Sandwich-type photoelectrochemical sensor for detecting acrylamide and its preparation method

Abstract

This invention discloses a sandwich-type photoelectrochemical sensor for detecting acrylamide and its preparation method. A TT-Por(Co)-COF modified ITO electrode is used as the first layer, which is then used to selectively adsorb AAM from the sample solution as the sandwich layer. After affinity adsorption, a CdTe quantum dot-labeled acrylamide aptamer is added to the photoelectrode surface as a signal incubation layer. This sandwich-type sensor design not only fully utilizes the properties of the TT-Por(Co)-COF framework to achieve specific adsorption of AAM, but also cleverly combines quantum dot modification to enhance the photoelectric conversion efficiency of the heterojunction, thereby effectively improving the detection signal and ensuring the sensor's rapid response and high selectivity to the target molecule. In practical samples, such as potato chips and coffee, the sensor exhibits excellent performance, achieving efficient and accurate detection, providing a new direction for developing novel sensing methods for hazardous byproducts in food processing.

Description

Technical Field

[0001] This invention relates to the field of photoelectrochemical sensor technology, and in particular to a sandwich-type photoelectrochemical sensor for detecting acrylamide and its preparation method. Background Technology

[0002] Food processing can generate various harmful byproducts, primarily originating from high-temperature treatment, fermentation, pickling, chemical reactions, and microbial contamination. Acrylamide (AAm), a typical contaminant formed in heat-processed foods, has been classified as a "probable human carcinogen" (Group 2A) by the International Agency for Research on Cancer. AAm is mainly formed through two reaction pathways: the Stryker reaction pathway and the acrolein pathway. The former involves the decarboxylation and deamination of amino acids under high-temperature conditions to form Stryker aldehyde, which is then converted into acrylamide. The latter involves the decarboxylation of organic acids at high temperatures to form acrolein, which is further converted into acrylic acid, and finally, under the action of asparagine, forms acrylamide. These reactions typically occur during high-temperature processing of food, such as grilling, baking, and frying, and are particularly common in snacks, fast food, and certain beverages, becoming a significant route of acrylamide intake. According to European Commission document 2017 / 2158, baseline levels for acrylamide content in various foods have been established. However, although acrylamide intake is within safe limits for most people, high-consumption groups may consume levels far exceeding recommended levels. Due to the potential hazards of acrylamide to humans, such as carcinogenicity and neurotoxicity, Europe and the United States have taken corresponding restrictive measures and require the labeling of its content on food labels. In order to reduce the risk of acrylamide in food, it is urgent to develop efficient and convenient detection methods to accurately monitor and quantify acrylamide in food.

[0003] Existing methods for acrylamide detection, such as enzyme-linked immunosorbent assay (ELISA), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS), provide reliable results. However, these methods generally suffer from limitations such as expensive equipment, complex operation, cumbersome sample pretreatment, and long detection cycles. Furthermore, current contaminant analysis methods primarily focus on the detection of contaminants in final bulk products during food production. Effective on-site and fixed-point contaminant detection methods have not yet been established in actual food processing stages. Therefore, there is an urgent need to develop sensor technologies with high sensitivity, rapid response, on-site operation capability, and low cost to achieve real-time monitoring of contaminants such as acrylamide during food processing.

[0004] In recent years, photoelectrochemical sensors have attracted widespread attention due to their unique signal-to-noise ratio advantage, resulting from the complete separation of input and output energy forms. Chinese patent CN114778638B discloses a photoelectrochemical sensor for detecting acrylamide. In the preparation process, a MoS2 / rGO composite material and a MoS2 / rGO / AuNPs nanocomposite material are prepared sequentially. Then, the MoS2 / rGO / AuNPs nanocomposite material is dissolved in a solution composed of ethylene glycol and Nafion to obtain an electroactive material dispersion. This dispersion is then drop-coated onto an ITO electrode and dried to serve as the MGA / ITO working electrode. Finally, using an electrochemical polymerization solution containing pyrrole, acrylamide, and LiClO4 as the electrolyte, platinum Pt as the counter electrode, and silver / silver chloride as the reference electrode, electrochemical polymerization is performed using cyclic voltammetry with the MGA / ITO as the working electrode to obtain a molecularly imprinted electrode (MIP). The acrylamide template is then removed by elution to obtain the rMIP electrode, which is used for acrylamide detection. Although the photoelectrochemical sensor obtained by the above patent can detect acrylamide, it uses the precious metal AuNPs, which increases the cost, and the detection range is not wide enough, the detection limit is high, and the sensitivity is low.

[0005] As the core building block of photoelectrochemical sensors, photoactive materials are fundamental to high-performance photoelectrochemical sensors. Among numerous types of photoactive materials, porphyrin-based covalent organic frameworks (COFs) have been widely used in the construction of photoelectrochemical sensors in recent years due to their excellent photon trapping ability, good carrier separation and transfer rate, porosity, and good chemical stability. For example, Li et al. synthesized a porphyrin-tetrathiazole COF (TT-COF(Zn)) photoelectrochemical sensor and successfully applied it to a high-sensitivity copper ion (Cu) sensor. 2+The detection of cardiac troponin I (cTnI) in clinical serum samples was achieved with a detection limit of 0.15 nM and excellent performance in water sample detection (X. Li et al., A molecular imprinting photoelectrochemical sensor modified by polymerbrushes and its detection for BSA, Chemical Engineering Journal, 2024). Li et al. developed a novel photocathode immunosensor that utilizes porphyrin covalent organic frameworks (p-COFs) and silicon nanowire arrays (p-SiNWs) to construct an S-type heterojunction, successfully achieving highly sensitive, rapid, and interference-resistant detection of cTnI in clinical serum samples (H.-J. Li et al., S-Scheme Porphyrin Covalent Organic Framework Heterojunction for Boosted Photoelectrochemical Immunoassays in Myocardial Infarction Diagnosis., ACS Sensors, 2023). However, most current photoelectrochemical sensors based on porphyrin COFs are still limited to using porphyrin COFs as photon trapping and electrical signal generation units, and the development and exploration of their targeting affinity are still very limited. The focus of this invention is how to utilize porphyrin COFs to construct a photoelectrochemical sensor for detecting acrylamide with higher sensitivity. Summary of the Invention

[0006] The purpose of this invention is to provide a sandwich-type photoelectrochemical sensor for detecting acrylamide and its preparation method, so as to solve the problem of low sensitivity of the aforementioned photoelectrochemical sensor for detecting acrylamide.

