Method for electrochemically detecting chloramphenicol by using molecular imprinting

Abstract

The invention relates to a method for electrochemically detecting chloramphenicol by using molecular imprinting. The method is characterized in that a chloramphenicol molecularly imprinted polymer prepared from a temperature-sensitive monomer N-isopropylacrylamide and Dawson type tungsten phosphate (P2W18) are jointly modified on a substrate electrode through a simple and controllable electrostatic layer-by-layer self-assembly method. The modified electrode synthesized by the method disclosed by the invention is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a platinum electrode is used as an auxiliary electrode, and the electrochemical sensing performance of the modified electrode on chloramphenicol is respectively evaluated by cyclic voltammetry, differential pulse voltammetry and square wave voltammetry. According to the modified electrode, the sensitivity of an electrochemical signal is synergistically enhanced through the high-selectivity imprinting cavity of the molecularly imprinted polymer and the rapid and reversible oxidation-reduction activity of P2W18, the detection linear range of chloramphenicol is 0.1 nM to 10 [mu] M, and the detection limit of differential pulse voltammetry is as low as 0.145 nM; and a good application prospect is provided for preparing a uniform and controllable molecularly imprinted polymer composite modified electrode material in the future. At present, an indissolvable molecularly imprinted polymer material is integrated into an electrochemical sensor through an electrostatic layer-by-layer self-assembly technology, so that the research on improving the selectivity of chloramphenicol detection and reducing the detection limit is rarely reported.

Description

Technical fields:

[0001] This invention belongs to the field of electrochemical detection technology, specifically involving the use of electrostatic layer-by-layer self-assembly to synthesize a molecularly imprinted polymer (MIP) from a temperature-sensitive monomer and a Dawson-type phosphotungstic acid (P2W). 18 It was synergistically modified onto the electrode and used for the electrochemical detection of trace amounts of chloramphenicol. Background technology:

[0002] Chloramphenicol (CAP) is a broad-spectrum antibiotic that easily accumulates in animals and enters the human body through the food chain, leading to health risks such as aplastic anemia and liver damage. Its use in food-producing animals has been banned in my country and the European Union. Developing efficient and sensitive chloramphenicol detection technologies is crucial for ensuring food safety and public health. Current detection technologies, such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), can achieve accurate detection, but they suffer from problems such as expensive instruments, complex operation, and long processing times, making them unsuitable for rapid on-site screening. Electrochemical sensors are considered the most promising alternative technology due to their low cost and fast response; however, traditional electrode materials lack sufficient sensitivity and have poor anti-interference capabilities, making it impossible to detect trace amounts of chloramphenicol in complex matrices. In recent years, molecularly imprinted polymers have been used as recognition elements due to their specific recognition functions for highly selective adsorption and recognition of substances. However, their poor solubility prevents electrode modification through electrostatic layer-by-layer self-assembly technology, severely limiting their widespread application in the field of electrochemical sensors. Therefore, various intelligent strategies can be used to optimize the application effect of molecularly imprinted polymers (MIPs), such as reducing the crosslinking density of the polymer, selecting monomers with stronger affinity for template molecules, and converting MIPs into hydrochloride salts to improve solubility in non-aqueous solvents. These strategies can enable MIPs to be applied more effectively in electrochemical sensors. To obtain optimal sensor performance, existing technologies attempt to combine polyanions with high charge transfer rates with MIPs during assembly to synergistically improve the sensitivity and selectivity of the sensing platform. However, while polyacids are widely used in electrochemical sensors due to their rapid and highly reversible redox activity, their detection sensitivity for low-concentration substances is very low, making it difficult to reach trace detection limits. Therefore, polyacids can be synergistically combined with MIPs through electrostatic layer-by-layer self-assembly to form a uniform and controllable composite modified electrode, thereby improving the sensor's detection performance. Based on the above considerations, this invention modifies electrodes by combining molecularly imprinted polymers with polyacids through electrostatic layer-by-layer self-assembly, thereby improving their selectivity and sensitivity. This not only enables the detection of trace chloramphenicol but also provides a new approach for the design of next-generation electrochemical sensors to solve various analytical challenges in environmental monitoring and food safety. Summary of the Invention:

[0003] This invention utilizes an electrostatic layer-by-layer self-assembly method to synthesize MIPs prepared from temperature-sensitive monomers and Dawson-type phosphotungstates (P2W). 18 The method involves co-modifying the electrode with MIP and then electrochemically detecting chloramphenicol. This approach aims to address the aforementioned issues of MIP modification and template elution, as well as the shortcomings of insufficient electrode selectivity and sensitivity, ultimately achieving precise trace-level detection of chloramphenicol.

[0004] The present invention achieves the above objectives through the following technical solutions:

[0005] Build [PSS / PAH / (P2W)] 18 / MIP) n The composite modified electrode, wherein the substrate is selected from quartz, silicon wafer, CaF2 and indium tin oxide glass (ITO), and ITO (effective area 2cm²) is selected. 2 The substrate is a working electrode; the bottom layer is a polyelectrolyte layer [polystyrene sulfonate (PSS) / polyallylamine hydrochloride (PAH)] used to regulate the uniformity of charge distribution on the substrate surface; the functional layer is an alternating layer of P2W. 18 (Dawson-type tungsten phosphate, negatively charged) and chloramphenicol MIP (positively charged after protonation, soluble in DMF); the number of layers n is a positive integer from 1 to 30, with n=30 preferred to balance membrane stability and detection performance. When n<10, the composite membrane thickness is insufficient, resulting in a weak electrochemical signal.

[0006] Furthermore, the preparation of key materials:

[0007] Preparation of chloramphenicol MIP: Using chloramphenicol (CAP) as the template molecule, N-isopropylacrylamide (NIPAM, a thermosensitive monomer with a minimum critical solution temperature of 32-35℃) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinking agent, and azobisisobutyronitrile (AIBN) as the initiator, it was prepared by in-situ polymerization: Polymerization system: 0.1g CAP and 0.2g NIPAM were dissolved in 670μL tetrahydrofuran (THF), 125μL EGDMA and 0.05g AIBN were added, and polymerization was carried out in a constant temperature water bath at 65℃ for 24h;

[0008] Template elution: The polymer was ground into a 200-mesh powder and extracted with methanol / 36% acetic acid (9:1, v / v) by Soxhlet extraction for 48 h (the eluent was changed every 12 h). After no CAP residue was detected at 275 nm by UV monitoring, the polymer was washed with methanol and deionized water and dried under vacuum at 60 °C for 2 h.

