Method for detecting and visually imaging tumor marker CEA by using integrated cathode shared bipolar electrode chip

Abstract

A novel shared cathode bipolar electrode chip is designed, and a dual-mode biosensing platform is constructed on the basis of the shared cathode bipolar electrode chip and used for realizing high-sensitivity detection and visual imaging of tumor marker carcino-embryonic antigen (CEA). According to the device, through series-parallel connection, the total resistance of a system is reduced, the total current is increased, an Au (at) NiCo2O4 (at) MnO2 nano-enzyme active probe is introduced into a shared cathode, the conductivity is enhanced, and meanwhile, the decomposition of a substrate H2O2 is accelerated and the transfer of electrons in the system is promoted by utilizing the excellent peroxidase activity (POD) of the Au (at) NiCo2O4 (at) MnO2 nano-enzyme active probe, so that the double amplification of an electrochemical luminescence signal is realized. The chip does not need to be regulated and controlled by an external resistor, the sensitivity of the immunosensor is improved, the requirement for accurate detection of disease markers on site is met, a new thought is provided for bipolar electrode devices, and the chip has wide application prospects in biosensing, clinical detection and environmental monitoring.

Description

Technical Field

[0001] In this invention, we designed a new cathode-sharing bipolar electrode chip based on Ohm's law and successfully constructed an ELISA / ECL dual-mode biosensor platform, namely DD-cBPE, which achieved dual-mode detection and visualization imaging of the tumor marker carcinoembryonic antigen (CEA). In addition to introducing nanocatalytic carriers for signal amplification, this system designed a series-parallel circuit based on Ohm's law to achieve dual amplification of ECL signals. In addition, we changed the current in the circuit by adjusting the concentration of TPrA to achieve [Ru(bpy)3] 2+ The ECL signal is close to luminol, and dual signal detection and visual imaging of CEA can be achieved at the same potential. The BPE chip breaks the traditional BPE concept and constructs a longitudinal BPE electrode. The cathode is located in the center of the BPE electrode, and the BPE anode is located at both ends of the electrode, which can realize dual-anode ECL signal detection. Parallel lateral resistors can solve the problem of low ECL signal caused by the high excitation potential and the difficulty in reaching the optimal potential, providing a new idea for timely detection of CEA (POCT). Background Art

[0002] Bipolar electrode electrochemiluminescence is an analytical method that combines bipolar electrodes and electrochemiluminescence technology. A bipolar electrode is a special electrode that can undergo redox reactions at both ends without the need for an external power supply. When a voltage is applied to both ends of a bipolar electrode, a potential difference is generated between the conductor and the solution interface. When the potential difference reaches a certain value, it triggers a redox reaction of the electroactive substances at both ends of the conductor, thereby producing electrochemiluminescence. This luminescence phenomenon does not require other light sources for light excitation, has no background light interference, and has high detection sensitivity. Bipolar electrode electrochemiluminescence technology is widely used and has been successfully applied to many fields, including bioanalysis, environmental monitoring, disease diagnosis, etc. It shows great potential in fields such as biomedicine and environmental science. With the continuous advancement of science and technology, bipolar electrode electrochemiluminescence technology is expected to play an important role in more fields in the future. Summary of the invention

[0003] Based on Ohm's law, the present invention designs a novel shared cathode bipolar electrode chip and successfully constructs a dual-mode biosensing platform, namely DD-cBPE. The device integrates ELISA, ECL, and ECL imaging to achieve highly sensitive detection and visual imaging of the tumor marker carcinoembryonic antigen (CEA).

[0004] To achieve the above purpose, the design idea of ​​the present invention is:

[0005] The total resistance of the system is reduced and the total current is increased by connecting BPE in series and parallel. The Au@NiCo2O4@MnO2 nanozyme activity probe is introduced into the shared cathode to enhance the conductivity. At the same time, its excellent peroxidase activity (POD) is utilized to accelerate the decomposition of the substrate H2O2, promote the transfer of electrons in the system, and realize the double amplification of the ECL signal. In addition, the chip does not require external resistance regulation, which improves the sensitivity of the immunosensor and meets the needs of timely detection. It provides a new idea for the bipolar electrode device and has broad application prospects in biosensing, clinical testing, and environmental monitoring.