[0007] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a sandwich-type photoelectrochemical sensor for detecting acrylamide, comprising the following steps:

[0008] (1) Preparation of Co-TAPP

[0009] 5,10,15,20-tetra[4-aminophenyl]-21H,23H-porphyrin and cobalt salt were dissolved in an organic solvent to obtain a mixture. The mixture was heated under reflux to react. After the reaction was completed, the precipitate was collected, washed and dried to obtain Co-TAPP powder.

[0010] (2) Preparation of TT-Por(Co)-COF

[0011] Co-TAPP and thiopheno[3,2-B]thiophene-2,5-dicarboxaldehyde were dispersed in an organic solvent, subjected to ultrasonic treatment and freeze-evacuate-thaw cycle treatment, heated to react, washed and dried to obtain TT-Por(Co)-COF powder;

[0012] (3) Preparation of CdTe QDs / Apt

[0013] Acrylamide aptamer, N-hydroxysuccinimide and 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and reacted, followed by the addition of carboxylated CdTe QDs and deionized water. The reaction was continued, and the precipitate was obtained by centrifugation. Deionized water was added to the precipitate again to obtain a CdTe QDs / Apt solution.

[0014] (4) Add methanol to TT-Por(Co)-COF powder, disperse by ultrasonication to obtain COF dispersion, drop the COF dispersion onto the surface of ITO glass electrode, and dry to obtain TT-Por(Co)-COF / ITO modified electrode.

[0015] The TT-Por(Co)-COF / ITO modified electrode was immersed in acrylamide solutions of different concentrations to complete the affinity adsorption of acrylamide. After the adsorption was completed, CdTe QDs / Apt solution was added to the surface of the TT-Por(Co)-COF / ITO modified electrode to continue incubation to form a sandwich structure. After washing, the photoelectrochemical sensor was obtained.

[0016] Inspired by the electron-deficient properties of the vinyl group in acrylamide and its terminal amino functional group, this invention designs a metal-coordinated porphyrin-based COF with selective affinity for acrylamide, based on an electron-donating thiophene unit and incorporating linking functional groups in an imine-bonded COF. Specifically, this invention synthesizes TT-Por(Co)-COF containing a DA conjugated system via an imine condensation reaction using thieno[3,2-B]thiophene-2,5-dicarboxaldehyde (TT) and 5,10,15,20-tetra[4-aminophenyl]-21H,23H-porphyrin cobalt (Co-TAPP) as raw materials. Further quantitative calculations and infrared spectroscopy confirm the weak interaction between the AAm fragment and the COF framework. A weak interaction exists between the sulfur (S) atom in the TT unit and the olefinic group in the AAm, involving both electrostatic and van der Waals forces. Furthermore, the amino terminus of AAM forms a significant NH…N hydrogen bond interaction with the central region of the imine bond in the COF framework. The weak interactions at these two locations jointly dominate the affinity adsorption of TT-Por(Co)-COF onto AAM.

[0017] Based on this, the present invention ultimately constructs a simple acrylamide sandwich-type photoelectrochemical sensor. First, a TT-Por(Co)-COF modified ITO electrode is used as the first layer, which is then used to selectively adsorb AAM from the sample solution as the sandwich layer. After affinity adsorption, a CdTe quantum dot (CdTe QDs) labeled acrylamide aptamer is added to the photoelectrode surface as a signal incubation layer. Due to the specific recognition of acrylamide by the AAM aptamer, CdTe QDs are introduced into the COF framework surface simultaneously with aptamer conformational folding, forming a heterojunction. This enhances the photoelectric conversion efficiency of the photoelectrode, resulting in a novel acrylamide turn-on photoelectrochemical sensor. This sandwich-type sensor design not only fully utilizes the properties of the TT-Por(Co)-COF framework to achieve specific adsorption of AAM, but also cleverly combines quantum dot modification to enhance the photoelectric conversion efficiency of the heterojunction, thereby effectively improving the detection signal and ensuring the sensor's rapid response and high selectivity to target molecules.

[0018] Preferably, in step (1), the cobalt salt is Co(OAc)2·2H2O, and the mass ratio of 5,10,15,20-tetra[4-aminobenzene]-21H,23H-porphyrin to the cobalt salt is 2:2 to 2.5;

[0019] Organic solvents, in a volume ratio of 8–10:1–3:1–5, include CHCl3, methanol, and DMF;

[0020] The mass-to-volume ratio of 5,10,15,20-tetra[4-aminophenyl]-21H,23H-porphyrin to CHCl3 is 200 mg: 80-100 mL.

[0021] Preferably, in step (1), the heating temperature is 75-85℃ and the reflux time is 20-30h; the drying temperature is 60-80℃ and the drying time is 10-15h.

[0022] Preferably, in step (2), the mass ratio of Co-TAPP to thiopheno[3,2-B]thiophene-2,5-dicarboxaldehyde is 10-20:5-10;

[0023] Organic solvents, in a volume ratio of 20–40:5–15:1–5, include benzyl alcohol, o-dichlorobenzene, and 6M acetic acid;

[0024] The mass-to-volume ratio of Co-TAPP to organic solvent is 10–20 mg: 500–600 μL.

[0025] Preferably, in step (2), the heating temperature is 100-150°C and the time is 48-96 hours.

