Preparation method and application of a photoelectrochemical immunosensor portable detector

Abstract

A preparation method and application of a photoelectrochemical immunosensor portable detector, belonging to the field of immunoassay and biosensor technology, specifically discloses the preparation of a new composite material and its further development into an application of a small portable detector, specifically based on the principle of photoelectrochemistry, involving the combination of two materials, MIL-101 (Cr) and cadmium telluride quantum dots, to selectively determine the cancer marker carcinoembryonic antigen in human serum samples or urine samples, and prepare it into a small detection instrument, the shell of which is designed using 3D printing technology, which is easy to use and has good help for the high-speed detection of carcinoembryonic antigen required by the application. The method has important scientific significance and application value for the early diagnosis of cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay and biosensor, and specifically relates to a method for preparing a portable photoelectrochemical immunosensor detector based on the photoelectrochemical principle, which is applied to the detection of carcinoembryonic antigen. Background Art

[0002] Cancer is the second leading cause of death among non-communicable diseases worldwide. Studies have shown that early diagnosis of cancer through the quantification of some biomarkers can be considered a golden means for timely treatment of cancer. Since the serum concentration of carcinoembryonic antigen (CEA) is closely related to colon cancer, rectal cancer, gastric cancer, lung cancer or breast cancer, CEA has been recognized as a broad-spectrum biomarker for cancer diagnosis and prognosis. The normal level of CEA in the serum of healthy people is generally lower than 5.0 ng / mL. Once this concentration threshold is exceeded, there may be a risk of cancer. Therefore, establishing a simple and sensitive detection method for CEA is of great significance for the early diagnosis, prevention and treatment of cancer.

[0003] At present, many methods have been developed for the detection of CEA, such as electrochemistry, fluorescence, chemiluminescence, surface enhanced Raman scattering, enzyme-linked immunosorbent assay, etc. However, most of these methods require expensive equipment and well-trained personnel, which increases the complexity of detection. Photoelectrochemistry is an emerging timely analytical technology in recent years. It has many attractive advantages in disease monitoring and immunoassay, such as good selectivity, high sensitivity, low cost, and simple operation. It can be used as a sensing platform for CEA detection. Metal-organic frameworks (MOFs) are porous materials assembled from organic ligands and metals or metal clusters. They are more developed and applied because of their large volume, large specific surface area, high porosity, and adjustable pore size. However, their wide band gap leads to a small photogenerated carrier density. Studies have shown that quantum dots, metal oxide semiconductors, and hybrid nanocomposites can promote the transfer of photogenerated electrons and amplify photoelectrochemical (PEC) signals due to their narrow band gaps. Therefore, narrow bandgap materials such as quantum dots (QDs) can be compounded with wide bandgap MOFs, and the active carboxyl and amino groups on the surface of MOFs can be covalently coupled with CEA antibodies (Abs), and CEA is further captured by its antibodies, blocking the transfer of photogenerated carriers through steric hindrance, resulting in a decrease in photocurrent. All of the above analysis processes can be integrated into a single small analytical instrument, and based on the above analysis, a portable CEA photoelectrochemical immunosensor is finally constructed.

[0004] In this study, a composite material M&C of MIL-101(Cr)&CdTe-QDs was designed and synthesized, and then connected to the Ab of CEA. The nonspecific binding site was blocked by bovine serum albumin (BSA), and finally modified with CEA. According to the specific recognition of antigen-antibody, a sensor for CEA detection was constructed. According to its characteristic of sensitive detection of CEA, it was further developed into a small analytical detector. Since the photocurrent change of MIL-101(Cr) changes with the change of the applied working voltage, a working system of "anode photocurrent-CEA concentration-cathode photocurrent" is formed; at the same time, the composite CdTe-QDs can improve the density of its photogenerated carriers. Therefore, the sensor can not only realize the real-time detection of CEA, but also realize its own correction. In addition, compared with the previous method of detecting CEA, only a small amount of blood or urine is needed to understand the CEA index, which reduces the pain of patients to a certain extent and reduces the related detection costs and health burden. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a method for preparing and applying a rapid, sensitive, portable and low-cost photoelectrochemical immunosensor portable detector to achieve ultra-sensitive detection of the cancer marker carcinoembryonic antigen.

[0006] The portable detector provided by the present invention can significantly reduce the influence of other complex matrices in the sample and perform accurate quantitative analysis of carcinoembryonic antigen based on the characteristics of antigen-antibody specific recognition. At the same time, the photoelectrochemical technology has high detection accuracy and low detection limit. The designed portable detector is small in size and easy to use. It is very suitable for the detection of CEA content, solves the problems of long detection time and low patient compliance, and sensitively determines CEA, a cancer marker.

[0007] The present invention is achieved through the following technical solutions:

[0008] A preparation method of a photoelectrochemical immunosensor portable detector, the preparation method is as follows:

[0009] (1) Synthesis and purification of MIL-101(Cr) material

[0010] MIL-101(Cr) is synthesized by a hydrothermal method in the absence of hydrofluoric acid. Chromium nitrate nonahydrate, terephthalic acid and deionized water are mixed in proportion, ultrasonicated, and mixed evenly to obtain a dark blue suspension, which is placed in a high-pressure reactor and heated in an oven. The obtained suspension is filtered to obtain a filter cake, which is a crude product of MIL-101(Cr). The crude product is washed with hot N,N-dimethylformamide, then washed with hot anhydrous ethanol, and dried to obtain a MIL-101(Cr) material.