[0006] The advantages of the present invention are:

[0007] The device affects the resistance of the system by reasonably regulating the concentration of the co-reactant, and then affects the current in the circuit, ultimately achieving the emission of different luminescent reagents at the same potential with similar luminescence efficiency. In addition, the chip does not require external resistance regulation, which improves the sensitivity of the immunosensor and meets the needs of timely detection. It provides a new idea for the bipolar electrode device and has broad application prospects in biosensing, clinical testing, and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a schematic diagram of the present invention.

[0009] Figure 2 This is a diagram of the experimental device of the present invention.

[0010] Figure 3 It is a schematic diagram of the cathode-sharing bipolar electrode structure and a simplified circuit diagram of the present invention.

[0011] Figure 4 The morphology of the nanozyme of the present invention is characterized. (A) SEM image of the template MnCO3, (B) SEM image of the intermediate NiCo2(OH)6@MnCO3, (C) SEM image of NiCo2O4@MnO2, (D) SEM image of the NiCo2O4@MnO2 area, (E) TEM image of NiCo2O4@MnO2, (F) TEM image of Au@NiCo2O4@MnO2.

[0012] Figure 5 The structural characterization images of the nanozyme NiCo2O4@MnO2 of the present invention. (A) XRD spectra of NiCo2O4@MnO2, NiCo2O4, and MnO2. (B) Fourier transform infrared (FTIR) spectra of NiCo2O4@MnO2, NiCo2O4, and MnO2. (C) XPS full spectrum of NiCo2O4@MnO2.

[0013] Figure 6Figure 1 shows the activity assay of the nanozyme of the present invention. (A) TMB is the reaction substrate, (B) OPD is the reaction substrate. (C) Conductive activity assay of Au@NiCo2O4@MnO2 / NiCo2O4@MnO2.

[0014] Figure 7 This is the ECL emission image of the present invention. The ECL emission wavelength of Luminol. (B)[Ru(bpy)3] 2+ The ECL emission wavelength.

[0015] Figure 8 ECL performance characterization of different modification stages of the shared cathode of BPE of the present invention, CEA concentration is 10 ng / mL. (A) ECL performance characterization of different stages at 425 nm. (B) ECL performance of different stages at 620 nm. (C) EIS characterization of different modification stages. (D) CV characterization of different modification stages.

[0016] Fig. 9 The experimental conditions of the present invention are optimized. (A) Driving voltage optimization, (B) TPrA concentration optimization.

[0017] Fig.10 The experimental conditions of the present invention are optimized. (A) Nanozyme NiCo2O4@MnO2 concentration optimization, (B) pH optimization, (C) reaction time optimization.

[0018] Fig.11 The linear analysis images of the dual-mode detection of CEA by the DD-cBPE biosensor of the present invention. (A) The linear range of CEA detection by ELISA method (0, 0.01, 0.05, 0.1, 1, 5, 10, 50, 100 ng / mL). (B) The corresponding relationship between CEA concentration and absorbance. (C) The linear relationship between logCEA and absorbance. (D) The ECL signal response of different concentrations of CEA at a wavelength of 425 nm (0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 50 ng / mL). (E) The ΔECL change value corresponding to different concentrations of CEA at a wavelength of 425 nm (ΔECL=ECLTarget-ECLBlank). (F) The linear relationship between logCEA and ΔECL change value (ΔECL=ECLTarget-ECLBlank) at a wavelength of 425 nm. (G) ECL signal response of different concentrations of CEA at 620 nm (0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 50 ng / mL). (H) ΔECL change corresponding to different concentrations of CEA at 620 nm (ΔECL = ECLTarget - ECLBlank). (I) Linear relationship between logCEA and ΔECL change value at 620 nm (ΔECL = ECLTarget - ECLBlank).