[0026] Preferably, in step (3), the concentrations of acrylamide aptamer, N-hydroxysuccinimide, 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride and carboxylated CdTe QDs are 8–12 nM, 0.05–0.15 M, 0.05–0.15 M, and 0.03–0.08 μM, respectively.

[0027] The volumes of acrylamide aptamer, N-hydroxysuccinimide, 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride, carboxylated CdTe QDs, and the first addition of deionized water were 1–2 mL, 200–300 μL, 200–300 μL, 0.8–1.2 mL, and 1–3 mL, respectively.

[0028] Preferably, in step (3), the mass-to-volume ratio of the precipitate and the second addition of deionized water is 0.05–0.1 μg: 5–10 mL.

[0029] Preferably, in step (4), the concentration of the COF dispersion is 3-8 mg / mL, the volume of the COF dispersion added to the surface of the ITO glass electrode is 10-50 μL, the volume of the CdTe QDs / Apt solution added to the surface of the TT-Por(Co)-COF / ITO modified electrode is 10-50 μL, and the incubation time is 10-30 min.

[0030] A second aspect of the present invention provides a sandwich-type photoelectrochemical sensor, which is prepared by the above-described method.

[0031] A third aspect of this invention provides an application of a sandwich-type photoelectrochemical sensor in the detection of acrylamide, with a detection range of 0.001–10. 4 The detection limit is 0.19 pM.

[0032] Therefore, the sandwich-type photoelectrochemical sensor for detecting acrylamide and its preparation method described above have the following beneficial effects:

[0033] (1) The sandwich-type photoelectrochemical sensor prepared in this invention has a detection limit of 0.19 pM for acrylamide and a detection range of 0.001–10 pM. 4 The nM value indicates that it has extremely high sensitivity. This high sensitivity is due to the specific adsorption capacity of TT-Por(Co)-COF for acrylamide and the signal amplification effect of CdTe quantum dot (CdTe QDs) labeled aptamers, which enables the detection of extremely low concentrations of acrylamide and is suitable for trace analysis.

[0034] (2) The sandwich-type photoelectrochemical sensor prepared in this invention is based on the dual recognition mechanism of the TT-Por(Co)-COF framework structure and the acrylamide aptamer, and can specifically recognize acrylamide molecules. There are electrostatic and van der Waals forces between the thiophene unit in TT-Por(Co)-COF and the olefin group of acrylamide, while the amino terminus of acrylamide forms hydrogen bonds with the imine bond in the COF framework. These weak interactions together ensure the high selectivity of the sensor for acrylamide and reduce the influence of other interfering substances.

[0035] (3) The photoelectrochemical sensor prepared in this invention has a sandwich structure (TT-Por(Co)-COF / ITO modified electrode + acrylamide + CdTe QDs / Apt), which makes the detection process simple and efficient. The specific recognition and conformational folding of the aptamer to acrylamide can be completed rapidly. Combined with the photoelectric signal amplification effect of CdTe quantum dots, a rapid response to the target molecule is achieved, and the incubation time is only 10-30 minutes.

[0036] (4) The photoelectrochemical sensor of the present invention has the functions of photoelectric signal amplification and stability enhancement. CdTe QDs form a heterojunction with TT-Por(Co)-COF, which improves the photoelectric conversion efficiency and thus amplifies the detection signal. In addition, the COF material itself has good stability and conductivity, which further ensures the long-term stability and reliability of the sensor.

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0038] Figure 1 The synthesis route and characterization results of TT-Por(Co)-COF are shown below, including (A) the synthesis route and unit cell diagram of TT-Por(Co)-COF; (B) PXRD pattern of TT-Por(Co)-COF: experimental spectrum (light blue), refined spectrum (red), Bragg position of AA stack (green), and error (purple); (C) FT-IR spectrum of TT-Por(Co)-COF and its ligand units; (D) TEM image of TT-Por(Co)-COF (inset: HR-TEM and fast Fourier transform and lattice distance map); (E) SEM image of TT-Por(Co)-COF.

[0039] Figure 2 The XPS characterization results of TT-Por(Co)-COF are shown below, where (A) is the full-spectrum XPS image of TT-Por(Co)-COF; (B) is the Co 2p of TT-Por(Co)-COF; and (C) is the high-resolution XPS image of N1s.

[0040] Figure 3 The UV-DRS characterization results of TT-Por(Co)-COF are shown, where (A) is the UV-Vis diffuse reflectance spectrum of TT-Por(Co)-COF; and (B) is the UV-Vis diffuse reflectance spectrum of TT-Por(Co)-COF. 2 With E g Correspondence diagram; (C) Mott-Schottky curve of TT-Por(Co)-COF;

[0041] Figure 4 The results represent the characteristics of the sensing mechanism, including (A) a visualization of the interaction force between TT-Por(Co)-COF and AAM in the IMH mapping; (B) a comparison of the FT-IR spectra of TT-Por(Co)-COF before and after adsorption of AAM; and (C) a scatter plot of the interaction force between TT-Por(Co)-COF and AAM in the IMH model.

[0042] Figure 5 The effect of quantum dot incubation on the photoelectric conversion effect of the photoelectrode is shown in (A) OCP plots of TT-Por(Co)-COF and TT-Por(Co)-COF / Apts; (B) LSV curves of TT-Por(Co)-COF and TT-Por(Co)-COF / Apts; and (C) photocurrent curves of TT-Por(Co)-COF and TT-Por(Co)-COF / Apts.

[0043] Figure 6 To characterize the sensor construction process, (A) cyclic voltammetry (CV) and (B) electrochemical impedance spectroscopy (EIS) were used (a. bare ITO electrode, b. TT-Por(Co)-COF / ITO, c. TT-Por(Co)-COF / ITO after immersion in AAM, and d. TT-Por(Co)-COF / ITO after adsorption of aptamers).

[0044] Figure 7 To analyze the results of condition optimization, the optimization included (A) electrolyte pH value; (B) AAM immersion time; and (C) aptamer adsorption time.