[0009] Protonation modification: Take 0.002g of MIP powder, protonate it with 2mL of concentrated hydrochloric acid, add 10mL of LDMF and sonicate for 30min, then add 10mL of HCl solution with pH=2 to prepare a 1×10-3M MIP solution with pH=2-3.

[0010] P2W 18 Preparation: The conventional heating and reflux method was used: 100g of Na2WO4·2H2O was dissolved in 350mL of boiling water, and 150mL of 85% concentrated phosphoric acid was added dropwise (dropping rate 2mL / min). The mixture was refluxed for 10-24h (preferably 24h to improve yield and purity).

[0011] Cool to room temperature, add 100g KCl and stir for 1 hour. Filter out the precipitate and dissolve it in distilled water. If the solution is turbid, filter again. Let stand overnight at 5°C to collect the crystals. Reflux the crystal solution for 24 hours (add hydrogen peroxide to adjust to bright yellow when the solution turns green). Cool for 3-4 hours, add 150g KCl and stir for 10 minutes. Filter and dry for 2 hours. Filter out the precipitate and dissolve it in distilled water at 80°C (filter while hot). Evaporate to 100-150mL at 80°C. Crystallize at 5°C for 3 days. Dry at 50°C overnight. Finally, dissolve in hot water at 95°C, cool for 3 hours, filter out snow-like crystals (about 57g), and air dry at room temperature for 2-3 days.

[0012] Composite modified electrode assembly:

[0013] Substrate pretreatment: Quartz / silicon wafers: boiled at 80℃ with H2SO4:H2O2 (7:3, V / V) for 20 min; CaF2 substrates: washed and dried, then sonicated with acetone for 15 min, then rinsed and dried; ITO glass substrates: boiled at 80℃ with H2O:H2O2:NH4OH (1:1:1, V / V / V); all substrates were rinsed with deionized water, dried, sonicated with acetone for 15 min, rinsed and dried, and finally immersed in 3-aminopropyltriethoxysilane (APTS) solution for 12±0.5 h.

[0014] Preparation of modified solution: PSS solution: Weigh 0.0103 g PSS and dissolve it in 50 mL of pH 2 HCl solution to prepare 1×10⁻⁶ solution. - 3 M; PAH solution: Weigh 0.0019 g PAH and dissolve it in 20 mL of pH 2 HCl solution, add 0.5844 g NaCl (1 M), and prepare a solution with 1 × 10⁻⁶ HCl. -3 M;P2W 18 Solution: Weigh 0.1213g P2W 18 Dissolved by ultrasound in 25 mL of distilled water to prepare a 1×10⁻⁶ solution. -3 MIP solution: Weigh 0.002 g MIP powder, add 2 mL concentrated hydrochloric acid for protonation, add 10 mL DMF and sonicate for 30 min, then add 10 mL pH=2 HCl solution to prepare a solution with a concentration of 1×10⁻⁶ MIP. -3 M, pH = 2-3 MIP solution.

[0015] Electrode assembly: Substrate activation: The APTS-modified substrate was immersed in an HCl solution with pH=2 for activation, forming an amino-cationic surface; Substrate deposition: The activated substrate was sequentially immersed in PSS solution (10 min), washed with water, and purged with nitrogen, and then immersed in PAH solution (10 min), washed with water, and purged with nitrogen to construct the (PSS / PAH) substrate.

[0016] Alternating functional layer deposition: Alternating immersion in P2W 18 After soaking in the solution (10 min) and MIP solution (10 min) for each step, rinse with deionized water for 10 s and dry with nitrogen flow, repeating n times to obtain [PSS / PAH / (P2W 18 / MIP) n Composite modified electrode.

[0017] Using [PSS / PAH / (P2W)] 18 / MIP) n Electrochemical tests were performed on the composite modified electrode in HAc-NaAc buffer (pH=3.8) with different concentrations of CAP.

[0018] Advantages of this invention:

[0019] The poorly soluble molecularly imprinted polymer was protonated and then dissolved in DMF, overcoming the previous problem that MIP could not be modified by electrostatic layer-by-layer self-assembly. By using the NIPAM temperature-sensitive monomer with a minimum critical dissolution temperature of 32-35℃, the template elution time was shortened from the traditional 72h to 48h, solving the problems of slow mass transfer and difficult elution of MIP.

[0020] MIP and P2W 18 Synergistic signal enhancement: Dawson-type P2W 18 With W 6+ / W 5+ The rapid and reversible redox pair can serve as an electron transport medium; when CAP molecules are enriched in the specifically imprinted cavity of MIP, the binding of CAP to MIP induces changes in local charge distribution, P2W 18 By rapidly transferring electrons through the "bridging oxygen-tungsten" structure, the "molecular recognition signal" is converted into an "electrochemical current signal," thereby amplifying the signal.

[0021] Controllable and stable electrode structure: P2W in the composite film is achieved through electrostatic alternating deposition self-assembly. 18 With uniform deposition of MIP (absorbance is linearly correlated with the number of layers), there is no aggregation on the membrane surface, ensuring the repeatability and stability of detection.

[0022] Excellent detection performance: MIP-P2W 18The composite modified electrode exhibits a linear detection range of 0.1 nM to 10 μM for chloramphenicol, with a detection limit as low as 0.145 nM (S / N = 3) using the DPV method, which can meet the requirements for trace chloramphenicol detection.