[0011] (2) Synthesis and purification of cadmium telluride quantum dots (CdTe-QDs)

[0012] Dissolve 2.5 hydrated chromium chloride in ultrapure water, add 3-mercaptopropionic acid, stir, adjust the pH to 10.5-11.0 with 1 mol / L NaOH solution, add potassium tellurite solution and sodium borohydride solution, stir and reflux to obtain CdTe-QDs, dialyze the obtained CdTe-QDs solution (500Da), freeze-dry and set aside;

[0013] (3) Preparation of composite modified electrodes

[0014] Disperse MIL-101(Cr) and CdTe-QDs in a water / anhydrous ethanol mixed solution with a volume ratio of (2-5):1, respectively, to obtain a MIL-101(Cr) dispersion with a concentration of 5 mg / mL and a CdTe-QDs dispersion with a concentration of 4 mg / mL, dry the above dispersions to obtain powders, take 70 mg±0.5 mg of CdTe-QDs powder and 30 mg±0.5 mg of MIL-101(Cr) powder, dissolve them in 0.5-3 mL of a water / anhydrous ethanol mixed solution with a volume ratio of (2-5):1, perform ultrasonic mixing to obtain an M&C suspension, take 40-80 μL of the M&C suspension and drop it on a FTO conductive electrode, dry it at 120-140°C for 30-90 min, and obtain a composite material M&C modified electrode (M&C / FTO);

[0015] (4) Preparation of photoelectrochemical (PEC) immunosensor

[0016] 30-50 μL Ab was drop-coated on the M&C / FTO electrode, incubated at 4°C for 90 min, and then the redundant Ab was washed off with PBS buffer, and 30-50 μL 1wt% bovine serum albumin (BSA) was drop-coated on the electrode to block nonspecific binding sites, incubated at 4°C for 60-120 min, and the electrode surface was washed with PBS buffer solution, and 30-50 μL of a series of CEA solutions of different concentrations were drop-coated, and incubated at 4°C for 60-120 min to produce specific immune binding of antigen and antibody, thereby preparing a photoelectrochemical immunosensor;

[0017] (5) Photoelectrochemical immunosensor portable detector

[0018] The three-electrode system (counter electrode, reference electrode, working electrode) is integrated into a system as the detector body. The minimum system of the stm32f103 microcontroller, a voltage regulator module (5V), a liquid crystal display, an analog-to-digital converter (external voltage is 12V), a battery pack motor, a transimpedance amplifier, an analog voltage module and a sealed shell, as well as a PBS solution and a simulated sunlight light source are selected to form a portable detector for portable and rapid detection of carcinoembryonic antigen.

[0019] In the step (3), the FTO is 2×2 cm 2 The circular slices were rinsed with acetone, ultrapure water and ethanol for 30 minutes before use. The mass proportion of CdTe-QDs in the M&C composite material is 10wt% to 80wt%, among which 70wt% CdTe-QDs has the best M&C photoelectric effect.

[0020] In the step (4), the pH of the PBS buffer is 7.4.

[0021] The preparation of a photoelectrochemical immunosensor portable detector for detecting carcinoembryonic antigen is as follows:

[0022] Relying on the specific binding of antigen and antibody, the prepared photoelectrochemical immunosensor portable instrument was used to analyze and detect CEA samples of different concentrations. The M&C / FTO electrode was incubated with CEA samples of different concentrations at room temperature for a period of time, and the prepared electrodes were labeled as CEA / M&C / FTO. The CHI-660E electrochemical workstation was used to perform photocurrent testing with a three-electrode system, with the M&C / FTO electrode as the working electrode, the platinum wire as the auxiliary electrode, and Ag / AgCl as the reference electrode. A xenon lamp with a 420nm filter was used as the light source (CEL-HXF300, Beijing). Using photoelectrochemical technology, the photocurrent changes after different concentrations of CEA standards were loaded onto the M&C / FTO electrode were recorded to obtain a standard curve, and then the CEA in the actual sample was selectively determined based on the standard curve. All tests were performed at room temperature and repeated three times.

[0023] The steps of determining the content of carcinoembryonic antigen and drawing a standard curve are as follows:

[0024] ① Use 5-15 mL, 0.1 M phosphate buffer solution with pH = 7.4 to prepare CEA standard solutions of different concentrations, and apply 20-50 μL of CEA solutions of different concentrations to the surface of the M&C / FTO electrode modified step by step with antibody (Ab) and bovine serum albumin (BSA), respectively. At this time, the electrode is marked as CEA / M&C / FTO, incubated at 4°C for 60-120 minutes, and connected to the electrochemical workstation after drying. The electrodes are immersed in a phosphate buffer solution with pH = 7.4, and the light source is turned on to irradiate the surface of the CEA / M&C / FTO electrode. At this time, the photocurrent intensity of CEA / M&C / FTO is measured;

[0025] ② Based on the linear relationship between the obtained current value and the logarithmic value of CEA concentration, a standard curve was drawn;

[0026] ③Use the standard curve method to obtain the concentration of CEA in the sample to be tested.

[0027] The operation steps of the PEC portable detector are as follows:

[0028] ① Use 5-15 mL, 0.1 M phosphate buffer solution with pH = 7.4 to prepare CEA standard solutions of different concentrations, and apply 20-50 μL of CEA solutions of different concentrations to the surface of the M&C / FTO electrode modified with antibody (Ab) and bovine serum albumin (BSA) step by step. At this time, the electrode is marked as CEA / M&C / FTO, incubate at 4°C for 60-120 minutes, and connect it to the detector after drying;

[0029] ② Immerse the PEC portable detector in step ① into the PBS solution, illuminate the surface of the CEA / M&C / FTO electrode with an external light source, and adjust the voltage;

[0030] ③ The photocurrent intensity at this time is displayed on the LCD screen, and the reading time is less than 30s. The read value corresponds to the concentration of carcinoembryonic antigen (0.0001ng / mL-10ng / mL), thereby realizing rapid and portable detection of carcinoembryonic antigen.

[0031] Compared with the traditional method for determining carcinoembryonic antigen, the present invention has the following advantages:

[0032] The present invention designs a photoelectrochemical (PEC) immunosensor that can generate dual photocurrent signals, which can be accurately used for rapid detection of carcinoembryonic antigen (CEA). At the same time, it is further developed into a small rapid detector for carcinoembryonic antigen to achieve portable and rapid detection of carcinoembryonic antigen.