[0045] Figure 8 The sensor performance analysis results include (A) the working curve of AAM-PECS; and (B) the fitted standard curve, with ah concentrations of: 0 nM (a), 0.001 nM (b), 0.01 nM (c), 0.1 nM (d), 1 nM (e), 10 nM (f), 10 2 nM(g), 10 3 nM(h);

[0046] Figure 9The diagram shows the anti-interference capabilities of AAM-PECS against other common interfering devices;

[0047] Figure 10 This is a schematic diagram illustrating the construction process of a sandwich-type photoelectrochemical sensor.

[0048] Figure 11 EDS plot of TT-Por(Co)-COF;

[0049] Figure 12 The TGA curve is for TT-Por(Co)-COF. Detailed Implementation

[0050] The present invention will be further described below. It should be noted that this embodiment is based on the present technical solution and provides detailed implementation methods and specific operation processes, but the present invention is not limited to this embodiment.

[0051] The reagents and instruments used in the following examples are as follows:

[0052] The AAm aptamer sequence (5ˋ-NH2-C6-ACG GCA ACA TGC CGA AGG AAC TACCGG AAA CGG CAA ATC CTC G-3ˋ) purified by HPLC was provided by Sangon Biotech (Shanghai) Co., Ltd. Thiophene[3,2-B]thiophene-2,5-dicarboxaldehyde (TT), 5,10,15,20-tetra[4-aminophenyl]-21H,23H-porphyrin (TAPP), cobalt acetate tetrahydrate, 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), acrylamide (AAm), carboxylated CdTe quantum dots (CdTe QDs), benzyl alcohol, o-dichlorobenzene, acetic acid, 1,4-dioxane, N,N-dimethylformamide (DMF), potassium ferricyanide, potassium ferrocyanide, potassium chloride, L-aspartic acid (L-Asn), glycine (GLy), L-serine (L-Serine), glucose, fructose, sodium chloride, copper sulfate, and methanol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Chloroform was provided by Guangdong Guangshi Reagent Technology Co., Ltd. Dopamine hydrochloride was supplied by Maclean Biotechnology Co., Ltd. Ferric chloride was supplied by Shanghai Wokai Biotechnology Co., Ltd.

[0053] The microstructure of TT-Por(Co)-COF materials was analyzed using scanning electron microscopy (SEM; ZEISS SUPRA 55, Germany) and transmission electron microscopy (TEM; FEITecnai G2 F20, USA). Crystal diffraction patterns of the prepared COF were obtained by X-ray diffraction spectroscopy (PXRD; Rigaku Ultima IV, Japan). The semiconductor properties, chemical bond information, and elemental valence distribution of the photoactive materials were investigated using ultraviolet-visible diffuse reflectance spectroscopy (UV-DRS), Fourier transform infrared spectroscopy (FT-IR; Thermo Nicolet 380, USA), and X-ray photoelectron spectroscopy (XPS; Thermo ESCALAB 250XI, USA). Thermogravimetric analysis (TGA; PerkinElmer TGA 8000, USA) was used to evaluate the thermal stability of the optoelectronic materials. Electrochemical studies included cyclic voltammetry (CV), differential pulse voltammetry (DPV), linear sweep voltammetry (LSV), open circuit potential (OCP) measurements, and Mott-Schottky curves (MS curves), all performed on a CHI600E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). Electrochemical impedance spectroscopy (EIS) was recorded using a Metrohm-Autolab PGSTAT302N electrochemical workstation. All electrochemical tests employed a three-electrode system, with the modified electrode serving as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. The light source for the photoelectrochemical tests was provided by China Education Jinyuan Co., Ltd. (CEL-PE300L-3A, 300W). Furthermore, comparative method analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS; AB Sciex Triple-TOF 5600+, USA).

[0054] Example 1

[0055] The preparation method of a sandwich-type photoelectrochemical sensor for detecting acrylamide includes the following steps:

[0056] (1) Preparation of Co-TAPP

[0057] 200 mg TAPP and 212 mg Co(OAc)₂·2H₂O were dissolved in 90 mL CHCl₃, followed by the addition of 20 mL methanol and 30 mL DMF solution. The resulting reaction mixture was heated to 80 °C and refluxed under nitrogen protection for 24 h. After the reaction was complete, the solvent was removed by rotary evaporation and the precipitate was collected. After washing three times with methanol, the resulting purplish-black powder was vacuum dried at 60 °C for 12 h to form Co-TAPP powder.

[0058] (2) Preparation of TT-Por(Co)-COF

[0059] TT-Por(Co)-COF material was prepared using a solvothermal method, detailed as follows: 14.67 mg Co-TAPP and 7.9 mg TT were added to 10 mL screw-capped Schott culture tubes and dispersed in a solvent mixture of benzyl alcohol, o-dichlorobenzene, and 6M acetic acid (volume ratio 30:10:4, added in 550 μL). The mixture was sonicated for 15 min and subjected to vacuum treatment through three cycles of freezing-evacuation-thawing. Subsequently, the reaction system was placed in an oven at 120 °C for 72 h. After the reaction, the mixture was cooled to room temperature, washed three times with 1,4-dioxane, and finally dried under vacuum at 80 °C for 12 h to obtain a purplish-black TT-Por(Co)-COF powder.

[0060] (3) Preparation of CdTe QDs / Apt

[0061] 1.5 mL of 10 nmol / mL AAm-Apt, 250 μL of NHS (0.1 mol / L), and 250 μL of LEDC (0.1 mol / L) were mixed and reacted for 1 h. Then, 1 mL of 0.05 μM carboxylated CdTe QDs solution and 2 mL of deionized water were added, and the reaction was continued for 12 h at 500 rpm at room temperature. Finally, the mixture was centrifuged at 6500 rpm for 10 min, and the precipitate was redispersed in 1 mL of deionized water and stored at 4 °C, labeled as CdTe QDs / Apt solution.