[0023] Outstanding selectivity and anti-interference capabilities: The specific recognition cavity of MIP is highly matched with CAP in terms of spatial structure and chemical action, which can effectively distinguish CAP from structurally similar antibiotics such as florfenicol and thiamphenicol, as well as other common antibiotics such as tetracycline and ampicillin; in the presence of 10μM ion interferences (such as NaCl and KCl), the relative standard deviation of the detection signal is less than 10%, which can adapt to the detection scenarios of trace CAP in complex matrices. Attached Figure Description

[0024] Figure 1 A schematic diagram illustrating the preparation and molecular recognition mechanism of molecularly imprinted polymers.

[0025] Figure 2 For [PSS / PAH / (P2W 18 / MIP) n [Schematic diagram of the internal multilayer structure of a multilayer film undergoing electrostatic self-assembly on a substrate]

[0026] Figure 3 The images show the infrared spectra of the polymers. a corresponds to the MIP before template extraction, b corresponds to the MIP after template extraction, and c corresponds to the non-imprinted polymer. NIP (Non-Ingredient Protease)

[0027] Figure 4 For [PSS / PAH / (P2W 18 / MIP) 10 Infrared spectrum of composite modified electrode

[0028] Figure 5 Image a shows the UV-Vis spectrum of protonated MIP dissolved in DMF, image b shows the UV-Vis spectrum of protonated NIP dissolved in DMF, and image c shows the spectrum of [PSS / PAH / (P2W)]. 18 The UV-Vis spectrum of the thin film modified on the quartz substrate is shown in Figure 5 (n=9). 18 / MIP) n (n=1-9) UV-Vis spectra on thin film modified quartz substrates; the inset shows the relationship between absorbance at 198nm, 228nm, and 288nm and the number of self-assembled layers n (x-axis: number of self-assembled layers n; y-axis: absorbance).

[0029] Figure 6 For [PSS / PAH / (P2W 18 / MIP) 10 X-ray photoelectron spectroscopy (XPS) of the composite modified electrode; a) XPS full scan spectrum of the corresponding composite film; b) W... 4f High-resolution spectrum, c corresponds to N 1s High-resolution spectrum, d corresponds to C 1s High-resolution spectrum, e corresponds to O 1s High-resolution spectrum.

[0030] Figure 7 For [PSS / PAH / (P2W 18 / MIP) 20 Composite modified electrode and [PSS / PAH / (P2W)] 18 / NIP) 20 Scanning electron microscope (SEM) images of the composite modified electrode; a) low magnification (×600, scale bar: 10 μm); b) medium magnification (×50,000, scale bar: 100 nm); c) high magnification (×200,000, scale bar: 100 nm); d) [PSS / PAH / (P2W] 18 / NIP) 20 Scanning electron microscope image of the composite modified electrode at high magnification (×200,000, scale bar: 100 nm).

[0031] Figure 8 In section a, the blank control for the cyclic voltammetry curve of the bare ITO electrode in a buffer solution containing 10 μM CAP is given. In section A, bg corresponds to [PSS / PAH / (P2W], respectively]. 18 / MIP) 30 In the modified ITO electrode, bg in B corresponds to [PSS / PAH / P2W] respectively. 18 ] 30 The modified electrode and bg in C correspond to [PSS / PAH / (P2W)], respectively. 18 / NIP) 30 Cyclic voltammetry curves of the modified ITO electrode in HAc-NaAc buffer solutions containing different CAP concentrations at pH 3.8 are shown. Where b is the buffer solution containing 0.1 nM CAP, c is 1 nM, d is 10 nM, e is 100 nM, f is 1 μM, and g is 10 μM. Figure D shows the [PSS / PAH / (P2W)] electrode. 18 / MIP) 30 The linear fitting curve of the oxidation peak current of the modified electrode versus the logarithm of the CAP concentration.

[0032] Figure 9 In section a, the blank control for the differential pulse voltammetry curve of the bare ITO electrode in a buffer solution containing 10 μM CAP is given. In section A, bg corresponds to [PSS / PAH / (P2W], respectively. 18 / MIP) 30 In the modified ITO electrode, bg in B corresponds to [PSS / PAH / P2W], respectively. 18 ] 30 The modified electrode and bg in C correspond to [PSS / PAH / (P2W)], respectively. 18 / NIP) 30 Differential pulse voltammetry curves of the modified ITO electrode in HAc-NaAc buffer solutions containing different CAP concentrations at pH = 3.8. Where b is the buffer solution containing 0.1 nM CAP, c is 1 nM, d is 10 nM, e is 100 nM, f is 1 μM, and g is 10 μM. Figure D shows the [PSS / PAH / (P2W)] electrode. 18 / MIP) 30 The linear fitting curve of the oxidation peak current of the modified electrode versus the logarithm of the CAP concentration.

[0033] Figure 10 In section a, the blank control for the square wave voltammetry curve of the bare ITO electrode in a buffer solution containing 10 μM CAP is given. In section A, bg corresponds to [PSS / PAH / (P2W], respectively. 18 / MIP) 30 In the modified ITO electrode, bg in B corresponds to [PSS / PAH / P2W] respectively. 18 ] 30 The modified electrode and bg in C correspond to [PSS / PAH / (P2W)], respectively. 18 / NIP) 30 Square wave voltammetry curves of the modified ITO electrode in HAc-NaAc buffer solutions containing different CAP concentrations at pH = 3.8. Where b is the buffer solution containing 0.1 nM CAP, c is 1 nM, d is 10 nM, e is 100 nM, f is 1 μM, and g is 10 μM. Figure D shows the [PSS / PAH / (P2W] electrode. 18 / MIP) 30 The linear fitting curve of the oxidation peak current of the modified electrode versus the logarithm of the CAP concentration.