[0033] In order to obtain the best sensor performance, the present invention evaluates the influence of various experimental parameters, including the optimal use volume of the M&C composite material, the ultrasonic mixing time of the composite material, the type of solution, the pH of the solution and the incubation time, etc. The results show that the drop volume of the M&C composite material is 75 μL, the ultrasonic mixing time of the composite material is 30 minutes, 0.1M PBS solution with a pH of 7.4 is determined as the base solution, and the antigen-antibody co-incubation is 90 minutes.

[0034] The photoelectric performance of the prepared PEC sensor was investigated. First, the optimal working voltage when MIL-101(Cr) / FTO was used as the working electrode was screened. The results showed that -0.3V and +0.3V were the optimal cathode voltage and anode voltage. Afterwards, the photocurrent changes of MIL-101(Cr) before and after CdTe-QDs modification were investigated. When the applied voltage was +0.3V and -0.3V, the anode photocurrent and cathode photocurrent of M&C showed an enhanced trend. Considering that the amount of CdTe-QDs added may affect the final photocurrent intensity of M&C, it was finally decided to select M&C at 70wt% (CdTe-QDs) as the final modified material for the working electrode.

[0035] The construction process of the PEC immunosensor was analyzed, and the photocurrent and electrochemical impedance with the gradual introduction of Ab, BSA and CEA were investigated. The results showed that with the gradual introduction of biomaterials, the electrochemical impedance value gradually increased and the photocurrent gradually decreased, proving the successful construction of the sensor.

[0036] The photoelectrochemical immunosensor prepared by the present invention was used to detect carcinoembryonic antigen, and the results showed that it had a good recovery rate. It was further developed into a small sensor detector for carcinoembryonic antigen, which is expected to be better applied in the rapid clinical detection process of carcinoembryonic antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 (A) Specific surface area and (B) pore size analysis of MIL-101(Cr);

[0038] Figure 2 This is the transmission electron microscope (TEM) spectrum of MIL-101(Cr);

[0039] Figure 3 This is the scanning electron microscope (SEM) image of MIL-101(Cr);

[0040] Figure 4 Transmission electron microscopy (TEM) of CdTe-QDs;

[0041] Figure 5 This is the high-resolution transmission electron microscopy (HR-TEM) image of CdTe-QDs;

[0042] Figure 6 is the scanning electron microscope (SEM) image of the composite material M&C;

[0043] Figure 7 UV-visible diffuse reflectance (DRS) spectra of MIL-101(Cr), CdTe-QDs and M&C;

[0044] Figure 8 Fourier transform infrared (FT-IR) spectra of MIL-101(Cr), CdTe-QDs and M&C;

[0045] Fig. 9 X-ray diffraction analysis (XRD) spectra of MIL-101(Cr), CdTe-QDs and M&C;

[0046] Fig.10 are the photoluminescence (PL) spectra of MIL-101(Cr), CdTe-QDs, and M&C;

[0047] Fig.11 Energy dispersive X-ray (EDS) layered images and spectra of MIL-101(Cr);

[0048] Fig.12 Energy dispersive X-ray (EDS) layered images and spectra of M&C;

[0049] Fig.13 X-ray photoelectron (XPS) spectroscopy for M&C;

[0050] Fig.14 is the photocurrent intensity of MIL-101(Cr) under different applied voltages;

[0051] Fig.15 To screen the photocurrent changes after combining different amounts of CdTe-QDs with MIL-101(Cr);

[0052] Fig.16 The photocurrent comparison and electrochemical impedance value after the optimal amount of CdTe-QDs combined with MIL-101(Cr);

[0053] Fig.17 The photocurrent and electrochemical impedance changes after step-by-step modification of Ab, BSA and CEA;

[0054] Fig.18 To assist experimental mechanism verification for Tauc plot and MS plot;

[0055] Fig.19 Optimize conditions for different experimental parameters;

[0056] Fig. 20 The photocurrent change and linearity graph of the detection of carcinoembryonic antigen standard;

[0057] Fig.21 To investigate the specificity and stability of PEC sensors;

[0058] Fig. 22 Schematic diagram of the prepared small sensor detector;

[0059] Fig.23 Schematic diagram of the internal circuit and product structure of the prepared small sensor detector. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but it does not constitute any limitation to the present invention. Any limited modifications made within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0061] Instruments, Equipment and Reagents

[0062] 1.1 Instrument:

[0063] Electronic analytical balance, kq5200 ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.), Fourier transform infrared spectroscopy (IFS-66 FTIR, Bruker, Germany), scanning electron microscope (FEI Helios Nanolab 600iSEM, USA), transmission electron microscope and high-resolution transmission electron microscope (JEOL Ltd. JEM-2100F TEM), UV-visible diffuse reflectance spectrophotometer (UV197 Vis DRS, Lambda1050), energy dispersive X-ray spectrometer (EDS), X-ray photoemission spectrometer (ESCALAB 250Xi, Thermo, USA), X-ray diffraction (XRD) was measured by using D8 Advance diffractometer (Bruker, Germany), and X-ray photoelectron spectroscopy (XPS) was measured on ESCALAB 250Xi The photoelectrochemical detection was carried out on an X-ray photoemission spectrometer, and the photoelectrochemical detection process was carried out on a photoelectrochemical workstation (CHI-660E, China). The light source used was a 500 W xenon lamp (CEL-HXF300, Beijing, China).

[0064] 1.2 Drug testing

[0065] Deionized water (Wahaha Group Co., Ltd.), sodium hydroxide and anhydrous ethanol (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.), acetone (Shandong Yuwang Industrial Co., Ltd.), terephthalic acid (Shanghai McLean Biochemical Co., Ltd.), chromium nitrate nonahydrate (Shanghai Nibo Biochemical Technology Co., Ltd.), cadmium chloride hemihydrate (Shanghai McLean Biochemical Co., Ltd.), potassium tellurite (Shanghai McLean Biochemical Co., Ltd.), 3-mercaptopropionic acid (Shanghai Bid Pharmaceutical), carcinoembryonic antigen standard (Shanghai Zeye Biotechnology Co., Ltd.).

