A method for detecting cocaine by electrochemical sensing

Abstract

The present invention discloses a method for electrochemical sensing detection of cocaine. The method modifies amino groups on the surface of a bare ITO electrode, and assembles a cocaine aptamer fragment 1 on the ITO electrode through an amino cross-linking agent. Since the end of the cocaine aptamer fragment 2 is modified with a ferrocene group, when cocaine is present, an Apt1-cocaine-Apt2 complex is formed on the electrode surface, Fc is introduced into the ITO electrode surface, and then through the host-guest recognition effect between CD-AgNPs and Fc, CD-AgNPs are anchored on the ITO electrode as an electrical signal label, thereby causing a large number of silver nanoparticles to be loaded on the electrode surface. Finally, a differential voltammetric pulse method is used to detect the electrical signal of the silver nanoparticles. The present invention is based on the AgNPs labeling strategy of the host-guest effect, which can achieve rapid and sensitive detection of cocaine.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical sensors and relates to an electrochemical sensing detection method for cocaine. Background Art

[0002] Cocaine, a common addictive stimulant that can cause severe addiction, is one of the most dangerous and illicitly abused drugs today. Long-term abuse of this narcotic can lead to adverse effects on the body, such as cardiovascular complications, pupil dilation, vasoconstriction, and multiple aneurysms. Therefore, developing methods for detecting cocaine is of great significance in forensic medicine, customs, and clinical medicine. Previous studies have primarily used specialized precision instruments such as mass spectrometers, gas chromatography, high-performance liquid chromatography, or enzyme-linked immunosorbent assay (ELISA) kits to analyze cocaine in samples. While these methods offer high accuracy, they are expensive and time-consuming to process.

[0003] Electrochemical sensors have been shown to be highly practical in detection due to their low cost, small size, high sensitivity and specificity, and fast detection speed. Electrochemically active labels are extremely important in cocaine detection. Ferrocene and methylene blue are early examples of such labels, and these molecules are covalently linked to capture molecules. Li et al. (Li X, Qi H, Shen L, et al. Electrochemical aptasensor for the determination of cocaine incorporating gold nanoparticles modification [J]. Electroanalysis, 2008, 20 (13): 1475-1482.) used ferrocene as an electrochemical label and a gold electrode modified with nanogold as a substrate to design an electrochemical aptasensor for the determination of cocaine. The ends of the cocaine aptamer were modified with the electroactive label ferrocene and a thiol group (thiol is used to self-assemble the aptamer onto the surface of the gold nanoparticles). The aptamer modified on the electrode folds in the presence of cocaine, bringing the ferrocene group closer to the electrode surface and changing the electrochemical signal of the sensor. The sensor uses a gold electrode assembled with gold nanoparticles, which provides a good platform for fixing the aptamer and improving sensitivity, achieving a detection limit of 0.5mM (approximately 0.15mg / mL). This form of labeling usually exists in a 1:1 ratio with the analyte, limiting their applicability in actual diagnosis. Because AgNPs have good biocompatibility, excellent stability and low toxicity, and through DPV electrochemical analysis, thousands of Ag can be detected. +Ions are released from each Ag nanoparticle tag. AgNPs have shown promise as labels for conductive sensitive detection. The preparation of nanoparticles typically involves reducing metal ions in a high-temperature environment, and most synthesis methods rely on organic solvents and toxic reducing agents. AgNPs synthesized in this way have potential health and environmental risks, and the synthesis conditions are harsh. In most synthetic systems, the surface of AgNPs is not passivated or protected, and the high surface energy of AgNPs makes them highly reactive, easily causing them to aggregate, affecting the reaction process. Summary of the Invention

[0004] The object of the present invention is to provide a method for detecting cocaine by electrochemical sensing.

[0005] The technical solutions for achieving the purpose of the present invention are as follows:

[0006] A method for detecting cocaine by electrochemical sensing comprises the following steps:

[0007] Step 1, preparing CD-AgNPs electroactive labels: After stirring and mixing a β-cyclodextrin (β-CD) solution and a silver nitrate solution, a sodium hydroxide solution is added and stirred for reaction. After the reaction is completed, the mixture is centrifuged and washed, and the resulting β-CD-coated silver nanoparticles (CD-AgNPs) are redissolved in water to form a CD-AgNPs solution;

[0008] Step 2, assembly of a cocaine electrochemical sensor: immersing a clean ITO electrode in a sodium hydroxide solution, reacting at room temperature to obtain a hydroxyl-activated ITO electrode, then immersing the ITO electrode in a (3-aminopropyl)triethoxysilane (APTES) solution, reacting at room temperature, rinsing and drying after the reaction, immersing the dried ITO electrode in a p-phenylene diisothiocyanate (PDITC) solution, reacting at room temperature, and washing with N,N-dimethylformamide (DMF) after the reaction, finally adding a PBS solution of aptamer fragment 1 (Apt1) dropwise onto the electrode surface and incubating at 37° C. to obtain a sensor for electrochemical detection of cocaine, wherein the APTES solution is composed of a mixture of APTES, ultrapure water, and anhydrous ethanol in a volume ratio of 1:1:98, and the PDITC solution is composed of PDITC, pyridine, and DMF, with the volume ratio of pyridine to DMF being 1:9;