[0034] Figure 11For the correlation spectrum of selectivity and anti-interference experiment: A is [PSS / PAH / (P2W 18 / MIP) 30 Comparison of differential pulse voltammetric responses of the modified electrode to CAP and structurally similar antibiotics; B represents [PSS / PAH / (P2W)]. 18 / NIP) 30 Comparison of differential pulse voltammetric responses of modified electrodes to CAP and structurally similar antibiotics; C represents [PSS / PAH / (P2W)]. 18 / MIP) 30 [Histogram of the response current of the modified electrode to CAP and interfering substances; D is [PSS / PAH / (P2W]] 18 / MIP) 30 The differential pulse voltammetry curve of the modified electrode against the CAP in the presence of interfering substances; E is a bar chart of the relative deviation of the CAP detection current in the presence of interfering substances. Detailed implementation method:

[0035] This invention is not limited to the following specific embodiments. Those skilled in the art can implement this invention using other specific embodiments based on the content disclosed herein. Any simple changes or modifications made to the design structure and concept of this invention fall within the protection scope of this invention.

[0036] The invention will now be described in detail with reference to the illustrations and specific implementation steps.

[0037] This invention utilizes an electrostatic layer-by-layer self-assembly method to prepare a MIP and Dawson-type polyacid (P2W) based on a temperature-sensitive monomer. 18 The method involves using a synergistic composite modified electrode to perform electrochemical detection of CAP, which includes the following steps: Unless otherwise specified, all reagents used in the steps can be purchased from the Aladdin website.

[0038] Step 1) Preparation of molecularly imprinted polymer: Dissolve 0.2g NIPAM (functional monomer) and 0.1g CAP (template molecule) in 670μL THF, sonicate for 15min (300W) until completely dissolved to form a homogeneous solution; add 125μL EGDMA was mixed with 0.05g AIBN (initiator), and sonicated for 15 min. The mixture was then transferred to a 65℃ constant temperature water bath and stirred for 24 h to obtain a block polymer. The polymer was ground to a particle size of 200 mesh and extracted with methanol / 36% acetic acid (9:1, v / v) using a Soxhlet extractor for 48 h, changing the eluent every 12 h. The extraction was monitored with a UV spectrophotometer (λ = 275 nm) until no CAP absorption was observed (absorbance < 0.01), confirming complete template removal. The sample was washed three times with methanol and twice with deionized water, and then vacuum dried at 60℃ for 2 h. 0.002g of the dried MIP powder was taken, protonated with 2mL concentrated hydrochloric acid (37%) for 30 min, then dissolved by sonication with 10mL DMF for 30 min. Finally, 10mL of pH = 2 HCl solution was added and mixed to prepare a 1×10⁻⁶ solution. -3 M, pH 2-3 MIP solution, for later use. Used in preparation and molecular recognition processes. Figure 1 To represent schematically.

[0039] Step 2) Preparation of polyacid P2W 18 Dissolve 100g of Na₂WO₄·2H₂O in 350mL of boiling water. Slowly add 150mL of 85% concentrated phosphoric acid (2mL / min) dropwise under magnetic stirring. After the addition is complete, heat to reflux (100±2℃) and reflux for 24h (the solution should remain bright yellow; if it turns green, add 10% hydrogen peroxide until it returns to bright yellow). Cool to room temperature (25±2℃), add 100g of KCl, stir for 1h until completely dissolved, let stand for 30min, and filter to obtain a yellow precipitate. Dissolve the precipitate in 200mL of distilled water (80℃), filter while hot to remove insoluble matter, cool the filtrate to room temperature, and let stand overnight at 5℃ to precipitate crystals. Filter the crystals, vacuum dry at 50℃ for 12h, and weigh approximately 57g for later use. Dissolve 0.1213g of the crystals in 25mL of distilled water using ultrasonication to prepare a 1×10⁻⁶ solution. -3 M P2W 18 Solution, pH = 2.5.

[0040] Step 3) [PSS / PAH / (P2W] 18 / MIP) n Preparation of composite modified electrodes

[0041] Substrate pretreatment: Quartz substrates and silicon wafers were boiled in a 7:3 (sulfuric acid:hydrogen peroxide, volume ratio) solution at 80°C for 20 minutes; ITO glass substrates were boiled in a 1:1:1 (water:hydrogen peroxide:ammonia, volume ratio) solution at 80°C for 20 minutes. The samples were then rinsed thoroughly with deionized water and dried completely. Next, the pre-cleaned quartz substrates, silicon wafers, ITO glass substrates, and cleaned CaF2 substrates were ultrasonically treated in acetone for 15 minutes. They were then repeatedly rinsed with deionized water and dried completely. Silanization: All treated substrates were immersed in a 3-aminopropyltriethoxysilane (APTS) solution for 12 ± 0.5 h.

[0042] Preparation of electrode modification solution:

[0043] Weigh 0.0103 g of PSS powder and dissolve it in 50 mL of HCl solution with pH = 2 to prepare a 1×10⁻⁶ solution with pH = 2. -3 MPSS solution.

[0044] Weigh 0.0019 g of PAH powder and dissolve it in 20 mL of HCl solution with pH = 2. Then add 0.5844 g of NaCl to prepare a solution containing 1 M sodium chloride and pH = 2, with a concentration of 1 × 10⁻⁶. -3 M PAH solution.

[0045] Weigh P2W 18 0.1213 g of powder was ultrasonically dissolved in 25 mL of distilled water to prepare 1×10⁻⁶ powder. -3 M's P2W 18 Solution.

[0046] 0.002 g of the chloramphenicol molecularly imprinted polymer after template extraction was protonated with 2 mL of concentrated hydrochloric acid, then dissolved by sonication for 30 min with 10 mL of DMF; finally, 10 mL of pH 2 HCl solution was added and mixed thoroughly to prepare a 1×10⁻⁶ polymer. -3 M, pH = 2-3 MIP solution.

[0047] Preparation of modified electrodes:

[0048] Substrate activation: The silanized ITO substrate was immersed in an HCl solution with pH=2 for 20 min for activation. After removal, it was washed with water and bleed with nitrogen to form amino cations on the substrate surface.

[0049] PSS layer deposition: Immerse in PSS solution for 10 min, remove and rinse with deionized water for 10 s (to remove weakly adsorbed PSS), then dry with nitrogen.