[0066] Example 1

[0067] A method for preparing a photoelectrochemical immunosensor, the preparation method is as follows:

[0068] (1) Synthesis and purification of MIL-101(Cr) material

[0069] MIL-101(Cr) was synthesized by hydrothermal method in the absence of hydrofluoric acid. Cr(NO3)·9H2O (2.0g, 5mmoL), terephthalic acid (H2BDC, 0.83g, 5mmoL) and 20mL deionized water were mixed and ultrasonically treated for 30min. After mixing evenly, a dark blue suspension with a pH of 2.58 was obtained. The suspension was placed in a 25mL stainless steel autoclave lined with polytetrafluoroethylene and heated at 218℃ in an oven for 18h. After the reaction was completed, it was cooled to room temperature to obtain a suspension with a pH of 0.5. The filter cake was collected by filtration to obtain a dark blue product. The product was washed with N,N-dimethylformamide at 60℃ for 3h each time to remove unreacted H2BDC, and then washed with anhydrous ethanol at 60℃ for 3h each time to replace the N,N-dimethylformamide molecules in the pores of the crude MIL-101(Cr). The sample was dried in an oven at 60°C for 2 h and then dried at 150°C for 10 h under vacuum conditions to remove the ethanol molecules adsorbed in the pores, thereby obtaining the MIL-101(Cr) material.

[0070] (2) Synthesis and purification of cadmium telluride quantum dots (CdTe-QDs)

[0071] Dissolve 0.2mmoL CdCl2·2.5H2O in 50mL ultrapure water, add 20μL 3-mercaptopropionic acid (MPA), stir vigorously, adjust pH to 10.5-11.0 with 1moL / L NaOH solution, add 50mL 0.8mmoL / L K2TeO3 solution. Then add 1mL 85mg / mL NaBH4 solution and stir for 5min, stir and reflux at 100℃ for 90min to obtain cadmium telluride carbon quantum dots. The obtained red carbon quantum dot solution is dialyzed (500Da), freeze-dried and set aside.

[0072] (3) Preparation of CdTe-QDs / FTO, MIL-101(Cr) / FTO and M&C / FTO

[0073] Cut FTO into 2×2cm 2 The circular slices were rinsed with acetone, ultrapure water and ethanol for 30 minutes. The prepared MIL-101(Cr) was dispersed in a water / anhydrous ethanol mixture with a volume ratio of 3:1, with a concentration of 5 mg / mL. 75 μL was applied on FTO and dried at 120°C to obtain MIL-101(Cr) / FTO, which was used for the subsequent test of the photocurrent ratio of MIL-101(Cr) and M&C composite materials; 70 mg of CdTe-QDs powder and 30 mg of MIL-101(Cr) powder (both of which were dissolved and dried in a 3:1 water / anhydrous ethanol mixed solution) were dissolved in 1 mL of a water / anhydrous ethanol solution with a volume ratio of 3:1, and an M&C suspension was obtained after ultrasonic dispersion for 30 minutes. 75 μL of the M&C suspension was applied on FTO and dried at 120°C to obtain M&C / FTO for later use.

[0074] (4) Preparation of PEC immunosensor

[0075] The M&C / FTO electrode prepared in step (3) was incubated with 45 μL Ab solution for 90 min, and then the redundant Ab was washed away with PBS buffer (0.1 M, pH = 7.4) to obtain an Ab / electrode. 45 μL BSA (1wt%) was drop-coated on the surface of the Ab / electrode to block nonspecific binding sites. After incubation for 90 min, the electrode was washed with PBS buffer solution to obtain a BSA / Ab / electrode. The BSA / Ab / electrode was incubated with 45 μL CEA for 90 min to produce specific immune binding of antigen and antibody to obtain a photoelectrochemical immunosensor. All incubation processes were carried out in a sealed container in a 4°C refrigerator.

[0076] Characterization and detection:

[0077] ① Characterization of the prepared MIL-101(Cr), CdTe-QDs and M&C materials

[0078] The size and morphology of the nanocomposites and the surface element distribution were characterized by specific surface area (BET) test, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, UV-visible diffuse reflectance (DRS) spectroscopy, X-ray diffraction analysis (XRD) spectroscopy, photoluminescence (PL) spectroscopy, energy dispersive X-ray (EDS) spectroscopy, and X-ray photoelectron (XPS) spectroscopy.

[0079] Through the specific surface area (BET) test, the trend of the adsorption / desorption isotherm of MIL-101(Cr) shows that MIL-101(Cr) belongs to mesoporous materials, and its pore size is mainly distributed in the area of ​​about 2.0nm. Figure 1 )BET test results show that its specific surface area is 2192.5604m 2 / g, average pore diameter 2.5964nm, pore volume 1.5257cm 3 / g. The above results show that the ordered pores of MIL-101(Cr) can be used as a "microreactor" in which uniform and stable cadmium telluride quantum dots with nanoscale can be assembled to form a composite material. The SEM of MIL-101(Cr) shows that it presents a regular octahedral structure. ( Figure 2 )TEM images showed that the particle size ranged from 180 to 360 nm (number of particles considered = 13), ( Figure 3 ) The edge roughening in several crystals seen in the image may be attributed to the presence of residual organic solvent in the pores during the synthesis of MIL-101(Cr).

[0080] Low-resolution TEM of CdTe-QDs ( Figure 4 ) showed that CdTe-QDs were smooth spherical, uniform in size, well dispersed, with high crystallinity, in a monodisperse state, with an average particle diameter of 2.77nm (the number of particles considered = 32), and distributed in a narrow diameter range (2.10nm-3.30nm). High-resolution TEM ( Figure 5 ) showed that CdTe-QDs have high crystallinity and its lattice parameter is 0.23nm.

[0081] No CdTe-QDs ( Figure 6 ), but the particles were obviously abundant, proving the preparation of M&C, and there was no obvious change in the morphology of MIL-101(Cr), indicating that ultrasonic mixing did not change the structure of MIL-101(Cr).