[0062] (4) Construction of sandwich-type AAm sensor

[0063] TT-Por(Co)-COF powder was added to methanol at a ratio of 5 mg: 1 mL and ultrasonically dispersed for 20 min to obtain a COF dispersion of 5 mg / mL. 30 μL of the COF dispersion was drop-coated onto the surface of a 10*20*1.1 mm ITO glass electrode and dried to obtain a TT-Por(Co)-COF / ITO modified electrode. The modified electrode was immersed in AAM solutions of different concentrations at room temperature for 40 min to complete the affinity adsorption of AAM. After adsorption was complete, 30 μL of CdTe QDs / Apt solution was drop-added to the surface of the modified electrode, and incubation was continued for 20 min to form a sandwich structure. The modified electrode was rinsed three times with deionized water to remove physical adsorption, yielding a photoelectrochemical sensor, namely the AAM-PECS sensor.

[0064] The modified electrode after incubation was placed in an electrolyte containing 0.1 mM dopamine hydrochloride (DA) and 0.1 M pH 5.6 citrate-sodium citrate buffer to measure the final photocurrent response of the sensor. All photocurrent tests were performed without bias.

[0065] Example 2

[0066] The photoelectrochemical sensor prepared in Example 1 was characterized.

[0067] (1) Preparation and characterization of TT-Por(Co)-COF

[0068] TT-Por(Co)-COF is prepared by solvothermal condensation of TT and Co-TAPP via imine, and its synthetic route is as follows: Figure 1 As shown in Figure A. PXRD of TT-Por(Co)-COF ( Figure 1 B) It exhibits typical AA-stacked COF diffraction peaks. A unit cell model of TT-Por(Co)-COF was constructed using Materials Studio; it belongs to the P1 space group of the mitsubishi crystal system, and the unit cell parameters are... ( Figure 1 A). Based on the experimental PXRD, after Pawley refinement of the unit cell model, the weighted graphical variance factor (R) is... wp The variance factor (R) was 8.08%, and the variance factor of the graph was 8.08%. p The percentage was 6.41%, indicating that the profile difference was reasonable. Its diffraction peaks at 3.24, 6.48, and 7.30° were attributed to the (100), (200), and (020) crystal planes, respectively. Fourier transform infrared spectroscopy (FT-IR) results showed that a C=N imine stretching vibration band appeared at 1583 cm⁻¹ in the TT-Por(Co)-COF material, while the C=O stretching vibration band at 1653 cm⁻¹ disappeared after polymerization, indicating that imine linkages were successfully formed in TT-Por(Co)-COF, completing the Schiff base condensation reaction. Further analysis using scanning electron microscopy (SEM)... Figure 1 D) and transmission electron microscopy (TEM). Figure 1 E) The microstructure of the prepared TT-Por(Co)-COF material was investigated. TT-Por(Co)-COF exhibits a layered nanosheet structure, which was further confirmed by TEM images. High-resolution transmission electron microscopy (HR-TEM) images showed characteristic lattice diffraction of the crystal, with a crystal plane spacing of 2.7 nm, corresponding to the (100) crystal plane of TT-Por(Co)-COF. In addition, the elemental mapping diagram of energy-dispersive X-ray spectroscopy (EDS) was also analyzed. Figure 11The results showed that C, N, O, and Co elements were uniformly distributed in TT-Por(Co)-COF, revealing the overall distribution of these elements in the material. The normalized mass percentage of Co was 7.02%. Further ICP-MS analysis revealed the Co content in TT-Por(Co)-COF to be 5.78%, consistent with the mapping results, indicating the successful introduction of numerous Co porphyrin metal active sites into the framework. Thermogravimetric analysis (TGA) curves were then used to evaluate the thermal stability and decomposition characteristics of TT-Por(Co)-COF. Figure 12 As shown, TT-Por(Co)-COF only shows a significant weight loss peak at 400℃, indicating that the material has good thermal stability.

[0069] (2) Characterization of elemental composition and bonding state of TT-Por(Co)-COF

[0070] XPS was used to further evaluate the elemental composition and bonding states of TT-Por(Co)-COF. Full-spectrum XPS ( Figure 2 A) The signals of C, N, O, and Co in TT-Por(Co)-COF are clearly displayed, which is consistent with the chemical composition of TT-Por(Co)-COF. In the Co2p spectrum ( Figure 2 In B), two peaks were observed at 780.30 eV and 795.70 eV, respectively, which were attributed to Co2p 3 / 2 and Co2p 1 / 2. In the N1s spectrum ( Figure 2 In C), the two peaks at 398.6 eV and 399.7 eV correspond to imine nitrogen and pyrrole nitrogen, respectively. These results fully demonstrate the successful synthesis of TT-Por(Co)-COF.

[0071] (3) Semiconductor property characterization of TT-Por(Co)-COF

[0072] The band gap affects the light absorption and electrical conductivity of a material and is an important parameter for evaluating its optoelectronic properties. This study analyzed the band gap of TT-Por(Co)-COF using ultraviolet-visible diffuse reflectance spectroscopy (UV-DRS). The band gap of the semiconductor can be calculated using the Tauc method, as shown in the following formula:

[0073] (αhv) 1 / n =A(hv-E) g (1)

[0074] Where h is Planck's constant (6.63 × 10⁻⁶). 34 J·s), ν is the frequency, A is a constant, E gLet n be the bandgap value of the semiconductor. For direct bandgap semiconductors, n takes the value of 1 / 2, while for indirect bandgap semiconductors, it takes the value of 2. Furthermore, it was found that when n = 1 / 2, the obtained value is (αhν). 2 The positive slope of hν is the same as that of typical direct bandgap semiconductors, which is consistent with previous studies reporting that porphyrin-imide-linked COFs are direct bandgap semiconductors. Finally, using (αhν)... 2 The relationship with hυ was used to calculate the Tauc curve of TT-Por(Co)-COF, showing a band gap of 1.60 eV. The semiconductor type of the composite material can be evaluated by analyzing the Mott-Schottky curve. The Mott-Schottky curve shows a positive slope (…). Figure 3 C), from which it can be deduced that TT-Por(Co)-COF is an N-type semiconductor. According to the Mott-Schottky formula (2) for N-type semiconductors.