[0009] Step 3, electrochemical detection of cocaine: A mixed solution of the cocaine to be tested and the ferrocene-modified aptamer fragment 2 (Fc-Apt2) in phosphate buffer saline (PBS) was added dropwise to the reaction area of the ITO electrode of the sensor prepared in step 2, and the reaction was carried out at 37° C. for 2 hours. The ITO electrode was then immersed in a CD-AgNPs solution at room temperature for host-guest reaction. Finally, a DPV test of the modified electrode was performed in 1M KCl solution with a scanning potential of -0.2 to 0.2 V. Ag / AgCl was used as a reference electrode. The concentration of the cocaine to be tested was calculated based on the linear relationship between the electrochemical signal and the logarithm of the cocaine concentration.

[0010] Preferably, in step 1, the molar ratio of β-CD to silver nitrate is 1:1; the stirring reaction time is more than 12 hours; the centrifugal speed is 8000 rpm, the centrifugal time is 10 minutes, and the number of centrifugal washings is more than 3 times.

[0011] Preferably, in step 2, the reaction time in the APTES solution is more than 4 hours; the drying temperature is 110°C, and the drying time is more than 30 minutes; the concentration of PDITC in the PDITC solution is 80 mM, and the reaction time in the PDITC solution is more than 2 hours; the incubation time at 37°C is more than 2 hours; and the concentration of Apt1 in the PBS solution of Apt1 is 1 μM.

[0012] Preferably, in step 3, the host-guest reaction time is 2.5 h.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] This invention uses a single-step synthesis of natural β-cyclodextrin-coated silver nanoparticles as an electroactive marker, with β-cyclodextrin acting as a reducing agent and surface stabilizer for the synthesis of AgNPs. β-cyclodextrin recognizes the Fc group on cocaine Apt2 through a simple and effective host-guest assembly strategy, and binds to cocaine via a two-stage aptamer recognition mechanism on an ITO electrode. The sensor exhibits a good linear relationship between the electrochemical signal and the logarithm of cocaine concentration within a cocaine concentration range of 1-100 μg / mL, achieving a detection limit of 0.53 μg / mL. It can detect cocaine in diluted normal human serum (NHS) and exhibits a certain degree of anti-interference capability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the method for detecting cocaine by electrochemical sensing according to the present invention.

[0016] Figure 2 This is the optimization curve of the host-guest reaction time between CD-AgNPs and Fc-Apt2.

[0017] Figure 3The differential pulse voltammograms of the ITO electrode surface lacking different components in 1 M KCl.

[0018] Figure 4 For [Fe(CN)6] 3- / 4- Nyquist plots of different surface-modified electrodes in electrolyte, (a) bare electrode, (b) ITO-OH, (c) ITO-OH-APTES, (d) ITO-OH-APTES-PDITC, (e) ITO-OH-APTES-PDITC-Apt1, (f) ITO-OH-APTES-PDITC-Apt1-Cocaine-Apt2 and (g) ITO-OH-APTES-PDITC-Apt1-Cocaine-Apt2-AgNPs.

[0019] Figure 5 Differential pulse voltammograms of cocaine in 1 M KCl at concentrations ranging from 1 to 100 μg / mL.

[0020] Figure 6 The graph shows the relationship between the peak value of the oxidation current and the logarithm of the cocaine concentration.

[0021] Figure 7 The bar graph shows the DPV analysis results of 100 μg / mL, 10 μg / mL, and 1 μg / mL cocaine in 0.1 M PBS and 10% NHS, respectively. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0023] Apt1 and Fc-Apt2 were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence of Apt1 is 5'-GGGAGTCAAGAACGAA-(CH2)6-NH2-3' (SEQ ID NO. 1); the sequence of Fc-Apt2 is 5'-TTCGTTCTTCAATGAAGTGGGACGACA-Fc-3' (SEQ ID NO. 2).

[0024] Preparation of Apt1 solution: 1 OD of purchased Apt1 was diluted to 1 μM with 5.74 mL of PBS (10 mM, pH=7.4).

[0025] Preparation of 10 μM Fc-Apt2 solution: 1 OD of purchased Fc-Apt2 was diluted to 10 μM with 360 μL of PBS (10 mM, pH = 7.4).