[0082] The optical properties of MIL-101(Cr), CdTe-QDs and M&C were analyzed by UV-vis DRS spectroscopy. Figure 7 ). MIL-101(Cr) exhibits two characteristic absorption bands centered at 432.5nm and 598.5nm, which is consistent with previous studies. The band of MIL-101(Cr) in the ultraviolet region belongs to the π-π* transition of the ligand, and the band in the visible region can be attributed to Cr 3+The dd spin allows transitions. It can be seen that the overall UV absorption trend of the composite material M&C is basically consistent with the UV absorption of MIL-101(Cr), which is attributed to the absorption of the MIL-101(Cr) matrix and is consistent with the SEM characterization, indicating that the basic structure of MIL-101(Cr) has not changed after the composite. In addition, CdTe-QDs have a wide absorption in the visible light region. These results show that the introduction of CdTe-QDs effectively expands the light absorption range of MIL-101(Cr). This also means that a heterojunction is formed between MIL-101(Cr) and CdTe-QDs, and the interaction between the two plays a role in enhancing the visible light absorption.

[0083] As shown by Fourier transform infrared spectroscopy (FT-IR) analysis ( Figure 8 ), 3345cm in the blue curve -1 、1628cm -1 The band at 590 cm indicates the presence of guest water molecules adsorbed in the MIL-101(Cr) cage. -1 and 1405cm -1 The bands at 1509cm correspond to the plane bending and symmetric vibration of OCO, respectively, indicating the presence of dicarboxylate linkers in the MIL-101(Cr) framework. -1 The band at 3446 cm is attributed to the C=C stretching vibration of the benzene ring in the MIL-101(Cr) framework. -1 The broad absorption peak at 1576 cm-1 is attributed to the -OH in the carboxyl group. In addition, -COO-(1576 cm-1) was observed in the spectrum of CdTe-QDs. -1 and 1449cm -1 ), indicating that there may be carboxyl groups on the surface of CdTe-QDs. -1 There are strong broad absorption bands of OH stretching mode at ~1500cm -1 and ~1600cm -1 There is a sharp peak of C=O at the bottom, which strongly confirms the presence of -OH and -COOH on the surface of MIL-101(Cr) and CdTe-QDs. The FT-IR of M&C (red curve, the bottom curve) is similar to that of MIL-101(Cr) and CdTe-QDs, indicating that there is no interaction between MIL-101(Cr) and CdTe-QDs.

[0084] XRD spectra of MIL-101(Cr), CdTe-QDs and M&C are shown in Figure 2. Fig. 9As shown. The lattice parameters obtained from XRD measurements are suitable for the cubic sphalerite structure of bulk CdTe-QDs crystals. The synthesized product has three diffraction peaks at 2θ=25.7°, 28.05° and 45.5°. The standard X-ray diffraction (XRD) spectrum of CdTe-QDs corresponds to the (111), (200) and (311) crystal planes of cubic CdTe-QDs, respectively, indicating that the prepared CdTe-QDs quantum dots have a relatively good crystal structure. The figure (red line) shows five diffraction peaks of MIL-101(Cr) (2θ=5.3°, 8.6°, 9.2°, 10.4°, 16.6°). The peak shape is consistent with the literature (Mohammadian, R.; Amini, MM; Shaabani, A. Thiourea-functionalized MIL-101(Cr)metal-organic framework as a hydrogen-bond-donating heterogeneousorganocatalyst for the Friedel-Crafts alkylation and Biginellireactions. Catal Commun 2020, 136, 1566-7367. DOI: 10.1016 / j.catcom.2019.105905), indicating the successful preparation of MIL-101(Cr). After MIL-101(Cr) was modified with CdTe-QDs, the basic structure of the parent MOF was retained after the approximate peak shape of CdTe-QDs was introduced into the final composite.

[0085] Fig.10 The photoluminescence spectra of the three materials excited at 437nm were recorded. All materials have absorption at 439nm, among which MIL-101(Cr) has the largest fluorescence intensity, CdTe-QDs has the smallest fluorescence intensity, and M&C is in between the two, indicating that CdTe-QDs inhibit the recombination of photogenerated carriers and holes in MIL-101(Cr), thereby improving the separation effect of photogenerated carriers.

[0086] EDS analysis can reveal the elemental composition of a sample. Fig.11 and Fig.12 The EDS images of MIL-101(Cr) and M&C respectively show that C, O, and Cr elements are distributed on the surface of MIL-101(Cr), and C, O, Cr, Cd, and Te elements are distributed on the surface of M&C. The inset shows the proportion of the content of each element in the selected area. This also strongly proves that the preparation of M&C is successful.

[0087] In addition, the XPS spectrum of M&C ( Fig.13 ) confirmed the presence of C (55.13%), Cd (0.2%), Te (0.2%), O (43.42%), and Cr (1.05%) elements, indicating that CdTe-QDs were successfully loaded on MIL-101 (Cr). The peaks of Cd3d appeared at 411.9 eV and 405.3 eV, corresponding to Cd 2+ Cd 3d 3 / 2 and Cd 3d 5 / 2 The binding energies at 582.48 and 572.14 eV in the Te 3d spectrum are attributed to the Te 3d 3 / 2 and Te 3d 5 / 2 The peaks of Cr 2p appear at 587.16eV and 577.69eV, respectively, which are attributed to Cr 2p 1 / 2 and Cr 2p 3 / 2 signal, proving that Cr in M&C 3+ The C1s spectrum shows three peaks at 284.46eV, 285.34eV and 288.57eV, which correspond to CC / C=O, CO and C=O in terephthalic acid in MIL-101(Cr) and CdTe-QDs, respectively. The above results indicate the successful preparation of M&C.

