Solid-state LEDs-based electrochemical luminescence closed bipolar electrode chip array for simultaneous detection and imaging of multiple tumor markers

Abstract

A novel bipolar electrode electrochemical luminescence array biosensing platform, namely ML-cBPE, is designed on the basis of a high-flux bipolar electrode chip and a light emitting diode (LED), and is used for realizing multi-target simultaneous detection and visual imaging. As a semiconductor material, the luminous efficiency of LEDs is far higher than that of a traditional luminous reagent. Miniaturization serves as the outstanding advantage of the LED, and seamless connection between the LED and the BPE can be achieved conveniently. An ECL probe Ab2 (at) Au (at) MnO2 with peroxidase activity is introduced into a BPE cathode array through enzyme-linked immunosorbent assay in a BPE sensing array, H2O2 can be accelerated under the action of enzyme, more electrons in a system can flow to an LED report array, and multi-target sensitive detection and visual imaging are achieved. Compared with a solution, the solid-state electrochemical luminescence has lower excitation potential and more obvious luminescence, and plays a role in protecting the electrode to a certain extent. The portable, integration and miniaturization are well embodied, and the requirement for clinical timely detection (POCT) is met.

Description

Technical Field

[0001] In the present invention, we designed a new bipolar electrode electrochemiluminescent biosensor platform, namely ML-cBPE, based on a high-throughput bipolar electrode array and LEDs for simultaneous detection and visualization of AFP, CEA, and PSA. Miniaturization, high luminescence efficiency, long duration, and stable luminescence are the outstanding advantages of LEDs. As a substitute for traditional luminescent reagents, the waste of luminescent reagents is reduced. The BPE cathode serves as the detection end and the anode serves as the reporting end. The ECL probe Ab2 / Au / MnO2 with peroxidase activity (POD) is introduced into the BPE cathode array by the sandwich method. Under the action of MnO2, it can catalyze the decomposition of H2O2, accelerate the transfer of electrons in the system, and make more electrons flow to the LEDs array, thereby realizing the simultaneous detection of multiple targets. The device integrates the wireless characteristics of bipolar electrodes and the stable luminescence characteristics of LEDs, realizes the deviceization of bipolar electrodes, and meets the needs of POCT detection. 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] The present invention designs a novel bipolar electrode electrochemiluminescent biosensing platform, namely ML-cBPE, based on a high-throughput bipolar electrode array and LEDs for simultaneous detection and visualization of AFP, CEA, and PSA.

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

[0005] As a semiconductor material, LEDs have a luminous efficiency far exceeding that of traditional luminescent agents. Miniaturization, as a prominent advantage of LEDs, facilitates the seamless connection between LEDs and BPE. In the BPE sensor array, the ECL probe Ab2@Au@MnO2 with peroxidase activity is introduced into the BPE cathode array through enzyme-linked immunosorbent assay. Under the action of the enzyme, H2O2 can be accelerated, so that more electrons in the system flow to the LED reporting array, achieving sensitive detection of multiple targets and visual imaging. Compared with solutions, solid-state electrochemiluminescence has a lower excitation potential and more obvious luminescence, which plays a role in protecting the electrode to a certain extent. Portability, integration, and miniaturization are well reflected, meeting the needs of timely detection (POCT) in clinical practice.

[0006] The advantages of the present invention are:

[0007] As a semiconductor material, LEDs have a luminous efficiency far exceeding that of traditional luminescent agents. Miniaturization, as a prominent advantage of LEDs, facilitates the seamless connection between LEDs and BPE. In the BPE sensor array, the ECL probe Ab2@Au@MnO2 with peroxidase activity is introduced into the BPE cathode array through enzyme-linked immunosorbent assay. Under the action of the enzyme, H2O2 can be accelerated, so that more electrons in the system flow to the LED reporting array, achieving sensitive detection of multiple targets and visual imaging. Compared with solutions, solid-state electrochemiluminescence has a lower excitation potential and more obvious luminescence, which plays a role in protecting the electrode to a certain extent. Portability, integration, and miniaturization are well reflected, meeting the needs of timely detection (POCT) in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0009] Figure 2 It is a diagram of the experimental device, a physical diagram and a visualization schematic diagram of the present invention.