[0026] Preparation of a mixed solution of cocaine and Fc-Apt2: 10 μL of 10× cocaine acetonitrile solution, 20 μL of Fc-Apt2 solution (10 μM), and 170 μL of PBS solution (10 mM, pH = 7.4) were added to make a 200 μL mixed solution of cocaine and Fc-Apt2.

[0027] Example 1

[0028] (1) First, prepare the CD-AgNPs electroactive label: add 4 mL of β-CD solution (5 mM) to 4 mL of silver nitrate aqueous solution (5 mM) and stir at room temperature for 60 seconds. Then stir the mixture with 8 mL of sodium hydroxide aqueous solution (20 mM) for 12 hours. Then centrifuge the mixture at 8000 rpm for 10 minutes, discard the supernatant, add 16 mL of water, and continue centrifuging at 8000 rpm for 10 minutes. Then discard the supernatant and add water... After centrifugation and washing the precipitate three times, dissolve the precipitated CD-AgNPs in 8 mL of water. Store the CD-AgNPs solution at 4°C until use.

[0029] (2) Assembly of cocaine electrochemical sensor: The ITO electrode was ultrasonically cleaned in ultrapure water for 30 min, then ultrasonically cleaned in acetone solvent for 30 min, and so on. The ITO electrode was sequentially soaked in ethanol, hydrogen peroxide and ultrapure water using an ultrasonic cleaner for 30 min each. The ITO electrode was then immersed in a 1M sodium hydroxide solution and reacted at room temperature for 10 hours to obtain a hydroxyl-activated ITO electrode. APTES, ultrapure water and anhydrous ethanol were then mixed in a volume ratio of 1:1:98, and the ITO electrode was immersed in the APTES solution and reacted at room temperature for 4 hours. The rinsed ITO electrode was then placed in a 110°C oven for 30 minutes. The ITO electrode was then immersed in an 80mM PDITC solution containing 2mL pyridine and 18mL N,N-dimethylformamide (DMF) at room temperature for 2 hours. The modified electrode was washed with DMF, and then 20μL of Apt1 solution (1μM) was added to the electrode surface and incubated at 37°C for 2 hours.

[0030] (3) Electrochemical detection of cocaine: A 20 μL mixture of cocaine and Fc-Apt2 (1 μM) was added dropwise to the reaction zone of the ITO electrode and allowed to react at 37°C for 2 h. The ITO electrode was then immersed in a 200 μL CD-AgNPs solution for a 2.5 h host-guest reaction at room temperature. Finally, a DPV test of the modified electrode was performed in a 1 M KCl solution, scanning the potential from -0.2 to 0.2 V (using Ag / AgCl as the reference electrode).

[0031] Example 2

[0032] Effect of host-guest reaction time between CD-AgNPs and Fc-Apt2 on current signal:

[0033] The construction process shows that the host-guest reaction time between CD-AgNPs and Fc immobilized on the electrode surface is closely related to the electrical signal intensity. To optimize analytical performance, the changes in the measured current signal were recorded after 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h of host-guest reaction. Figure 2 The results show that the current signal shows a relatively stable platform after 2.5 hours, so 2.5 hours is selected as the optimal host-guest reaction time.

[0034] Example 3

[0035] Sensor feasibility study

[0036] Figure 3 The current of AgNPs on different modified electrode surfaces was measured by DPV. It can be seen that no obvious electrochemical signal was observed in any of the cases without the addition of Apt1 (curve b), Apt2 (curve d), and CD-AgNPs (curve e). A small current signal appeared in the absence of cocaine (curve c). This is because in the absence of the target, there is a very small probability that the two aptamer fragments will directly bind through base complementary pairing. However, when they are all modified to the electrode surface in sequence (curve a), compared with the signals of other missing components, it can be observed that Ag is electrochemically oxidized to Ag. + There is an obvious oxidation current peak, and its potential is about 0.04V.

[0037] Figure 4 The electrochemical characterization of the gradually modified ITO electrode surface was carried out by electrochemical impedance spectroscopy (EIS). The specific operation is that each time a component is modified on the ITO electrode, the electrode is immersed in a potassium ferricyanide solution. The semicircular diameter of the high-frequency region in the EIS corresponds to the charge transfer resistance (Rct) of the electrode surface. Figure 4 The Rct values of the various modified electrode surfaces can be seen. Because electron transfer between the electrode surface and the electrolyte is hindered by the molecular layer gradually deposited on the electrode, the Rct values gradually increase (curves a, b, c, d, e, and f). However, after silver nanoparticles were immobilized on the electrode surface, the Rct value (12.41Ω) was observed to decrease (curve g). This is because the introduction of AgNPs on the electrode surface facilitates electron transfer between the medium and the electrode. Figure 4 Each component is shown connected to the electrode surface as described above.

[0038] Example 4

[0039] PBS solutions containing different concentrations of cocaine (1 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL) were added dropwise to the ITO electrode surface of the sensor constructed in Example 1, and DPV patterns were obtained.