[0088] ② Photocurrent performance test of MIL-101(Cr), CdTe-QDs and M&C materials

[0089] After MIL-101(Cr) was prepared, the optimal working voltage when it was used as a working electrode was first screened. Because high applied potential (anode or cathode potential) will have an adverse effect on the surface state of biomaterials and photoelectrodes, it is not conducive to the further preparation of PEC immunosensors. Therefore, the applied voltage range of -0.3V to +0.3V was mainly studied. Fig.14 It shows that the photocurrent of MIL-101(Cr) changes with the applied voltage. The cathode photocurrent and anode photocurrent reach the maximum values ​​when the applied voltage is -0.3V and +0.3V, which are -(34.99±0.02)nA and +(40.13±0.06)nA respectively. Therefore, -0.3V and +0.3V are finally determined to be the optimal cathode voltage and anode voltage.

[0090] Considering that the amount of CdTe-QDs added may affect the final photocurrent intensity of M&C, it was investigated. Fig.15 It shows that the photocurrent change trend of M&C containing 70wt% CdTe-QDs is the most obvious. Combined with the most suitable amount of the material, it is decided to select M&C containing 70wt% CdTe-QDs as the final modification material for the working electrode.

[0091] Next, the photocurrent changes of MIL-101(Cr) before and after modification with CdTe-QDs were investigated. Fig.16 It can be seen that when the CdTe-QDs accounted for 70wt% and the applied voltage was +0.3V, the anode photocurrent of M&C was enhanced, which was 14 times that of MIL-101(Cr); when the applied voltage was -0.3V, the cathode photocurrent of M&C was enhanced, which was 12 times that of MIL-101(Cr). This result is also consistent with the electrochemical impedance spectroscopy ( Fig.16 ) are confirmed.

[0092] ③Construction process of PEC immunosensor

[0093] During the preparation of photoelectrochemical sensors, the changes in photocurrent and electrochemical impedance spectroscopy (EIS) are effective methods for monitoring them. Fig.17 As shown, the anode and cathode photocurrents decrease successively. This may be because the steric hindrance of the material interface on the surface of the FTO electrode is continuously reduced, resulting in a decrease in the excitation efficiency of CdTe-QDs, a gradual increase in the inhibition rate of CEA on photoelectric conversion, a gradual increase in photoelectrochemical impedance, and a gradual decrease in the photocurrent signal.

[0094] ④Analysis of experimental mechanism

[0095] In order to further explore the working mechanism of the prepared PEC immunosensor, the band gap, flat band (E fb ) and the Fermi level. Fig.18 As shown, the E of MIL-101(Cr) g is 2.38 eV, and the E g The MS curve shows that compared with the standard hydrogen electrode, the E of MIL-101(Cr) is 1.83eV. fb is -0.31 V vs. RHE, and the E fb The slope of the MS curve of MIL-101(Cr) is positive, indicating that it is an n-type semiconductor. Its ECB value is higher than its E fb On the contrary, the slope of the MS curve of CdTe is negative, indicating that it is a p-type semiconductor. VB Value than its E fb The value is 0.2eV higher. After calculation, the E CBThe value is -0.51eV vs.RHE, and the E VB The value is 1.29eV vs.RHE. According to the formula: E VB =E CB +E g (E CB is the conduction band (CB) edge potential, E VB is the valence band (VB) edge potential, E g is the band gap energy. ), the E of MIL-101(Cr) is obtained VB The value is 1.87eV vs.RHE, and the E CB The value is -0.54eV vs. RHE. Therefore, the potential electron transfer pathway is deduced, such as Fig.18 When the applied voltage is -0.3 V, the electron transport path may conform to "Type II" or "Type Z". However, the UPS test results show that the Fermi E of MIL-101(Cr) f is -0.39 eV, and the E f Lower than MIL-101(Cr), which is -0.27eV. f The levels are different and the charge is redistributed to achieve E f That is, electrons from n-type MIL-101(Cr) (higher E f ) diffuses into p-type CdTe-QD (lower E f ), while holes move in the opposite direction. Therefore, the potential electron transfer path is as follows Fig.18 When the applied voltage is -0.3V, the electron transfer path may follow the "Z-type" electron transfer path. Photogenerated electrons are given from the FTO electrode, and the FTO detects a relatively positive charge, generating a cathode photocurrent. After that, the electrons are transferred from the VB of MIL-101(Cr) to its CB, and then to the VB of CdTe-QDs, and finally to the CB of CdTe-QDs. Due to its CB value is higher than O2 / O2 - The potential of (-0.33 eV) is more negative, so it can reduce O2 in the electrolyte to O2 -. When the applied voltage is 0.3V, it is inferred that the driving force of the applied voltage on the electrons is greater than the driving force of the external light source, and the Fermi level is not considered, forming a "Type II" electron transfer path. The photogenerated electrons are transferred from the CB of CdTe-QDs to the CB of MIL-101 (Cr) that is relatively closest to it, and the electrons are transferred to the FTO surface, and the output is in the form of anode photocurrent. At the same time, electrons are transferred on the surface of CdTe-QDs, leaving electron holes (h+), and the VB value of CdTe-QDs is more positive than the potential of H2O / O2 (1.23eV), thereby oxidizing H2O in the electrolyte solution to O2. Both photocurrent generation processes can be changed by changing the applied voltage. Through the "II" type and "Z" type electron transfer pathways, the rate of electron-hole recombination is reduced, and the photoelectric performance is improved.

[0096] ⑤ Optimization of experimental conditions

[0097] In order to obtain the best sensor performance, the optimal volume of the composite M&C, the ultrasonic mixing time of the composite, the type of solution, the pH of the solution and the incubation time were optimized ( Fig.19 ). The experiment was carried out at room temperature (25±0.5℃). First, the effect of the amount of the composite material M&C on the performance of the sensor was investigated. In the composite material M&C, when the concentration of CdTe-QDs was 70mg / mL and the concentration of MIL-101(Cr) was 30mg / mL, the photocurrent response change trend was the largest when the drop volume was 75μL, so 75μL was selected as the optimal amount of the composite material M&C. Secondly, the effect of the ultrasonic mixing time of the composite material on the performance of the sensor was investigated. After 30min of ultrasound treatment of the MIL-101(Cr) solution and the CdTe-QDs solution, the photocurrent reached the maximum value, and with the extension of the ultrasound time, the photocurrent intensity tended to be stable. After that, the type of solution was investigated to select the best base solution. The photocurrent intensity of M&C in 0.1M PBS solution was the largest, and finally 0.1M PBS solution was determined as the base solution. The photocurrent intensity of M&C in PBS solutions under different pH conditions was further investigated, and finally the base solution was determined to be a PBS solution with pH=7.4 as the best buffer solution. Since whether the antigen and antibody can effectively bind has a significant impact on the successful use of the sensor, the optimal incubation time of the antigen and antibody was investigated and selected. Fig.19 As shown, the photocurrent reached the best quenching effect when the antigen and antibody were co-incubated for 90 minutes, and the photocurrent intensity no longer changed significantly with the extension of the incubation time.