[0010] Figure 3 The morphology and structure characterization images of the nanozyme of the present invention. (A) SEM image of MnCO3. (B) Scanning electron microscope (SEM) image of MnO2. (C) TEM image of MnO2. (D) Transmission electron microscope (TEM) image of Au@MnO2. (E) XRD spectrum of MnO2. (F) Infrared spectrum of MnO2. (G) Full XPS spectrum of MnO2. (H) High-resolution XPS spectrum of Mn 2p. (I) High-resolution XPS spectrum of O 1s.

[0011] Figure 4 This is a graph showing the activity determination of the nanozyme of the present invention.

[0012] Figure 5The ECL emission images of the present invention. (A) ECL emission wavelength of red light. (B) ECL emission wavelength of green light. (C) ECL emission wavelength of yellow light

[0013] Figure 6 Feasibility characterization of the ML-cBPE biosensor platform of the present invention, CEA / PSA / AFP: 0.1 ng / mL. (A) 555nm feasibility characterization (AFP). (B) 590nm feasibility characterization (PSA). (C) 640nm feasibility characterization (CEA). (a) Bare (b) Bare / Ab1 (c) Bare / Ab1 / BSA (d) Bare / Ab1 / BSA / (CEA / PSA / AFP) (e) Bare / Ab1 / BSA / (CEA / PSA / AFP) / Ab2@Au@MnO2.

[0014] Figure 7 The experimental conditions of the present invention are optimized. (A) (B) (C) APTES incubation time optimization. (D) (E) (F) Glutaraldehyde (GA) incubation time optimization. (G) (H) (I) pH optimization.

[0015] Figure 8 The linearity, stability, selectivity and reproducibility analysis images of the ML-cBPE biosensor for detecting AFP. (A) The linear range of AFP detected by ECL method at a wavelength of 555 nm (0, 0.001, 0.01, 0.1, 1, 10, 50 ng / mL). (B) The ECL change value corresponding to the AFP concentration (ΔECL=

[0016] ECL Target -ECL Blank ). (C) The relationship between the logarithm of AFP concentration and the ECL change value. (D) The stability of 0.1 ng / mL AFP after 10 cycles. (E) Validation of ML-cBPE selectivity using AFP as the target. (F) ML-cBPE reproducibility.

[0017] Fig. 9 The linearity, stability, selectivity and reproducibility analysis images of the ML-cBPE biosensor for detecting PSA. (A) The linear range of PSA detected by ECL method at a wavelength of 590 nm (0, 0.001, 0.01, 0.1, 1, 10, 50 ng / mL). (B) The ECL change value corresponding to the PSA concentration (ΔECL=

[0018] ECL Target -ECL Blank). (C) The relationship between the logarithm of PSA concentration and the ECL change value. (D) The stability of 0.1 ng / mL PSA after 10 cycles. (E) Verification of ML-cBPE selectivity using PSA as the target. (F) ML-cBPE reproducibility.

[0019] Fig.10 The linearity, stability, selectivity and reproducibility analysis images of the ML-cBPE biosensor for detecting CEA of the present invention. (A) The linear range of CEA detected by ECL method at a wavelength of 590 nm (0, 0.001, 0.01, 0.1, 1, 10, 50 ng / mL). (B) The ECL change value corresponding to the CEA concentration (ΔECL=

[0020] ECL Target -ECL Blank ). (C) The relationship between the logarithm of CEA concentration and the change in ECL value. (D) The stability of 0.1 ng / mL CEA after 10 cycles. (E) Verification of ML-cBPE selectivity using CEA as the target. (F) ML-cBPE reproducibility.

[0021] Fig.11 This is the determination of the AFP recovery rate in serum of the present invention.

[0022] Fig.12 This is the determination of the PSA recovery rate in serum of the present invention.