[0050] PAH layer deposition: Immerse in PAH solution for 10 min, wash with water for 10 s, dry with nitrogen to construct (PSS / PAH) bottom layer;

[0051] Alternating deposition of functional layers: Repeat the following steps 30 times: ① Immersion in P2W 18 ① Immerse in MIP solution for 10 min → rinse with water → blow with nitrogen; ② Immerse in MIP solution for 10 min → rinse with water → blow with nitrogen;

[0052] Finished product: [PSS / PAH / (P2W)] 18 / MIP) 30 Composite-modified ITO electrode, ready for use, assembly process corresponding to Figure 2 .

[0053] Step 4) The prepared imprinted polymer and [PSS / PAH / (P2W)] on different substrates 18 / MIP) n The composite modified electrodes were characterized separately.

[0054] Infrared spectrum of polymer

[0055] Sample preparation: Take the “MIP before template elution”, “MIP after template elution” and “NIP without CAP (prepared under the same conditions, only CAP is omitted)” from step 1), and mix them with KBr to compress tablets;

[0056] Test conditions: Perkin-Elmer 580B infrared spectrometer, scanning range 4000-500 cm⁻¹ -1 ;

[0057] MIP after template extraction Figure 3 a) MIP before template extraction Figure 3 b) and non-imprinted polymers (NIP) Figure 3 The infrared spectrum of c) was analyzed, and the results are as follows:

[0058] MIP without eluting template ( Figure 3 b) at 1649cm -1 and 1544cm -1 The presence of broad absorption peaks at these locations corresponds to the amide I band (C=O stretching vibration) and the amide II band (NH bending vibration), respectively, confirming that the functional monomer NIPAM and the template molecule CAP pre-assemble into a complex through hydrogen bonding (the -OH and -NH2 functional groups of CAP are involved in the process).

[0059] After template washing ( Figure 3 a) The above absorption peaks were blue-shifted to 1661 cm⁻¹. -1 and 1549cm -1 This indicates that the hydrogen bond network is disrupted and the template molecule CAP has been removed;

[0060] Figure 3 In AC, 1638cm -1The C=C vinyl stretching vibration peak disappears, and at the same time, the peak at 2975 cm⁻¹ disappears. -1 (methyl CH stretching), 1730cm -1 (C=O stretching), 1461cm -1 (CH bending vibration) and 1172cm -1 The presence of a characteristic absorption peak of the crosslinking agent EDGMA at the (COC stretching) point proves that EGDMA has been successfully polymerized and integrated into the MIP and NIP networks;

[0061] Compared to NIP, template-eluted MIP ( Figure 3 a) at 2975cm -1 The presence of a broad absorption peak indicates that the CAP-induced cross-linked structure exhibits amorphous characteristics; the uneluted MIP ( Figure 3 b) at 811cm -1 The presence of the characteristic peak of the C-Cl asymmetric stretching vibration of CAP at the position, which disappears after elution, and the MIP spectrum tends to be consistent with that of NIP, confirms that the template molecule was completely removed and the MIP retained the specific recognition cavity, that is, the MIP was successfully synthesized.

[0062] Infrared spectrum of composite modified electrode

[0063] Sample: Take the [PSS / PAH / (P2W)] sample prepared on the CaF2 substrate in step 2). 18 / MIP) 10 Infrared spectroscopy testing of composite modified electrodes

[0064] Test conditions: Perkin-Elmer 580B infrared spectrometer, scanning range 4000-500 cm⁻¹ -1

[0065] result: Figure 4 The characteristic peak of the MIP component is clearly visible in the spectrum: 1654 cm⁻¹ -1 Corresponding to the C=O stretching vibration of NIPAM, 1561 cm -1 Corresponding to the NH bending vibration of NIPAM; 2877cm -1 (CH telescopic), 1702cm -1 (CO telescopic) and 1220cm -1 (CO stretching) is a characteristic peak of the crosslinking agent EGDMA, confirming the stable existence of the MIP network in the composite film;

[0066] Dawson-type P2W detected 18 Characteristic vibration peak: 1084 cm⁻¹ -1 [Vas(PO a )]、979cm -1 [Vas(WOd [Terminal oxygen stretching], 929cm -1 [Vas(WO b -W), bridged oxygen stretching] and 841cm -1 [Vas(WO c -W), keratin stretching], indicating P2W 18 Successfully incorporated into the composite membrane;

[0067] 1084cm -1 The strongest peak is P2W 18 PO a The stretching vibration is generated by the overlap of the COC stretching vibration of EGDMA;

[0068] and Figure 3 Compared to the infrared peaks of MIP in a, 2877, 1654, 1561, 1220, 1084 and 702 cm⁻¹ -1 A slight red or blue shift at the peak position is attributed to MIP and P2W. 18 The charge redistribution driven by intermolecular interactions at the interface is manifested through the coupling effect of vibrational dipoles and infrared radiation.

[0069] In summary, infrared spectroscopy confirms the relationship between MIP and P2W. 18 Anions have been successfully integrated into the multilayer membrane system, and the interfacial interactions are stable.

[0070] Polymer solutions and [PSS / PAH / (P2W)] 18 / MIP) n UV spectrum of composite modified electrode

[0071] Sample preparation

[0072] MIP solution: Take 0.002 g of MIP powder eluted from the template in step 1), protonate it with 2 mL of concentrated hydrochloric acid for 30 min, add 10 mL of N,N-dimethylformamide (DMF) and sonicate for 30 min, then add 10 mL of pH=2 HCl solution to prepare a solution with a concentration of 1×10⁻⁶ MIP powder. -3 M, pH=2-3 MIP-DMF solution.

[0073] NIP solution: Prepare the non-imprinted polymer (NIP, template molecule CAP omitted) according to step 1). Take 0.002 g of NIP powder and prepare it as described above. The method for preparing MIP solution yields 1×10⁻⁶ -3 M, NIP-DMF solution with pH = 2-3 (control sample).

[0074] Composite membrane sample: Take the [PSS / PAH / (P2W)] membrane prepared on the quartz substrate in step 2). 18 / MIP)9] Composite modified electrode; pretreated blank quartz substrate as reference.