[0098] Example 2

[0099] The specific operation of using the photoelectrochemical immunosensor to detect carcinoembryonic antigen is as follows:

[0100] Relying on the specific binding of antigen and antibody, the prepared M&C / FTO electrode was used to analyze CEA samples of different concentrations. The M&C / FTO electrode was incubated with CEA samples of different concentrations at room temperature for 90 minutes, and the prepared electrodes were labeled as CEA / M&C / FTO. After cleaning, it was used for photocurrent testing. The photocurrent test was carried out using a CHI-660E electrochemical workstation with a three-electrode system, with the M&C / FTO electrode as the working electrode, the platinum wire as the auxiliary electrode, and Ag / AgCl as the reference electrode. A xenon lamp with a 420nm filter was used as the light source. All tests were carried out at room temperature, and the photoelectrochemical measurements were repeated three times.

[0101] Based on the above optimal conditions, a series of concentrations of CEA markers were tested. The results showed that Fig. 20 As shown in the figure, when the applied voltage is 0.3V and -0.3V respectively, the photocurrent response changes with the increase of CEA concentration, and shows good linearity in the range of 0.0001ng / mL to 10ng / mL. The anode photocurrent conforms to the linear equation I(nA)=-100.19log C CEA (ng / mL)+59.31(R 2 =0.9968, n=18), the detection limit is 0.00018ng / mL (S / N=3). The cathode photocurrent conforms to the linear equation I (nA) = 76.17log C CEA (ng / mL)-107.62(R 2 =0.9910, n=18), the detection limit is 0.00023ng / mL (S / N=3). In clinical medicine, the critical value of CEA is 3ng / mL. Therefore, the photoelectrochemical immunosensor constructed by the present invention has high sensitivity and can meet the detection requirements.

[0102] Specificity, reproducibility and stability of PEC sensors:

[0103] 5ng / mL bovine serum albumin (BSA), α-fetoprotein (AFP) and 5ng / mL prostate antigen (PSA) were selected as interfering substances. Under the same conditions, the above interfering substances were detected by the same method. The experimental results are as follows: Fig.21 As shown in Figure 2, only the mixed solution of 1 ng / mL target protein CEA and all substances showed a large photocurrent change, and the photocurrent response values ​​of other interfering substances were basically consistent with the background photocurrent. Therefore, it can be seen that the prepared immunosensor has good selectivity.

[0104] Stability is an important indicator for evaluating sensor performance. Fig.21As shown in the figure, when measuring 1 ng / mL CEA (anode) and 0.1 ng / mL CEA (cathode), the anode and cathode photocurrents did not decay significantly within 600 s. In addition, the prepared immunosensor was stored in a refrigerator (4°C) for 15 days, and its photoelectrochemical response value could still be preserved by 92% after 15 days. The above results show that the prepared sensor has good stability during use. Next, its repeatability was evaluated. Under the same conditions, 6 electrodes were prepared and cultured with 1 ng / mL CEA. When the electrode was used as an anode, the relative standard deviation (RSD) of the test results was 2.41%; when the electrode acted as a cathode, the RSD value was 1.20%.

[0105] Carcinoembryonic antigen test:

[0106] The photoelectric properties of the M&C PEC sensor in the detection of CEA in serum samples were analyzed by the standard addition method. 0.0005 ng / mL, 0.01 ng / mL, and 5.00 ng / mL of CEA were added to serum samples and urine (as shown in Tables 1 and 2). The results showed that the prepared M&C PEC sensor had high sensitivity in the detection of CEA in serum and urine samples.

[0107] Table 1 Anodic determination of CEA in human serum and urine samples (n=6)

[0108]

[0109] Table 2 Cathodic determination of CEA in human serum and urine samples (n=6)

[0110]

[0111] Example 3

[0112] A photoelectrochemical immunosensor portable detector and preparation method:

[0113] The photoelectrochemical immunosensor portable detector is as follows: Figure 22-23 As shown, it includes: a detector body, a liquid crystal display, a battery pack motor, a transimpedance amplifier, an analog voltage conversion module, three electrodes (counter electrode, reference electrode, working electrode), a voltage regulator module (5V), a minimum system of a single-chip microcomputer of stm32f103, an analog-to-digital converter (external voltage is 12V) and a sealed shell.

[0114] Among them, the battery pack motor is installed inside the detector body, and the output shaft of the battery pack motor is connected to the single-chip microcomputer through a voltage stabilizing module to generate a stable external voltage. The analog voltage conversion module converts the stable voltage output by the single-chip microcomputer. An analog-to-digital converter is provided on one side of the detector to generate a stable digital signal with a specific duty cycle, and a transimpedance amplifier component is connected to it to amplify the low current signal originally detected. A liquid crystal display is provided in the upper and middle part of the main body of the detector for reading the test results. The sealed shell is installed on the detector body, and the detector battery pack motor and all microprocessors connected to the detector battery pack motor are arranged in the sealed shell. Three electrodes (counter electrode, reference electrode, working electrode) are connected to the analog voltage conversion module and the transimpedance amplifier to ensure the input of the detection signal. The detector battery pack motor is embedded in the sealed shell of the detector through transition matching, and all microprocessors are fixed by metal clips and black tapes, and the liquid crystal display is fixed on the back by black tape, so as to achieve the effect of combining with the shell of the detector.