[0023] Fig.13 This is the determination of the recovery rate of CEA in serum of the present invention. DETAILED DESCRIPTION

[0024] 1. Synthesis of Nanozyme NiCo2O4@MnO2

[0025] 1. Preparation of MnO2

[0026] MnCO3 was synthesized according to previous literature

[157] . 5mmol MnSO4·H2O was dissolved in 350mL secondary water and added to 350mL ethanol under continuous stirring until it was completely dissolved. 50mmol (NH4)2SO4 was added, recorded as liquid a, and 50mmol NH4HCO3 aqueous solution was added to 350mL secondary water, recorded as liquid b. Liquid b was added to liquid a under continuous stirring, and the reaction was continued at a constant temperature of 50℃ for 9h. Centrifuged at 8500rpm for 10min, and finally dried under vacuum at 50℃. The final product was calcined at 350℃ in air atmosphere at a heating rate of 1℃ / min for 3h to obtain the final product MnO2.

[0027] 2. Preparation of Ab2 / Au / MnO2

[0028] 3 mg of MnO2 was dissolved in 1.5 mL of ethanol solution, and 50 μL APTES was added after ultrasonication for 5 min, and stirred at 800 rpm for 30 min, and washed with ethanol for 3 times. Then, it was dispersed in 1 mL of secondary water, and 250 μL of 1% HAuCl4 aqueous solution was added. After vigorous stirring for 10 min, freshly prepared 0.5 mM ascorbic acid (AA) was added, and stirring was continued for 5 min. After purification in secondary water for three times, it was dispersed in 1 mL of 0.1 M PBS buffer. Then, 100 μg / mL of Ab2 (CEA, PSA, AFP) was added to the Au / MnO2 PBS mixture, incubated at 37 ° C for 12 h, purified by PBS for three times, and finally dispersed in 0.01 M PBS buffer containing 0.05% Tween 20 and 0.001% BSA.

[0029] 2. Nanozyme Activity Assay

[0030] Peroxidase activity (POD) assay: 50 μL 5 mM H2O2, 50 μL 5 mM TMB (DMSO), and finally 50 μL 3 mg / mL MnO2 were added to 200 μL 0.1 M HAc-NaAc (pH 3) buffer. After a period of reaction, centrifuge, take the supernatant, and measure the absorbance at 652 nm using a UV-visible spectrophotometer.

[0031] 3. Biosensor Construction and Detection

[0032] 1. ITO chip pretreatment

[0033] ITO (square resistance of about 15 Ohm) chips were purchased from Liaoning Youxuan. The ITO electrodes were washed with acetone, ethanol, and water in turn, and then dried with nitrogen. The cleaned electrodes were soaked in a 1:1:5 H2O2 / NH3·H2O / H2O mixture for 30 minutes, and then rinsed with water three times. Then, they were immersed in a 1% APTES ethanol solution and incubated overnight, rinsed with ethanol three times, and fixed at 50°C for 30 minutes.

[0034] 2. Construction of ML-cBPE biosensor platform

[0035] PDMS was fixed on the electrode surface. The detailed preparation process of PDMS can be found in Chapter 2. In the high-throughput BPE cathode array reservoir, 40 μL of 5% GA aqueous solution was added, incubated at room temperature for 1 hour, and rinsed with water twice. Then 30 μL (CEA, PSA, AFP) of Ab1 (100 μg / mL) was added to different reservoirs of the BPE cathode array, incubated at 37°C for 3.5 hours, and the reservoir was washed 3 times with PBS. 0.01% BSA blocked nonspecific active sites for 30 minutes. Then different concentrations of CEA, PSA, and AFP were added respectively, incubated at 37°C for 2 hours, and the reservoir was washed with PBS. Finally, the ECL probe Ab2 / Au / MnO2 (CEA, PSA, AFP) was introduced, incubated at 37°C for 2 hours, the reservoir was washed with PBS, and dried at room temperature.

[0036] 3. ML-cBPE system can simultaneously detect CEA, PSA, AFP and visualize images

[0037] After the immunosensor was successfully prepared, the red, yellow, and green light-emitting diodes were fixed to the BPE anode with conductive silver tape, and 100 μL of 0.25 mM, 2.5 mM, and 25 mM H2O2 were introduced into the BPE cathode reservoir to detect CEA, PSA, and AFP. The photomultiplier tube PMT was set to 800 V, the drive voltage was set to 0-3.5 V, and the scan speed was set to 0.2 V / s. ECL measurements were performed using filters of 640 nm, 590 nm, and 555 nm. The drive voltage was set to 3.5 V, and a mobile phone was used to image CEA, PSA, and AFP at different concentrations.