[0075] Test conditions

[0076] A Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, California, USA) was used; the scanning wavelength range was 200–400 nm, the scanning rate was 600 nm / min, and the bandwidth was 2 nm; blank DMF was used as the solution sample reference, and blank quartz substrate was used as the composite film sample reference. Each sample was scanned three times, and the average spectrum was used for analysis.

[0077] Results and Analysis

[0078] UV-Vis spectra of MIP and NIP solutions

[0079] MIP-DMF solution: A strong absorption peak appears at 228 nm (originating from the n→π transition of the carbonyl group (C=O) in NIPAM, protonation induces electron redistribution and enhances the transition activity), and a weak absorption peak appears at 288 nm (corresponding to the localization of lone pair electrons in the MIP recognition cavity, which is a symmetric forbidden transition), confirming that MIP retains the specific recognition cavity.

[0080] NIP-DMF solution: Weaker peaks appear at 228 nm and 288 nm compared to MIP (because there is no CAP template molecule, there is no specific cavity matching CAP, and no "ordered electronic structure" due to template interaction. Therefore, the chemical environment of the chromophore is more random, and the efficiency of electronic transition (absorbance) is lower than that of MIP).

[0081] [PSS / PAH / (P2W 18 The UV-Vis spectrum of the composite film [ / MIP)9]

[0082] MIP characteristics are preserved: the peak shapes at 228 nm and 288 nm are consistent with those of the MIP solution, with no shift or distortion, confirming that the MIP structure has not been destroyed.

[0083] P2W 18 Feature detection: Peaks at 198 nm (charge transfer from terminal oxygen to tungsten) and 290 nm (charge transfer from bridging oxygen to tungsten) confirmed P2W. 18 Successfully incorporated and structurally intact.

[0084] [PSS / PAH / (P2W 18 / MIP) n Layer dependency of (n = 1-9)

[0085] Absorbance trend: 198nm (P2W)18 The absorbance at 228 nm (MIP) and 288 nm (MIP cavity) increases linearly with the number of layers n, without a plateau. Furthermore, each straight line satisfies R... 2 >0.99, confirming that the composite film is uniform and can be deposited in a controllable manner.

[0086] In summary, UV-Vis spectroscopy confirms that the MIP retains the recognition cavity and P2W. 18 The structure is intact, the two components are well compatible, and the composite membrane is uniformly assembled, providing structural support for the high selectivity and high sensitivity detection of chloramphenicol.

[0087] [PSS / PAH / (P2W 18 / MIP) n X-ray photoelectron spectroscopy of composite modified electrode

[0088] Sample: The n=10 composite film prepared using a silicon wafer in step 2);

[0089] Test: ESCALAB 250Xi spectrometer, Al Kα rays (1486.6 eV);

[0090] Results (corresponding to Figure 6):

[0091] The wide-scan spectrum (Figure 6a) shows the coexistence of W, N, C, and O elements, with the W element originating from P2W. 18 The N element comes from NIPAM of MIP, confirming that the expected component was successfully incorporated;

[0092] High resolution W 4f spectrum( Figure 6 b) at 35.25 eV (W) 4f7 / 2 ) and 37.35eV (W 4f5 / 2 A characteristic double peak appears at position ) and the P:W molar ratio is 2:18, similar to the Dawson type P2W. 18 The stoichiometry is consistent, confirming that P2W 18 The structure was not damaged;

[0093] N 1s The spectra (Figure 6c) are at 399.14 eV (neutral amide nitrogen - CONH-) and 401.27 eV (protonated amide nitrogen - CONH-). 2+ The appearance of a double peak at position ) corresponds to the chemical state of NIPAM in MIP, confirming that MIP has been successfully protonated;

[0094] C 1sThe spectrum (Fig. 6d) shows characteristic peaks at 284.80 eV (CC / CH), 286.39 eV (CO / CN) and 288.40 eV (C=O), providing direct evidence for the copolymerization of NIPAM and EGDMA to form a MIP network;

[0095] O 1s The spectrum (Figure 6d) shows a peak at 531.85 eV, which is attributed to the carbonyl group (C=O) and ether bond (COC) in the MIP framework, further verifying the structural integrity of the MIP.

[0096] In summary, the XPS results confirm P2W. 18 It has been successfully compounded with the MIP components, and the chemical states of each component meet the design expectations.

[0097] [PSS / PAH / (P2W 18 / MIP) n Composite modified electrode and [PSS / PAH / (P2W)] 18 / NIP) n Scanning electron microscope image of composite modified electrode

[0098] Sample: The n=20 composite modified silicon wafer prepared in step 2);

[0099] Tested with a JSM-7610FPlus scanning electron microscope, accelerating voltage 5.0kV;

[0100] Results (corresponding to Figure 7):

[0101] Low magnification (×600, Fig. 7a, scale bar 10 μm): The polymer cross-linking modules on the surface of the multilayer film are evenly distributed, with no obvious agglomeration or gaps, confirming that the composite film has complete macroscopic coverage.

[0102] Medium magnification (×50,000, Fig. 7b, scale bar 100 nm): The membrane surface exhibits a significantly rough morphology. This structure can increase the specific surface area and the density of MIP recognition sites, thereby improving the binding efficiency of template molecules.

[0103] High magnification (×200,000, Figures 7c-7d, scale bar 100nm): Figure 7 c. A continuous cloud-like MIP overlying layer is visible, partially encapsulating spherical P2Ws. 18 Nanoparticles (particle size approximately 50-80 nm); Figure 7 d ratio Figure 7 The surface of c is smoother, and nanoscale fine cavities are distributed on the MIP-modified electrode, corresponding to the chloramphenicol-specific recognition sites of MIP. These cavities achieve selective binding to CAP through the synergistic effect of complementary geometric configurations and chemical forces (such as hydrogen bonds).

[0104] In summary, SEM observations confirm P2W 18 The MIP is uniformly co-assembled with the electrode surface without phase separation or aggregation, providing structural protection for the synergistic function of "specific recognition-electron transfer" during the sensing process.