[0115] The sealed shell of the photoelectrochemical immunosensor portable detector is realized by 3D printing. The model of the 3D printer is Chuangxiang 3D ender-3, and the printing material used is polylactic acid (PLA), commonly known as corn material, which is a fully biodegradable and environmentally friendly material. The parameters for the sealed shell of the detector using 3D printing are as follows: Slicing parameters: (1) Layer height: 0.2mm; (2) First layer height: 0.2mm; (3) Vertical shell: Number of outer circles: 3; Spiral structure: None; (4) Horizontal shell: Physical layer: Top layer 3; Bottom layer 3; (5) Order of printing the innermost or outermost circle: Print the innermost circle first; (6) Seam position: Align all seams; (7) Filling density: 30%; (8) Filling structure: Honeycomb filling; Printing parameters: (1) Printing speed: Circumferential speed: 30mm / s; Outer Circumferential speed: 70%; (2) Filling speed: 60 mm / s; (3) Solid speed: 60 mm / s; (4) Top solid filling speed: 60 mm / s; (5) Support material speed: 60 mm / s; (6) Bridge speed: 60 mm / s; (7) Groove speed: 20 mm / s; Printer settings: (1) Extruder head: 0.4; (2) Retraction length: 3 mm; (3) Retraction speed: 40 mm / s; (4) Layer change and wire withdrawal: None; (5) Extruder head temperature: 190°C; (6) Hot bed temperature: 50°C.

[0116] The advantages and significance of the photoelectrochemical immunosensor portable detector are as follows: (1) In the present invention, the analog voltage conversion module and the transimpedance amplifier are respectively embedded on both sides of the three electrodes to realize the functions of short-time rapid power supply and amplification of the detection signal; (2) An analog-to-digital converter is installed inside the detector to realize the function of real-time tracking and detection of early cancer-related indicators by converting the detected current signal with the concentration signal of carcinoembryonic antigen; (3) In combination with 3D printing technology, polylactic acid material is used for the design of the sealed shell of the detector, which realizes the advantages of good dimensional stability, non-toxicity and environmental friendliness of the sealed shell of the detector, while ensuring the wear resistance of the detector during use.

[0117] During the carcinoembryonic antigen detection process, PBS solution and external light source can be prepared in advance, the PEC portable detector is immersed in the PBS solution, the external light source is irradiated on the surface of the working electrode, and the voltage is adjusted. The photocurrent intensity at this time can be displayed on the LCD screen, which corresponds to the concentration of carcinoembryonic antigen, thereby realizing rapid and portable clinical detection of carcinoembryonic antigen.

[0118] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a photoelectrochemical immunosensor portable detector, characterized in that: The following steps are involved: (1) Synthesis and purification of MIL-101(Cr) material Chromium nitrate nonahydrate, terephthalic acid and deionized water are mixed in proportion, ultrasonicated and uniformly mixed to obtain a dark blue suspension, the suspension is placed in a high pressure reactor, heated in an oven, the obtained suspension is filtered to obtain a filter cake, i.e. a crude product of MIL-101(Cr), the crude product is washed with hot N,N-dimethylformamide, then washed with hot anhydrous ethanol, and dried to obtain a MIL-101(Cr) material; (2) Synthesis and purification of CdTe-QDs Dissolve 2.5 hydrated chromium chloride in ultrapure water, add 3-mercaptopropionic acid, stir, adjust the pH to 10.5-11.0 with 1 mol / L NaOH solution, add potassium tellurite solution and sodium borohydride solution, stir and reflux to obtain CdTe-QD, dialyze the obtained CdTe-QDs solution, freeze-dry and set aside; (3) Preparation of composite modified electrodes MIL-101(Cr) and CdTe-QDs were respectively dispersed in a water / anhydrous ethanol mixed solution with a volume ratio of (2-5):1 to obtain a MIL-101(Cr) dispersion with a concentration of 5 mg / mL and a CdTe-QDs dispersion with a concentration of 4 mg / mL. The above dispersions were dried to obtain powders. 70 mg±0.5 mg of CdTe-QDs powder and 30 mg±0.5 mg of MIL-101(Cr) powder were dissolved in 0.5-3 mL of a water / anhydrous ethanol mixed solution with a volume ratio of 0.5-3 mL, and ultrasonically mixed to obtain a M&C suspension. 40-80 μL of the M&C suspension was dropped on a FTO conductive electrode and dried at 120-140°C for 30-90 min to obtain M&C / FTO. (4) Preparation of photoelectrochemical immunosensor 30-50 μL Ab was drop-coated on the M&C / FTO electrode, incubated at 4°C for 90 min, and then the redundant Ab was washed off with PBS buffer, and 30-50 μL 1wt% bovine serum albumin was drop-coated on the electrode to block nonspecific binding sites, incubated at 4°C for 60-120 min, and the electrode surface was washed with PBS buffer solution, and 30-50 μL of a series of CEA solutions of different concentrations were drop-coated, and incubated at 4°C for 60-120 min to produce specific immune binding of antigen and antibody, thereby preparing a photoelectrochemical immunosensor; (5) Photoelectrochemical immunosensor portable detector The three-electrode system is integrated into a system as the detector body, and the stm32f103 microcontroller minimum system, a voltage regulator module, a liquid crystal display, an analog-to-digital converter, a battery pack motor, a transimpedance amplifier, an analog voltage module and a sealed housing, as well as a PBS solution and a simulated sunlight light source are selected to form a portable detector.

2. The method for preparing the photoelectrochemical immunosensor portable detector according to claim 1, characterized in that: In the step (3), the FTO is 2×2 cm 2 The circular slices were rinsed with acetone, ultrapure water and ethanol for 30 minutes in sequence before use.

3. The method for preparing the photoelectrochemical immunosensor portable detector according to claim 1, characterized in that: In the step (3), the mass proportion of the CdTe-QDs in the M&C composite material is 10wt% to 80wt%.

4. The method for preparing the photoelectrochemical immunosensor portable detector according to claim 1, characterized in that: In the step (4), the pH of the PBS buffer is 7.4.

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