[0038] 4. Nanozyme Characterization

[0039] 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 3 shown.

[0040] 5. Feasibility Characterization of Biosensing Platform

[0041] Feasibility of ML-cBPE biosensing platform Figure 6As shown. By introducing different concentrations of H2O2 into the BPE sensor array, the LED reporter array performs signal readout. With the gradual modification of Ab1, BSA, and target (CEA, PSA, AFP), the electrode surface impedance increases, resulting in a slower transfer rate of electrons on the electrode surface and a gradual decrease in the ECL signal. After the ECL probe Ab2@Au@MnO2 is immunoadsorbed on the electrode surface, MnO2 can catalyze the decomposition of H2O2, accelerate electron transfer, and allow more electrons to flow to the LED, thus enhancing the ECL signal, proving that the ML-cBPE biosensor platform was successfully prepared.

[0042] 6. Optimization of experimental conditions

[0043] The experimental conditions of the present invention are optimized as follows Figure 7 In order to improve the sensitivity of the immunosensor, the incubation time of APTES, the incubation time of glutaraldehyde (GA), and the pH were optimized. The experimental results are shown in Figure 7 Under the best experimental conditions, linear analysis was performed.

[0044] 7. Linear and Visual Imaging Analysis

[0045] Under the optimal experimental conditions, the ECL signal was used to measure the different concentrations of AFP. The relationship between the AFP and ECL changes at 555nm wavelength is as follows: Figure 8 As shown in A. In the range of 0.001-50 ng / mL (ag), the ECL signal is positively correlated with the concentration of the target AFP. The higher the concentration, the larger the corresponding ECL signal value. Figure 8 B shows the change in AFP and ECL (ΔECL = ECL Target -ECL Blank ) correspondence and visualization imaging. Figure 8 C is the fitting curve of AFP concentration logarithm and ECL change value, regression coefficient R2 = 0.991, linear regression equation y1 = 10902.91 + 2498.10logC, detection limit LOD = 90fg / mL. The relationship between PSA and ECL change at 590nm wavelength is as follows Fig. 9 As shown in A. In the range of 0.001-50 ng / mL (ag), the ECL signal is proportional to the concentration of the target PSA. Fig. 9 B shows the change of PSA and ECL (ΔECL = ECL Target -ECL Blank ) correspondence. Fig. 9 C is the fitting curve of the logarithm of PSA concentration and ECL change value, regression coefficient R2 = 0.996, linear regression equation y2 = 10177.49 + 2234.75logC, detection limit LOD = 63.1fg / mL. The relationship between CEA and ECL change at 640nm wavelength is as follows Fig.10 As shown in A. In the range of 0.001-50 ng / mL (ag), the ECL signal is proportional to the concentration of the target CEA. Fig.10 B shows the change in CEA and ECL (ΔECL = ECL Target -ECL Blank ) correspondence. Fig.10 C is the fitting curve of CEA concentration logarithm and ECL change value, and the regression coefficient R 2 =0.996, linear regression equation y3=11087.16+1910.38logC, detection limit LOD=3.98fg / mL.