[0019] Fig.12 The dual-mode visual biosensor platform of the present invention detects CEA. (A) Visual detection of CEA by ELISA, (B) Dual-wavelength ECL detection of CEA.

[0020] Fig.13 The stability of the immunosensor of the present invention. (A) The stability of luminol after 12 consecutive cycles, (B) The stability of [Ru(bpy)3] after 12 consecutive cycles 2+ The corresponding stability.

[0021] Fig.14 The selectivity of the DD-cBPE biosensor platform of the present invention (1 ng / mL). (A) Response to different targets in ELISA mode, (B) ECL response of luminol detection of different targets, (C) [Ru(bpy)3] 2+ Detect ECL responses of different targets.

[0022] Fig.15 The reproducibility of the DD-cBPE biosensor platform of the present invention (1 ng / mL). (A) The reproducibility of the ECL signal of luminol between groups (left) and within groups (right). (B) [Ru(bpy)3] 2+ The inter-group reproducibility (left) and intra-group reproducibility (right) of the ECL signal.

[0023] Fig.16 This is the actual sample recovery rate determination in serum of the present invention. DETAILED DESCRIPTION

[0024] 1. Synthesis of Nanozyme NiCo2O4@MnO2

[0025] 1. Preparation of MnCO3

[0026] Dissolve 5mmol MnSO4·H2O in 350mL secondary water, add to 350mL ethanol under continuous stirring until completely dissolved. Add 50mmol (NH4)2SO4, recorded as liquid a. At the same time, add 50mmol NH4HCO3 aqueous solution to 350mL secondary water, recorded as liquid b. Add liquid b to liquid a under continuous stirring, and react at a constant temperature of 50℃ for 9h. Centrifuge at 9000rpm for 10min, and finally dry under vacuum at 50℃.

[0027] 2. Preparation of NiCo2O4@MnO2

[0028] Under continuous stirring, 45 mL of secondary water was mixed with 15 mL of ethanol, followed by the addition of 0.15 mmol NiCl2·6H2O, 0.3 mmol CoCl2·6H2O, 9 mmol CO(NH2)2, and then 0.1 mmol MnCO3. After stirring for 1 h, the white suspension was transferred to a 100 mL reactor and reacted at 90 °C for 5 h. The suspension was washed three times with ethanol and water respectively, and dried at 60 °C overnight. Finally, the suspension was calcined at 350 °C for 3 h at a heating rate of 1 °C / min.

[0029] 3. Preparation of Ab2@Au@NiCo2O4@MnO2

[0030] In order to ensure that the surface of the material is rich in amino groups, 3 mg of NiCo2O4@MnO2 was ultrasonically dispersed in 1.5 mL of ethanol, and then 50 μL of APTES was added, stirred for 30 min, centrifuged, and washed with ethanol three times. The obtained NH2-NiCo2O4@MnO2 was redispersed in 1 mL of secondary water, 250 μL of HAuCl4·3H2O was added, stirred for 10 min, and 530 μL of freshly prepared 0.5 mM ascorbic acid aqueous solution (AA) was quickly added. After vigorous stirring for 5 min, centrifuged, washed with water twice, and finally dispersed in 1 mL of water. 200 μL of Ab2 (10 μg / mL) was added to the above solution and stirred overnight at 4°C. In order to block the nonspecific active sites on the surface of the material, 100 μL of 0.01% BSA aqueous solution was added and stirred at 4°C for 2 h. The product was centrifuged, washed with water, dispersed in 1 mL of 0.01 M PBS (PH 7.4), and stored at 4°C for use.