[0075]

[0076] Where: C sc Let ε0 be the space charge capacitance, and ε0 be the vacuum permittivity (8.854 × 102). -12 F / m), ε r Let N be the relative permittivity of the semiconductor at room temperature, A be the contact area between the electrode and the electrolyte solution, and N be the relative permittivity of the semiconductor at room temperature. D E is the donor concentration, and E is the applied voltage. FB The potential is the flat-band potential, and k is the Boltzmann constant (1.38 × 10⁻⁶). -23 J / K), T is the thermodynamic temperature, and e is the electron charge (1.602 × 10⁻⁶). - 19 C). When the ordinate of the Mott-Schottky curve is 0, the value of E can be approximated as the flat-band potential E. FB By extending the curve to intersect the X-axis, the flat-band potential E can be deduced. FB The value is -1.10 eV (vs Ag / AgCl, i.e., -0.90 eV relative to NHE). Theoretically, the flat band potential of an N-type semiconductor is equal to its conduction band potential. The actual valence band potential is studied according to formula (3):

[0077] E VB =E CB +E g (3)

[0078] Its valence band potential E can be obtained from calculation. VB =0.70eV (vs NHE), therefore the band gap range of TT-Por(Co)-COF is -0.90eV to 0.70eV.

[0079] (4) Research on sensing mechanisms

[0080] The novel sandwich-type sensing strategy proposed in this study is based on the affinity recognition mechanism of acrylamide (AAm) by the COF framework and the enhancement effect of the photoelectric conversion effect by the quantum dot-tagged aptamer after incubation. First, the affinity adsorption of AAM by TT-Por(Co)-COF was confirmed by FT-IR. Figure 4 As shown in B, the TT-Por(Co)-COF / AAm modified electrode after affinity adsorption of AAM retains the characteristic infrared peaks of COF, and at 1665 cm⁻¹... -1 The appearance of an olefin stretching vibration peak attributed to AAM suggests a possible affinity recognition effect of the COF framework on AAM. To further elucidate the affinity adsorption mechanism of TT-Por(Co)-COF on AAM, an independent gradient model (IGMH) based on Hirshfeld partitions was employed to investigate the non-covalent interactions between the AAM fragment and TT-Por(Co)-COF. Considering the periodic nature of the system, the CP2K first-principles program was used, which offers more efficient optimization capabilities compared to traditional Gaussian packages. Specifically, the model for AAM adsorption by TT-Por(Co)-COF was geometrically optimized using Quickstep code in CP2K v2023.1 at the DFT PBE-D3(BJ) level. During this process, the Perdew-Burke-Ernzerhof (PBE) exchange-related functional was employed, combined with DFT-D3(BJ) dispersion correction, to accurately describe the intermolecular interactions. The calculations used the GTH pseudopotential and the DZVP-MOLOPT-SR-GTH basis set, with the plane wave cutoff energy set to 400 eV. The convergence criteria for geometry optimization were: SCF convergence criterion of 2.0E-06 eV, and geometry optimization convergence criteria of maximum displacement (MAX_DR) 3E-3 and maximum force (MAX_FORCE) 4.5E-4.

[0081] IGMH analysis was performed on the optimized structure using Multifum wavefunction analysis software combined with VMD visualization. The IGMH mapping revealed the distribution of δg_inter isosurfaces in each expected interaction region. Figure 4As shown in Figure A, two main weak interaction regions exist between TT-Por(Co)-COF and the AAm segment. In these two regions, a weak interaction exists between the sulfur (S) atom in the TT unit and the olefinic group in AAm, characterized by both electrostatic and van der Waals forces. Furthermore, hydrogen bonding interactions also form distinct blue elliptical δg_inter isosurfaces between the amino terminus in AAm and the central region of the imine bond in the COF framework, indicating a high electron density in the NH…N interaction region. These weak interactions (such as… ... Figure 4 (As shown in C) These interactions jointly dominate the affinity adsorption of AAM by TT-Por(Co)-COF and AAM. Further analysis of the mechanism of these interactions reveals that the coordination between electrostatic forces and van der Waals forces can stabilize the interactions between molecules over a large range; while the formation of hydrogen bonds helps to accumulate high electron density in local areas, enhancing the selective adsorption performance of TT-Por(Co)-COF for AAM.

[0082] The enhanced photoelectric conversion effect of quantum dot-tagged aptamers after hatching is another key factor in the successful construction of the sensor. To further analyze the changes in the properties of the photoelectrode after the quantum dots are hatched, we studied the behavior and performance changes of the photoelectrode in detail through open circuit potential (OCP) curves, linear sweep current-voltage (LSV) curves, and photocurrent response tests. OCP testing is an effective means of analyzing the photoelectric properties of semiconductor materials. By measuring the open circuit potential of the material, the separation and recombination efficiency of charge carriers can be evaluated. During the test, the voltage change of the material under illumination was observed by controlling the switching of the light source. The photovoltage decay process reflects the recombination of photogenerated charge carriers and can be used to estimate the average lifetime of photogenerated charge carriers. Formula (4) is as follows:

[0083]

[0084] Where, k B Where is Boltzmann's constant, T is the Kelvin temperature, and e is the positive elementary charge. The switching response of the open-circuit voltage (OCV) of the photoelectrode prepared under illumination was measured by testing the incubated photoelectrode (see...). Figure 5 A) shows that the photoelectrode after hatching quantum dots significantly promotes the generation of electron-hole pairs due to the formation of heterojunctions, resulting in a significantly increased photoelectric potential compared to the original COF photoelectrode. Furthermore, LSV ( Figure 5 B) and PEC curve ( Figure 5C) also exhibits the same trend. These results show that the photoelectrode after hatching quantum dots demonstrates stronger photon trapping ability and stronger photogenerated carrier separation and migration capabilities under illumination, thereby effectively improving photoelectric performance. The introduction of quantum dots not only optimizes the light absorption characteristics of the photoelectrode but also enhances the carrier separation efficiency through the heterojunction effect. After the quantum dots form a heterojunction with the substrate material, the quantum dots can provide additional bandgap modulation at the interface, thereby accelerating the separation of photogenerated electrons and holes. These experimental data confirm the enhancing effect of quantum dot hatching on the photoelectric conversion effect of the photoelectrode, especially under the synergistic effect of the heterojunction, which improves the charge separation and migration performance of the photoelectrode, thereby greatly enhancing the efficiency of the photoelectrochemical reaction. Based on this, this invention constructs a novel sandwich-type photoelectrochemical sensing strategy for acrylamide.