[0105] Step 5): Electrochemical detection of chloramphenicol

[0106] Construction of detection system

[0107] Three-electrode system: The working electrode is [PSS / PAH / (P2W)] prepared in step 2). 18 / MIP) 30 ] and [PSS / PAH / (P2W 18 / NIP) 30 Modify ITO (effective area 2cm) 2 The auxiliary electrode is a platinum wire, and the reference electrode is a saturated calomel electrode (SCE).

[0108] Detection solution: Dissolve 0.0323g CAP in 100mL of deionized water to prepare a 1×10⁻⁶ solution. -3 The stock solution M was diluted with HAc-NaAc buffer at pH 3.8 to prepare CAP detection solutions of 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM.

[0109] Instruments: Admiral Squidstat electrochemical workstation; testing methods: cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV).

[0110] Cyclic voltammetry (CV) detection results

[0111] Experimental conditions: Potential scan range -1.5 to +1.5 V, scan rate 100 mV / s, detection solution was HAc-NaAc buffer solution of different concentrations of CAP (pH = 3.8).

[0112] Performance comparison analysis:

[0113] Bare ITO electrode: As shown in Figure 8A-a, Ba, Ca, even in a 10 μM high-concentration CAP solution, there are almost no obvious redox peaks, indicating that its electrocatalytic activity for CAP is extremely weak and it cannot achieve CAP detection.

[0114] MIP-P2W 18 Electrode: As shown in Figure 8A, the oxidation peak current shows a significant increasing trend with increasing CAP concentration (0.1 nM ~ 10 μM). The linear regression equation for the logarithmic model across the entire concentration range is y = 9.0100x + 1.527, R0 2=0.9938, excellent linearity; according to the IUPAC formula LOD=3σ / S, LOQ=10σ / S (standard deviation of blank measurement σ=1μA), calibration slope (S=0.285μA / nM), the detection limit is calculated to be LOD=0.33nM (S / N=3), and the quantitation limit LOQ=1.08nM.

[0115] P2W 18 -only electrode / NIP-P2W 18 Electrode: As shown in Figures 8B / 8C, a weak anodic peak current appears only when the CAP concentration is ≥100nM, and there is no effective response in the low concentration (0.1~100nM) range; the detection limit is conservatively estimated to be 1μM based on reliable signals (nonlinear response in the low concentration range makes it impossible to accurately calculate the signal-to-noise ratio).

[0116] Performance advantage mechanism: The specific recognition cavity of MIP can pre-enrich CAP molecules on the electrode surface, P2W 18 It provides a reversible multi-electron redox center, and the two work together to catalyze the CAP redox reaction through efficient interfacial charge transfer, amplifying the response signal and significantly enhancing the electrocatalytic activity.

[0117] Detection results of differential pulse voltammetry (DPV) and square wave voltammetry (SWV)

[0118] Experimental conditions for DPV: potential scan range -1.0 to 0V, step potential 4mV, pulse height 50mV, pulse width 50ms, pulse period 200ms; buffer solution as in CV experiment.

[0119] SWV: Potential scan range -1.0 to 0V, step potential 4mV, pulse amplitude 25mV, frequency 15Hz; buffer solution as in CV experiment.

[0120] Performance Comparison Analysis

[0121] Differential pulse voltammetry (DPV)

[0122] Bare ITO electrode: As shown in Figure 9A-a, Ba, and Ca, there is no obvious current change in 10 μM CAP solution, indicating weak electrocatalytic activity;

[0123] MIP-P2W 18 Electrode: As shown in Figure 9A, the oxidation peak current increases significantly with CAP concentration (0.1 nM to 10 μM), and the linear fitting R... 2 =0.9948; Based on LOD=3σ / S, LOQ=10σ / S (σ=0.001μA, S=0.0207μA / nM), the detection limit is as low as 0.145nM (S / N=3), the quantitation limit is 0.477nM, the background suppression effect is excellent, and the quantitation accuracy is high.

[0124] P2W 18 -only electrode / NIP-P2W 18 Electrode: As shown in Figures 9B / 9C, a sharp oxidation peak appears only in the 10 μM CAP solution (the interfacial charge transfer rate is higher at high concentrations), while the response is weak at low concentrations (0.1–100 nM). Based on reliable signals, the detection limit is conservatively estimated to be 1 μM (nonlinear response in the low concentration range makes it impossible to accurately calculate the signal-to-noise ratio).

[0125] Square wave voltammetry (SWV)

[0126] Bare ITO electrode: As shown in Figure 10A-a, Ba, and Ca, there is no obvious current change in 10 μM CAP solution, indicating weak electrocatalytic activity;

[0127] MIP-P2W 18 Electrode: As shown in Figure 10A, the peak current response is stable across the entire concentration range, and the linear fitting R0 is satisfactory. 2 =0.9986; Based on LOD=3σ / S, LOQ=10σ / S (σ=0.003μA, S=0.0116μA / nM), the detection limit is 0.774nM (S / N=3), and the quantitation limit is 2.55nM; Moreover, the SWV analysis time is only 10 seconds, which is 90% shorter than DPV (100 seconds), taking into account both practical sensitivity and high-throughput detection requirements.

[0128] P2W 18 -only electrode / NIP-P2W 18 Electrode: As shown in Figure 10B / 10C, ​​the response is weak in the low concentration range. Based on reliable signals, the detection limit is conservatively estimated to be 1 μM (nonlinear response in the low concentration range, signal-to-noise ratio cannot be accurately calculated).

[0129] In summary, [PSS / PAH / (P2W)] 18 / MIP) 30 The composite modified electrode recognizes P2W via MIP specificity. 18 The synergistic effect of rapid electron transfer significantly improves the selectivity and sensitivity for chloramphenicol (CAP) detection: the DPV method has a detection limit as low as 0.145 nM and a linear range of 0.1 nM to 10 μM, meeting the requirements for accurate detection of trace CAP; the SWV method enables rapid high-throughput analysis, and both methods can be flexibly adapted to different application scenarios. This performance verifies the effectiveness of the composite electrode preparation scheme described in claim 1, providing an efficient technical path for trace chloramphenicol detection.