[0031] 2. Nanozyme Activity Assay

[0032] Peroxidase activity (POD) assay. 50 μL 5mM H2O2, 50 μL 5mM TMB (DMSO), and finally 50 μL 3 mg / mL NiCo2O4@MnO2 were added to 200 μL 0.1M HAc-NaAc (pH 3) buffer. After a period of reaction, centrifugation was performed, and the supernatant was taken. The absorbance at 652 nm was measured using a UV-visible spectrophotometer. In addition, the POD activity of NiCo2O4@MnO2 was further demonstrated by o-phenylenediamine (OPD). To 200 μL 0.1 M HAc-NaAc (pH 5) buffer, 50 μL 5 mM H2O2, 50 μL 10 mM OPD, and finally 50 μL 3 mg / mL NiCo2O4@MnO2 were added in sequence. After a period of reaction, the mixture was centrifuged, the supernatant was taken, and the absorbance at 450 nm was measured using a UV-visible spectrophotometer.

[0033] A three-electrode system was constructed with a gold electrode as the working electrode, a platinum electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode to verify the conductivity of Au@NiCo2O4@MnO2. 3mg / mL of NiCo2O4@MnO2 and Au@NiCo2O4@MnO2 were dispersed in 350μL of secondary water, 135μL of ethanol, and 15μL of a 5% nafion mixed solution. 15μL of the solution was dropped on the surface of the gold electrode and dried at room temperature. 5mM K3[Fe(CN)6] / K4[Fe(CN)6] was used as the electrolyte, and an external voltage of -0.35 to 0.6V was applied to test the conductivity.

[0034] 3. Biosensor Construction and Detection

[0035] 1. ITO chip pretreatment

[0036] ITO (square resistance of about 15Ohm) chips were purchased from Liaoning Youxuan. After the etched electrodes were ultrasonically cleaned with acetone, ethanol and water in turn, the electrodes were immersed in a 1M potassium alcohol solution (isopropanol solution of KOH) and boiled for 20 minutes. After cooling to room temperature, the secondary water was washed three times to remove the loosely bound hydroxyl groups on the electrode surface. The hydroxylated modified chip was then immersed in a 1% APTES ethanol solution and incubated overnight at 4°C. Rinse three times with ethanol to remove the loosely bound amino groups on the surface. Then heat at 50°C for 30 minutes to ensure that the amino groups are firmly adsorbed on the chip surface.

[0037] 2. Construction of DD-cBPE biosensor platform

[0038] The prepared PDMS membrane was covered on the electrode surface, and 40 μL of 5% GA aqueous solution was introduced into the BPE shared cathode reservoir, incubated at room temperature for 1 h, and the reservoir was washed with 0.01 M PBS. Then 40 μL Ab1 (100 μg / mL) was introduced into the BPE shared cathode reservoir, incubated at 37 ° C for 3.5 h, and washed three times with PBS. Then 40 μL 0.1% BSA was added to seal the plate for 1 h to block nonspecific active sites. After washing with PBS, different concentrations of CEA were introduced and incubated at 37 ° C for 2 h. After washing the reservoir with PBS, 40 μL of ECL probe Ab2@Au@NiCo2O4@MnO2 was introduced and incubated at 37 ° C for 2 h. The reservoir was washed three times with PBS and dried at room temperature for ECL measurement.

[0039] 3. DD-cBPE biosensor platform dual signal detection of CEA

[0040] After the immunosensor was successfully prepared, 100 μL of 100 mM H2O2 was introduced into the BPE shared cathode reservoir, and 100 μL of 2.5 mM [Ru(bpy)3] 2+ / 25mM TPrA. b1, b2 Introduce 100μL 2.5mM Luminol / 0.25mM H2O2 into the BPE anode cell. The PMT is set to 850V, the drive voltage is set to 0-6.5V, the scan rate is set to 0.2V / s, and ECL measurements are performed under 425nm and 620nm filters.

[0041] 4. DD-cBPE system visual detection of CEA

[0042] ECL imaging visualized detection of CEA. After the immunosensor was successfully prepared, the driving voltage was set to 6.5V, and different concentrations of CEA were imaged using a mobile phone.