[0085] (5) Electrochemical characterization during sensor fabrication

[0086] In PEC sensors, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are important tools for evaluating the electrochemical properties of the electrode interface, effectively revealing reaction kinetics, mass transfer processes, and charge transfer efficiency. (Using 5 mM Fe(CN)6...) 3- / 4- As an electron probe, the sensor fabrication process was characterized by CV and EIS. Impedance changes were compared between the bare ITO electrode, TT-Por(Co)-COF / ITO, TT-Por(Co)-COF / ITO immersed in AAM, and TT-Por(Co)-COF / ITO electrodes after aptamer adsorption. In the impedance diagram, R... ct The radius of the semicircle corresponding to charge transfer impedance is a key parameter for assessing the ease of electron transfer. A larger radius indicates higher impedance and a more difficult electron transfer process. Figure 6 As shown in Figure B, the ITO electrode modified with TT-Por(Co)-COF exhibits lower impedance (curve b), indicating that TT-Por(Co)-COF accelerates electron transport on the electrode surface, thereby improving the electrode's conductivity. Immersion of TT-Por(Co)-COF in AAM solution slightly increases the impedance (curve c). However, the TT-Por(Co)-COF material with adsorbed aptamers shows a significant increase in impedance (curve d). This may be because the adsorbed substances form a physical or chemical barrier on the electrode surface, hindering efficient electron transport and thus affecting the interaction between electrons and the electrode, limiting electron migration on the electrode surface, increasing interfacial resistance, and leading to an increase in impedance. Furthermore, the changes in the redox peaks in the CV plot show a good correlation with the EIS plot results. Figure 6A). Compared to the bare ITO electrode, the ITO electrode modified with TT-Por(Co)-COF exhibited a lower redox peak potential difference (curves a and b), indicating that the modified electrode has stronger electron transport capability. With the immersion of AAM and adsorption of the aptamer in TT-Por(Co)-COF, the redox peak decreased, consistent with the increase in impedance in the EIS plot, while the current decreased slightly. These results fully demonstrate the successful construction of the AAM-PECS sensor.

[0087] Example 3

[0088] (1) Optimize the analysis conditions

[0089] To obtain optimal performance of the AAM-PECS sensor, a series of key experimental parameters were optimized, including the pH of the electrolyte, the adsorption time of the aptamer, and the soaking time of the AAM solution. Figure 7 As shown in Figure A, the photocurrent intensity of the sensor reaches its maximum value when the pH value is 5.6, indicating that the sensor has the best sensitivity at this time. Figure 7 B shows that, within the immersion time range of 10 to 50 minutes, the photocurrent of TT-Por(Co)-COF in AAM solution peaks at 20 minutes. Figure 7 C indicates that the photocurrent gradually increases with the extension of aptamer adsorption time, reaching a maximum at 40 minutes and then decreasing. Therefore, a citrate-sodium citrate buffer solution at pH 5.6, immersion in AAM solution for 20 minutes, and an aptamer adsorption time of 40 minutes were ultimately selected as the optimal conditions for subsequent experiments.

[0090] (2) Sensor analytical performance

[0091] Under optimal conditions, the analytical performance of the AAM-PECS sensor based on TT-Por(Co)-COF was evaluated. Figure 8 As shown, in the range of 0.001 to 10 3 Within the concentration range of nM, the photocurrent intensity gradually increases with increasing AAM solution concentration. A good linear relationship is observed between the photocurrent and the logarithm of the AAM concentration, with the linear equation: ΔI = 0.3846lg(C) + 4.4728(R). 2=0.991), and calculated using 3σ / S (where n = 11, σ is the slope of the calibration curve at low concentration, and S is the blank standard deviation), the limit of detection (LOD) is 0.19 pM. To ensure the sensor provides accurate and reliable results, response stability is crucial. To evaluate signal stability, eight consecutive photocurrent tests were performed, showing that the photocurrent remained highly stable with a relative standard deviation (RSD) of 0.93%. Furthermore, five AAM-PECS samples were prepared in parallel and treated with PEC using the same concentration of DIM, and the photocurrent response of each sensor was recorded, with a maximum RSD of 6.75%. Finally, the performance of this sensor was compared with other AAM sensors (see Table 1). The results show that this sensor performs excellently over a wider linear range and has a lower limit of detection, demonstrating its superior analytical performance.

[0092] Table 1 compares the developed method with other methods for determining AAM.

[0093]

[0094]

[0095]

[0096] (3) Cross-interference study

[0097] In actual testing, the anti-interference ability of AAM-PECS against interfering substances is crucial. Further testing was conducted to evaluate the cross-interference between AAM-PECS and common interfering substances. The tests included L-aspartic acid (L-Asn), L-serine, glycine (Gly), glucose (Glu), fructose (Fru), and common ions (Cu). 2+ Na + Fe 3+ The concentrations of these organic interfering substances were set to 100 times the AAM concentration, while the concentrations of the ionic interfering substances were set to 1000 times the AAM concentration. From Figure 9 As can be seen from the test results, AAM-PECS exhibits good anti-interference capability when facing these interfering objects, with no significant changes in response. This indicates that AAM-PECS has excellent selectivity and recognition characteristics for AAM, effectively avoiding the influence of common interfering objects and ensuring its reliability and accuracy in practical applications.