[0040] Figure 5 The following are differential pulse voltammograms for cocaine concentrations ranging from 1 to 100 μg / mL in 1 M KCl. The oxidation potential of silver nanoparticles in KCl solution is approximately 0.04 V (vs. Ag / AgCl), reflecting the electrochemical conversion of silver nanoparticles to silver ions. A distinct oxidation peak is observed at approximately 0.04 V, and the current intensity increases linearly with increasing cocaine concentration.

[0041] Figure 6 The figure shows the fitting curve of the electrochemical signal of the ITO electrode and the logarithm of the cocaine concentration in the cocaine concentration range of 1-100 μg / mL. It can be seen that the fitting linear regression equation of the oxidation current signal and the logarithm of the cocaine concentration is: I (μA) = 1.65 + 3.681 g [C cocaine / (μg / mL)], with a correlation coefficient of 0.9989. The detection limit (LOD) of the sensor is 0.53 μg / mL.

[0042] Example 5

[0043] Detection of different concentrations of cocaine in 10% NHS:

[0044] The concentrations of cocaine in the diluted serum samples were 100 μg / mL, 10 μg / mL, and 1 μg / mL, respectively. The oxidation peak currents at 0.4 V were 84.6%, 86.1%, and 97.2% of the oxidation peak currents in the PBS group, respectively. Figure 7 The detection effect of cocaine in 10% NHS shows the anti-interference ability of the electrochemical biosensor in complex human serum, which has great application potential in clinical applications.

Claims

1. A method for detecting cocaine by electrochemical sensing, characterized in that: The following steps are involved: Step 1, preparing CD-AgNPs electroactive labels: After stirring and mixing a β-cyclodextrin solution and a silver nitrate solution, a sodium hydroxide solution is added and stirred for reaction. After the reaction is completed, the mixture is centrifuged and washed, and the obtained β-cyclodextrin-coated silver nanoparticles are redissolved in water to form a CD-AgNPs solution; Step 2, assembly of a cocaine electrochemical sensor: immersing a clean ITO electrode in a sodium hydroxide solution, reacting at room temperature to obtain a hydroxyl-activated ITO electrode, then immersing the ITO electrode in an APTES solution, reacting at room temperature, rinsing and drying after the reaction, immersing the dried ITO electrode in a PDITC solution, reacting at room temperature, and rinsing with DMF after the reaction, and finally adding a PBS solution of Apt1 dropwise to the electrode surface and incubating at 37°C to obtain a sensor for electrochemical detection of cocaine, wherein the APTES solution is composed of a mixture of APTES, ultrapure water, and anhydrous ethanol in a volume ratio of 1:1:98, and the PDITC solution is composed of PDITC, pyridine, and DMF, with the volume ratio of pyridine to DMF being 1:9; Step 3, electrochemical detection of cocaine: A mixed PBS solution of cocaine and Fc-Apt2 was added dropwise to the reaction area of the ITO electrode of the sensor prepared in step 2, and the reaction was carried out at 37°C for 2 hours. The ITO electrode was then immersed in a CD-AgNPs solution at room temperature for host-guest reaction. Finally, the modified electrode was subjected to a DPV test in a 1M KCl solution with a scanning potential of -0.2 to 0.2 V. Ag / AgCl was used as a reference electrode. The concentration of the cocaine to be measured was calculated based on the linear relationship between the electrochemical signal and the logarithm of the cocaine concentration.

2. The electrochemical sensing detection method according to claim 1, characterized in that: In step 1, the molar ratio of β-CD to silver nitrate is 1:

1.

3. The electrochemical sensing detection method according to claim 1, characterized in that: In step 1, the stirring reaction time is more than 12 hours.

4. The electrochemical sensing detection method according to claim 1, characterized in that: In step 1, the centrifugal speed is 8000 rpm, the centrifugal time is 10 min, and the number of centrifugal washings is more than 3 times.

5. The electrochemical sensing detection method according to claim 1, characterized in that: In step 2, the reaction time in the APTES solution is more than 4 hours.

6. The electrochemical sensing detection method according to claim 1, characterized in that: In step 2, the drying temperature is 110° C. and the drying time is more than 30 minutes.

7. The electrochemical sensing detection method according to claim 1, characterized in that: In step 2, the concentration of PDITC in the PDITC solution is 80 mM, and the reaction time in the PDITC solution is more than 2 hours.

8. The electrochemical sensing detection method according to claim 1, characterized in that: In step 2, the incubation time at 37°C is more than 2 hours.

9. The electrochemical sensing detection method according to claim 1, characterized in that: In step 2, the concentration of Apt1 in the Apt1 PBS solution is 1 μM.

10. The electrochemical sensing detection method according to claim 1, characterized in that: In step 3, the host-guest reaction time is 2.5 h.

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