60 min, the signal inhibition rate gradually increases with the increase of the incubation time, and tends to stabilize at 60 min.

[0097] (3) Establishment of a tetracycline electrochemical antifouling aptamer sensor detection method

[0098] The electrochemical antifouling aptamer sensor prepared in Example 3 was tested in a series of tetracycline solutions with concentration gradients (from 0.01 to 100 ng / mL). -1 The electrochemical antifouling aptamer sensor was incubated in the solution for 60 min, and the response signals to different concentrations of tetracycline were recorded. The results are as follows: Figure 10 As shown, the linear fitting function of the sensor can be obtained as ΔI(μA)=2.90435lg(C)+8.91302(R). 2 =0.997), ΔI refers to the current signal before incubation minus the current signal after incubation (I0-I), and C refers to the concentration of the tetracycline solution (ng / mL). -1 The calculated limit of detection (LOD) for tetracycline by this sensor is 0.0053 ng / mL. -1 (S / N=3) This method is expected to enable ultrasensitive detection of tetracycline in complex food matrices.

[0099] (4) Specific detection

[0100] The electrochemical antifouling aptamer sensor prepared in Example 3 was used to detect tetracycline, three other tetracycline analogs (chlortetracycline, doxycycline, and oxytetracycline), and a mixed solution containing these four antibiotics to verify the sensor's specificity. The concentration of each antibiotic was 100 ng / mL. -1 The specificity of the sensor was evaluated by recording the current change of the sensor when detecting tetracycline analogs using DPV and then calculating the signal suppression rate. The results are as follows: Figure 11 As shown, the signal inhibition rates were very low when detecting chlortetracycline, doxycycline, and oxytetracycline, at 9.61%, 5.56%, and 7.16%, respectively. However, when detecting tetracycline solution, the signal inhibition rate was 51.67%, and the current changed significantly, indicating that the sensor has excellent selectivity for tetracycline detection. Detection of a mixed solution containing these four antibiotics showed a signal inhibition rate of 47.45%, which was not significantly different from the signal inhibition rate of tetracycline solution. This indicates that even in the presence of interfering substances, the sensor can still specifically bind to tetracycline, demonstrating the good specificity of this electrochemical antifouling aptamer sensor.

[0101] (5) Repeatability test

[0102] According to Example 3, seven identical batches of electrochemical antifouling aptamer sensors were constructed under the same experimental conditions. Each batch contained three electrochemical antifouling aptamers, and their measurements at 100 ng / mL were recorded under the same conditions. -1 DPV current response value after tetracycline solution. For example... Figure 12 As shown, the RSD of these 7 batches of sensors was 3.20%, indicating that the electrochemical antifouling aptamer sensor based on Y-peptide has good repeatability in detecting tetracycline.

[0103] (6) Stability testing

[0104] In 100ng mL -1 In a tetracycline solution, the electrochemical antifouling aptamer sensor prepared in Example 3 was used to record the DPV current response value after seven consecutive stability tests under the same conditions. Figure 13 As shown in (A), after seven consecutive DPV tests, the current values ​​showed good consistency, with an RSD of 2.58%. To further investigate the long-term storage stability of the electrochemical antifouling aptamer sensor prepared in Example 3, it was stored at 4°C after preparation, and then 100 ng / mL was measured every three days under the same conditions. -1 A tetracycline solution was used, and the DPV current response value was recorded after detection. Figure 13 As shown in (B), even after 15 days, the DPV current response value remained relatively stable. These experimental results demonstrate that the constructed sensor exhibits excellent continuous testing stability and long-term storage stability.

[0105] Example 5: Detection of tetracycline in milk

[0106] Milk was selected as the sample to be tested, and the spiked recovery experiment was used to verify the practical application performance of the constructed sensor in the sample.

[0107] Use 0.02 mol L -1 The milk sample was diluted 100-fold with PBS, and three different concentrations (5 ng / mL) were added to the diluted milk sample in equal amounts. -1 25ng mL -1 and 50 ng mL -1The tetracycline standard solution was used, and then the electrochemical antifouling aptamer sensor prepared in Example 3 was used for detection to obtain the spiked recovery rate in the milk sample. The same sample was analyzed by high-performance liquid chromatography (HPLC) to verify the results obtained by the constructed sensor and ensure the accuracy of the analysis. The results are shown in Table 1. The recovery rate obtained by the sensor was 91.0%–103.1%, and the RSD was less than 3.9%, which is consistent with the results obtained by HPLC. This indicates that the method has good detection accuracy, and the newly designed electrochemical antifouling aptamer sensor based on Y-peptide modification has broad application prospects in food matrices.

[0108] Table 1. Results of Spike Recovery in Milk Samples

[0109]

[0110] Based on the analysis of the above experimental results, it can be seen that the preparation method described in this invention has the advantages of excellent antifouling performance, simple preparation process, and strong practicality.

[0111] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An anti-fouling Y peptide, characterized in that, The anchor end is cysteine, the connecting end is polyproline and two amino acid residues, and the anti-fouling sequence comprises a zwitterionic peptide; The anchor end comprises one or more cysteines; The polyproline is PPPP; The two amino acid residues comprise glutamic acid and lysine; The zwitterionic peptide is RSERSERSE; The anchor end and the connecting end form a main chain of a Y-shaped peptide, and the two anti-fouling sequences are two branches of the Y-shaped peptide.

2. An electrochemical antifouling sensing surface, characterized in that, The electrochemical anti-fouling sensing surface is an electrode modified with the Y-shaped peptide of claim 1.

3. The electrochemical antifouling sensing surface according to claim 2, wherein, The electrode is a glassy carbon electrode.

4. An electrochemical anti-fouling aptamer sensor, characterized in that, The electrochemical anti-fouling sensor is modified with the anti-fouling Y-shaped peptide of claim 1 and an aptamer corresponding to a target to be detected.

5. The electrochemical antifouling aptamer sensor of claim 4, wherein, The target to be detected comprises a small molecule compound.

6. The electrochemical antifouling aptamer sensor of claim 5, wherein, The small molecule compound comprises tetracycline.

7. The electrochemical antifouling aptamer sensor according to any one of claims 4-6, wherein, The 5' end of the aptamer is modified by thiol and methylene, and the sequence of the aptamer is: 5'-SH-(CH2)6-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTGCGCGTGGATCCGAGCTCCACGTG-3'.

8. A method of detecting tetracycline, characterized by, The method is detection by using the electrochemical anti-fouling aptamer sensor of claim 6 or 7.

9. The method of claim 8, wherein, The specific steps of the method are: (1) The electrochemical anti-fouling aptamer sensor of claim 6 or 7 is incubated in tetracycline standard solution of different concentrations for more than 60 min, and the current response value is recorded by differential pulse voltammetry, and a standard curve is drawn; (2) The electrochemical anti-fouling aptamer sensor of claim 6 or 7 is incubated in a sample solution added with tetracycline of a specific concentration for more than 60 min, and the current response value is determined by differential pulse voltammetry, and the standard recovery rate is calculated according to the standard curve drawn in step (1).

10. The anti-fouling Y-shaped peptide of claim 1, or the electrochemical anti-fouling sensing surface of claim 2, or the electrochemical anti-fouling aptamer sensor of any one of claims 4-7 is applied in the field of biological detection.

11. Use according to claim 10, characterized in that, The field of biological detection comprises the field of food detection and the field of environmental detection.

12. The anti-fouling Y-shaped peptide of claim 1, or the electrochemical anti-fouling sensing surface of claim 2, or the electrochemical anti-fouling aptamer sensor of any one of claims 4-7 is applied in the detection of small molecule compounds.

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