[0043] Enzyme-linked immunosorbent assay (ELISA) colorimetric visualization detection of CEA. After the ECL imaging detection is completed, rinse the BPE shared cathode pool and add 200μLTMB colorimetric solution (100μL HAc-NaAc, 20μL 5mM TMB, 20μL 5mM H2O2). After incubation at 37℃ for 10min, the absorbance of different concentrations of CEA at 652nm was measured using a UV spectrophotometer. Transfer to a 96-well plate and use a mobile phone for imaging.

[0044] 4. Nanozyme Characterization

[0045] The morphology, size, lattice structure, peak position, and elements of the nanozyme were characterized by SEM, TEM, XRD, infrared, and XPS to prove that the material was successfully prepared. Figure 4 and Figure 5 shown.

[0046] 5. Feasibility Characterization of Biosensing Platform

[0047] luminol / H2O2 and [Ru(bpy)3] 2+ / TPrA are both anode luminescent. After applying the forward driving voltage, a signal is generated at the same time. In order to facilitate the distinction and meet the detection requirements, they are distinguished under the filter and subsequent measurements are performed. Figure 7 As shown, the ECL emission wavelength of luminol is 425nm, [Ru(bpy)3] 2+ The ECL emission wavelength is 620nm. The ECL method, EIS and CV were used to characterize the different modification stages of the shared cathode to prove that the biosensor was successfully prepared. Figure 8 shown.

[0048] 6. Optimization of experimental conditions

[0049] The experimental conditions of the present invention are optimized as follows Fig. 9 , Fig.10 As shown. In order to improve the sensitivity of the immunosensor, the driving voltage, the concentration of the co-reactant TPrA, the concentration of the nanozyme NiCo2O4@MnO2, the system pH and the reaction time were optimized. Under the optimal experimental conditions, a linear analysis was performed.

[0050] 7. Linear and Visual Imaging Analysis

[0051] In the ELISA method for determining CEA, the linear range of CEA concentration is as in the present invention. Fig.11 As shown in A. Fig.11 B and C represent the relationship between CEA concentration and absorbance and the relationship between the logarithm of CEA concentration and absorbance, respectively. Linear equation I1 = 0.516 + 0.149logC, R 2 =0.991, detection limit LOD = 2.5pg / mL. The linear range of luminol detection of CEA is as follows Fig.11 As shown in D, the ECL signal depends on the CEA concentration. The higher the concentration, the more ECL probes are introduced and the more electrons in the system flow to the BPE anode. Fig.11 E and F respectively show the CEA concentration and ΔECL change value (ΔECL = ECL Target -ECL Blank ) and the relationship between the logarithm of CEA concentration and the ΔECL change value. Regression coefficient R 2 =0.992, regression equation y1=13375.87+3053.92logC, detection limit LOD=72.4fg / mL. Fig.11 G introduced [Ru(bpy)3] 2+ The linear range of CEA detection. Fig.11 H and I respectively introduce the CEA concentration and ΔECL change value (ΔECL = ECL Target -ECL Blank ) and the relationship between the logarithm of CEA concentration and the ΔECL change value. Regression coefficient R 2 =0.991, regression equation y1=15026.58+3261.02logC, detection limit LOD=43.65fg / mL. Dual-mode detection visualization imaging Fig.12 As shown in A and B.

[0052] 8. Immunosensor Performance Evaluation

[0053] Stability is an important indicator for evaluating the ECL performance of biosensors. When the CEA concentration is 1 ng / mL, luminol, [Ru(bpy)3] 2+ The corresponding ECL signal is as follows Fig.13 As shown in A and B. After 12 consecutive cycles, the RSD of luminol was 2.94%, [Ru(bpy)3] 2+ The RSD of the results was 1.01%. These results show that the immunosensor has good stability. The selectivity of the DD-cBPE immunosensor was studied using 100 ng / mL PSA, AFP, BSA, and HSA as interferents and 1 ng / mL CEA as the target. Fig.14 As shown in A, B, and C. The ECL signal response and absorbance of the interfering substance are close to the blank value, and the ECL signal and absorbance after the components are mixed are close to the pure CEA signal response. This shows that the biosensor has good selectivity. Reproducibility is another important indicator for evaluating immunosensors. The repeatability of the DD-cBPE biosensor platform was verified by conducting inter-group and intra-group experiments under the same conditions. The results are shown in Fig.15 As shown in A and B, the ECL response of luminol was 2.08% for the inter-group experiment and 2.25% for the intra-group experiment. 2+ The ECL responses corresponded to an inter-group RSD of 2.23% and an intra-group RSD of 2.07%, indicating that the biosensing platform had excellent reproducibility.