[0098] Example 4

[0099] Acrylamide was detected in the actual sample.

[0100] To evaluate the actual detection capability of AAM-PECS, coffee and potato chips were selected as actual samples. The preparation process of the two actual samples is as follows:

[0101] The potato chip samples were purchased from a supermarket in Haikou City. 4g of potato chips ground into powder were mixed with 100mL of deionized water. The resulting solution was centrifuged at 8000rpm for 10 minutes. The supernatant was collected and defatted in 10mL of hexane. Then, 5g of QuECHERS buffer salt was added and filtered. The filtrate was measured.

[0102] The coffee sample was selected from the Aroma and Beverage Research Institute of the Chinese Academy of Tropical Agricultural Sciences. 1g of coffee powder was mixed with 30mL of deionized water, the resulting solution was centrifuged for 10 minutes, and the supernatant was used as the sample to be tested.

[0103] The actual detection performance of AAM was analyzed by performing tests on the samples themselves and supplementing them with spiked recovery tests. The experimental results are shown in Table 2. The spiked recovery rate of AAM ranged from 84.3% to 118.3%, and the maximum RSD did not exceed 7.4%, indicating the potential practicality of AAM-PECS based on TT-Por(Co)-COF / CdTe-COOH QDs / Apt for the detection of AAM in actual samples.

[0104] Table 2. Detection of AAM in potato chip and coffee samples.

[0105]

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a sandwich-type photoelectrochemical sensor for detecting acrylamide, characterized in that: Includes the following steps: (1) Preparation of Co-TAPP 5,10,15,20-tetra[4-aminophenyl]-21H,23H-porphyrin and cobalt salt were dissolved in an organic solvent to obtain a mixture. The mixture was heated under reflux to react. After the reaction was completed, the precipitate was collected, washed and dried to obtain Co-TAPP powder. (2) Preparation of TT-Por(Co)-COF Co-TAPP and thiopheno[3,2-B]thiophene-2,5-dicarboxaldehyde were dispersed in an organic solvent, subjected to ultrasonic treatment and freeze-evacuate-thaw cycle treatment, heated to react, washed and dried to obtain TT-Por(Co)-COF powder; (3) Preparation of CdTe QDs / Apt Acrylamide aptamer, N-hydroxysuccinimide and 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and reacted, followed by the addition of carboxylated CdTe QDs and deionized water. The reaction was continued, and the precipitate was obtained by centrifugation. Deionized water was added to the precipitate again to obtain a CdTe QDs / Apt solution. (4) Add methanol to TT-Por(Co)-COF powder, ultrasonically disperse to obtain COF dispersion, drop the COF dispersion onto the surface of ITO glass electrode, and dry to obtain TT-Por(Co)-COF / ITO modified electrode; The TT-Por(Co)-COF / ITO modified electrode was immersed in acrylamide solutions of different concentrations to complete the affinity adsorption of acrylamide. After the adsorption was completed, CdTe QDs / Apt solution was added to the surface of the TT-Por(Co)-COF / ITO modified electrode to continue incubation to form a sandwich structure. After washing, the photoelectrochemical sensor was obtained.

2. The method for preparing the sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (1), the cobalt salt is Co(OAc)2·2H2O, and the mass ratio of 5,10,15,20-tetra[4-aminobenzene]-21H,23H-porphyrin to the cobalt salt is 2:2~2.5; Organic solvents, in a volume ratio of 8~10:1~3:1~5, include CHCl3, methanol, and DMF; The mass-to-volume ratio of 5,10,15,20-tetra[4-aminophenyl]-21H,23H-porphyrin to CHCl3 is 200 mg: 80~100 mL.

3. The method for preparing the sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (1), the heating temperature is 75~85 ℃ and the reflux time is 20~30 h; the drying temperature is 60~80 ℃ and the drying time is 10~15 h.

4. The method for preparing the sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (2), the mass ratio of Co-TAPP to thiopheno[3,2-B]thiophene-2,5-dicarboxaldehyde is 10~20:5~10; The organic solvents, in a volume ratio of 20-40:5-15:1-5, include benzyl alcohol, o-dichlorobenzene, and 6 M acetic acid; The mass-to-volume ratio of Co-TAPP to organic solvent is 10~20 mg: 500~600 µL.

5. The method for preparing a sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (2), the heating temperature is 100~150 ℃ and the time is 48~96 h.

6. The method for preparing the sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (3), the concentrations of acrylamide aptamer, N-hydroxysuccinimide, 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride and carboxylated CdTe QDs are 8~12 nM, 0.05~0.15 M, 0.05~0.15 M, and 0.03~0.08 µM, respectively. The volumes of acrylamide aptamer, N-hydroxysuccinimide, 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride, carboxylated CdTe QDs, and the first addition of deionized water were 1~2 mL, 200~300 µL, 200~300 µL, 0.8~1.2 mL, and 1~3 mL, respectively.

7. The method for preparing a sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (3), the mass-to-volume ratio of the precipitate and the second addition of deionized water is 0.05~0.1 µg: 5~10 mL.

8. The method for preparing a sandwich-type photoelectrochemical sensor for detecting acrylamide according to claim 1, characterized in that: In step (4), the concentration of COF dispersion is 3~8 mg / mL, the volume of COF dispersion added to the surface of ITO glass electrode is 10~50 µL, the volume of CdTe QDs / Apt solution added to the surface of TT-Por(Co)-COF / ITO modified electrode is 10~50 µL, and the incubation time is 10~30 min.

9. A sandwich-type photoelectrochemical sensor, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the sandwich-type photoelectrochemical sensor according to claim 9, characterized in that: Application of sandwich-type photoelectrochemical sensor in the detection of acrylamide, with a detection range of 0.001~10. 4 The detection limit is 0.19 pM.

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