[0130] Step 6): Selectivity and Anti-interference Experiment

[0131] Experimental Design

[0132] Interfering substance selection: Structurally similar antibiotics: florfenicol (FF), thiamphenicol (TAP); Common coexisting antibiotics and ions: tetracycline (TC), ampicillin (AMP), NaCl, KCl;

[0133] Experimental system: The three-electrode system and HAc-NaAc buffer solution with pH=3.8 were used as the main test method, and differential pulse voltammetry was used.

[0134] Test group settings: Single CAP group: CAP concentration of 0.1 μM; Single interfering agent group: concentration of each antibiotic interfering agent of 1 μM and concentration of ion interfering agent of 10 μM; Mixed group: 0.1 μM CAP + 1 μM antibiotic interfering agent + 10 μM ion interfering agent;

[0135] Evaluation metrics: Calculate the relative deviation of the oxidation peak current between the mixed group and the single CAP group, and compare the current response intensity between the single interfering agent group and the single CAP group.

[0136] Results and Analysis

[0137] Selective verification: As shown in Figure 11A, [PSS / PAH / (P2W 18 / MIP) 30 The modified electrode showed a significantly higher oxidation peak current response to 0.1 μM CAP than to 1 μM FF, TAP, TC, and AMP, with response intensity ratios all greater than 5:1; while [PSS / PAH / (P2W 18 / NIP) 30 The modified electrodes showed no significant difference in response to the above substances (Figure 11B), confirming that the specific recognition cavity of MIP has a precise selective binding capability to CAP.

[0138] Anti-interference capability verification: The relative standard deviation (RSD) of the oxidation peak current of CAP in the mixed group was 3.2% to 8.7% (n=3) compared with that of the single CAP group, all of which were less than 10% (Figure 11C, E); the differential pulse voltammetry curves showed that the presence of interfering substances did not cause the characteristic peak of CAP to shift or significantly distort the peak shape (Figure 11D), indicating that the composite modified electrode can still stably detect CAP in complex matrices;

[0139] Mechanism analysis: The specific recognition cavity of MIP is highly matched with CAP in terms of spatial configuration and chemical interaction (such as hydrogen bonding), while it is significantly different from the interfering substances. Therefore, it can preferentially bind to CAP and generate specific electrochemical signals, thereby resisting the influence of interfering substances.

[0140] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for electrochemical detection of chloramphenicol using molecular imprinting, characterized in that: A MIP-POM composite modified electrode was prepared using an electrostatic layer-by-layer self-assembly method, and electrochemical detection of chloramphenicol in solution was performed. The process included the following steps: Polyelectrolyte layers [polystyrene sulfonate (PSS) / polyallylamine hydrochloride (PAH)] and polyoxometalate (P2W) are alternately deposited on a substrate (the substrate is quartz, silicon wafer, CaF2, or indium tin oxide glass ITO). 18 ) and chloramphenicol molecularly imprinted polymer (MIP) are used to form a composite modified film, which is assembled into [PSS / PAH / (P2W 18 / MIP) n ] Composite modified electrode, where n is a positive integer from 1 to 30; the [PSS / PAH / (P2W 18 / MIP) n A composite modified electrode was used as the working electrode, a platinum electrode as the auxiliary electrode, and a saturated calomel electrode as the reference electrode to form a three-electrode system for electrochemical detection of chloramphenicol CAP.

2. The method for using molecular imprinting for electrochemical detection of chloramphenicol according to claim 1, characterized in that: The MIP-POM composite modified electrode used was prepared by electrostatic layer-by-layer self-assembly and has [PSS / PAH / (P2W)] 18 / MIP) n [A composite modified membrane structure, where n is a positive integer from 1 to 30.] 3. The method for using molecular imprinting for electrochemical detection of chloramphenicol as described in claim 1, characterized in that... The MIP is prepared by in-situ polymerization at 60°C using chloramphenicol as the template molecule, N-isopropylacrylamide (a temperature-sensitive monomer with a minimum critical solution temperature of 32-35°C) as the functional monomer, ethylene glycol dimethacrylate as the crosslinking agent, and azobisisobutyronitrile as the initiator.

4. The method for using molecular imprinting for electrochemical detection of chloramphenicol as described in claim 1, characterized in that... The preparation of the MIP includes: polymerizing the template molecule, functional monomer, crosslinking agent and initiator in tetrahydrofuran; removing the template by Soxhlet extraction with a methanol / acetic acid mixture for 48 hours; washing and drying to obtain MIP powder; adding the powder to 2 mL of concentrated hydrochloric acid for protonation for 30 min; and finally dissolving it in N,N-dimethylformamide (DMF) to form a positively charged MIP solution.

5. The method for using molecular imprinting for electrochemical detection of chloramphenicol as described in claim 1, characterized in that... The P2W of the MIP-POM composite modified electrode used 18 It is a Dawson-type 2:18 phosphotungstic acid, which dissolves in water to form a negatively charged P2W. 18 Solution.

6. The electrode as described in claim 2, characterized in that... The electrostatic layer-by-layer self-assembly method includes the following pretreatment steps: the substrate is sequentially cleaned with acid and alkali, sonicated with acetone, and immersed in 3-aminopropyltriethoxysilane (APTS) solution for 12±0.5h, then immersed in HCl solution with pH=2 to activate and form an amino cationic surface; subsequently, under constant temperature conditions of 25±2℃, the substrate is alternately immersed in a negatively charged PSS solution (1×10⁻⁶). -3 M), positively charged PAH solution (1×10) -3 M, containing 1M NaCl, pH=2), negatively charged P2W 18 Solution (1×10) -3 M, pH = 2-3), positively charged MIP solution (1×10⁻⁶) -3 M, pH=2-3, solvent is DMF), after each soaking for 10 minutes, rinse with deionized water for 10 seconds, and then dry with nitrogen gas flow.

7. The method for using molecular imprinting for electrochemical detection of chloramphenicol as described in claim 1, characterized in that: When the prepared MIP-POM composite modified electrode was used for electrochemical detection of CAP, the concentrations of CAP were 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM, respectively, and the buffer solution was 0.1 M HAc-NaAc (pH = 3.8).

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