[0054] IX. Determination of real samples in serum

[0055] In order to evaluate the ability of DD-cBPE immunosensor to detect real samples, the samples were measured using the spike recovery method. Fig.16 As shown, the serum recovery rates of ELISA and ECL were between 95-99.6%, and the RSDs were less than 5%. The results showed that the DD-cBPE biosensor platform has a good ability to detect real samples and is expected to be used clinically in the near future.

Claims

1. A method for using an integrated cathode-sharing bipolar electrode chip for CEA detection and visualization imaging is characterized by: The device integrates ELISA, ECL, and ECL imaging to achieve highly sensitive detection and visual imaging of carcinoembryonic antigen (CEA). The unique circuit structure of the device not only achieves multiple detections of the target, but also breaks the traditional signal amplification strategy. The total resistance of the system is reduced through BPE series-parallel connection, thereby increasing the total current. The Au@NiCo2O4@MnO2 nanozyme activity probe is introduced into the shared cathode to enhance the conductivity while utilizing its excellent peroxidase activity (POD) to accelerate the decomposition of the substrate H2O2, promote the transfer of electrons in the system, and achieve dual amplification of the ECL signal. The device affects the resistance of the system by rationally regulating the concentration of the co-reactant, thereby affecting the current in the circuit, and ultimately achieving luminescence of different luminescent reagents at the same potential with similar luminescence efficiency.

2. The method for detecting CEA by bipolar electrode electrochemiluminescence according to claim 1, characterized in that: Under the action of glutaraldehyde (GA), Ab1 is coupled to the electrode surface, and the ECL probe with POD activity is fixed on the electrode surface through antigen-antibody specific recognition to form a stable sandwich structure. Under the excellent peroxidase (POD) activity of Au@NiCo2O4@MnO2, TMB is oxidized to generate blue oxTMB, thereby realizing ELISA detection and visualization. In addition, NiCo2O4@MnO2 can accelerate the decomposition of H2O2 and the flow of electrons in the system, so that more electrons flow to the BPE anode, thereby realizing ECL detection and visualization imaging of CEA.

3. The method for preparing a highly sensitive bipolar electrode for electrochemiluminescence detection of CEA according to claim 1-2, characterized in that: The optimal driving voltage of the bipolar electrode is 6.5V.

4. The method for preparing a highly sensitive bipolar electrode for electrochemiluminescence detection of CEA according to claims 1 to 2, characterized in that: The optimal concentration of the coreactant TPrA was 25 mM.

5. The method for preparing a highly sensitive bipolar electrode for electrochemiluminescence detection of CEA according to claims 1 to 2, characterized in that: The optimal concentration of nanozyme NiCo2O4@MnO2 is 3 mg / mL.

6. The method for preparing a highly sensitive bipolar electrode for electrochemiluminescence detection of CEA according to claims 1 to 2, characterized in that: The optimal pH of the system is 6.

7. The method for preparing a highly sensitive bipolar electrode for electrochemiluminescence detection of CEA according to claims 1 to 3, characterized in that: The optimal reaction time between the probe and the co-reactant H2O2 is 10 min.

8. A method for detecting CEA by high-sensitivity bipolar electrode electrochemiluminescence according to claims 1 to 2, characterized in that: The sensitivity, flexibility, controllability and selectivity of the sensor are significantly improved.

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