[0046] 8. Immunosensor Performance Evaluation

[0047] Stability is an important indicator for evaluating the ECL performance of biosensors. Under the optimal experimental conditions, different concentrations of AFP were measured by ECL signals. Figure 8 D is the stability corresponding to 10 cycles of continuous circulation of 0.1 ng / mL AFP. The fluctuation range of the ECL signal is not obvious, indicating that the immunosensor has good stability. In order to verify the selectivity of the immunosensor, 0.1 ng / mL AFP was used as the target, and 50 ng / mL PSA, HSA, CEA, and BSA were used as interferences. The results are shown in Figure 2. Figure 8 E. The ECL signal of the interfering substance did not change significantly, indicating that the sensor had good selectivity. Finally, in order to verify the reproducibility of the immunosensor, under the same conditions, 6 groups of ML-cBPE sensing platforms with the same concentration from different batches were constructed for verification. The ECL change values ​​of sensors from different batches were similar, indicating that the sensor had good reproducibility. The results are shown in Figure 8 As shown in F. Fig. 9 D is the stability corresponding to 10 cycles of continuous cycling of 0.1 ng / mL PSA. The ECL signal does not change significantly, indicating that the immunosensor has good stability. In order to verify the selectivity of the immunosensor, 0.1 ng / mL PSA was used as the target, and 50 ng / mL AFP, HSA, CEA, and BSA were used as interferences. The results are shown in Fig. 9 As shown in E, the interference signal is close to the blank value, indicating that the biosensor has good selectivity. Fig. 9 As shown in Figure F, the ECL change values ​​of sensors from different batches are similar, indicating that the sensor has good reproducibility. Fig.10D is the stability corresponding to 10 cycles of continuous cycling of 0.1 ng / mL CEA. The ECL signal did not change significantly, indicating that the immunosensor has good stability. In order to verify the selectivity of the immunosensor, 0.1 ng / mL CEA was used as the target, and 50 ng / mL PSA, HSA, HSA, and BSA were used as interferences. The concentration of the interferences was close to the blank value, indicating that the platform has good selectivity. The results are shown in Fig.10 As shown in E. Fig.10 F shows the reproducibility of the immunosensor at this wavelength, and the results indicate that the platform has good reproducibility.

[0048] IX. Determination of real samples in serum

[0049] In order to evaluate the ability of ML-cBPE immunosensor to detect real samples, the samples were measured by spike recovery method, and the results are shown in Tables 11, 12, and 13. The ECL method was used to detect the actual samples of AFP, PSA, and CEA, and the serum recovery rate was between 95-99.7%, and the RSD was less than 5%. The results show that the ML-cBPE biosensor platform has a good ability to detect real samples and is expected to be used in clinical practice.

Claims

1. Based on solid-state LEDs electrochemiluminescence closed bipolar electrode chip array for simultaneous detection and imaging of multiple tumor markers, the characteristics are: The bipolar electrode electrochemiluminescence array biosensor platform, namely ML-cBPE, is used to achieve simultaneous detection of multiple targets and visualized imaging. As a semiconductor material, LEDs have a luminescence efficiency far exceeding that of traditional luminescent reagents. Miniaturization, as a prominent advantage of LEDs, facilitates the seamless connection between LEDs and BPEs. In the BPE sensor array, the ECL probe Ab2@Au@MnO2 with peroxidase activity is introduced into the BPE cathode array through enzyme-linked immunosorbent assay. Under the action of the enzyme, H2O2 can be accelerated, so that more electrons in the system flow to the LED reporter array, achieving sensitive detection of multiple targets and visualized imaging. Compared with solutions, solid-state electrochemiluminescence has a lower excitation potential and more obvious luminescence, which plays a role in protecting the electrode to a certain extent. Portability, integration, and miniaturization are well reflected, meeting the needs of clinical timely detection (POCT).

2. The method for simultaneous detection of multiple targets by bipolar electrode electrochemical luminescence according to claim 1, characterized in that: Under the action of glutaraldehyde (GA), Ab1 is coupled to the electrode surface, and a stable sandwich structure is formed through specific recognition between antigen and antibody. The light-emitting diode (LED) has unidirectional conductivity and is fixed to the BPE anode by conductive silver tape. The ECL probe Ab2 / Au / MnO2 with peroxidase catalytic activity (POD) is introduced to the electrode surface at the BPE cathode by the sandwich method, catalyzing the decomposition of H2O2, accelerating more electrons in the system to flow to the light-emitting diode, and realizing signal amplification. Due to the different luminous intensities of LED lights, by changing the H2O2 concentration, the simultaneous detection and visualization of CEA, PSA, and AFP under the same voltage and the same photomultiplier tube are achieved.

3. The method for preparing a highly sensitive bipolar electrode for electrochemical luminescence multi-target simultaneous detection according to claims 1 to 2, characterized in that: The optimal incubation time of APTES was 8h.

4. The method for preparing a highly sensitive bipolar electrode for electrochemical luminescence multi-target simultaneous detection according to claims 1 to 2, characterized in that: The optimal incubation time for glutaraldehyde (GA) was 60 min.

5. The method for preparing a highly sensitive bipolar electrode for electrochemical luminescence multi-target simultaneous detection according to claims 1 to 2, characterized in that: The optimal pH of the system is 6.

6. A method for simultaneous detection of multiple targets 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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