Five-path three-electrode electrochemical luminescence detector

Abstract

The five-path three-electrode electrochemical luminescence detector comprises an electrochemical luminescence chip and a constant potential excitation device, the electrochemical luminescence chip comprises an electrode plate and a sample adding plate, and a shared counter electrode, a first working electrode, a first reference electrode, a second working electrode and a second reference electrode are arranged on the front face of the electrode plate; a driving electrode of the shared counter electrode is arranged on the back surface of the electrode plate, the shared counter electrode is connected with the driving electrode, and the sample adding plate covers the shared counter electrode, the first working electrode, the first reference electrode, the second working electrode and the second reference electrode; the bottom ends of the first working electrode, the first reference electrode, the second working electrode and the second reference electrode and the bottom end of the driving electrode are used as electric contact areas, and sample adding sheets corresponding to the shared counter electrode, the first working electrode and the first reference electrode or the second working electrode and the second reference electrode form a first reaction tank and a second reaction tank; the constant potential excitation device is connected with the electric contact area to excite the first reaction tank and the second reaction tank to generate electrochemical luminescence reaction.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence detection, and more particularly, to a five-channel three-electrode electrochemiluminescence detector. Background Art

[0002] Electrochemiluminescence (ECL) technology, as an analytical method with high sensitivity and high selectivity, has been widely used in the fields of biomedicine, environmental monitoring, food safety, etc. An electrochemiluminescence detection system generally consists of a detection unit, an excitation unit, a photoelectric detection unit, and a data processing unit. In a traditional three-electrode electrochemiluminescence detection system, the detection unit usually includes a working electrode, a reference electrode, and a counter electrode; the excitation unit is used to control the potential between the reference electrode and the working electrode to keep it relatively constant, so as to drive the electrochemiluminescence reaction on the surface of the working electrode.

[0003] The traditional three-electrode electrochemiluminescence detection system shows good performance in a single reaction cell, but there are many problems in a dual reaction cell. Currently, there is a three-electrode electrochemiluminescence sensor based on sharing a working electrode, a reference electrode, and a counter electrode. The detection area and the quality control area (i.e., the two reaction cells) it contains are integrated on the chip surface in a parallel structure. During the electrochemistry excitation process, the two reaction cells share a negative feedback control loop composed of a working electrode and a reference electrode, and the current provided by the excitation unit to the dual reaction cells needs to flow through the same working electrode.

[0004] This excitation method limits the current distribution of the two reaction cells to the shared working electrode, and the current cannot be independently regulated. This current coupling effect will cause the electrochemical processes in the two reaction cells to interfere with each other, thereby affecting the stability and controllability of the detection system. Currently, there is a portable electrochemistry excitation device that adopts the potentiostat architecture of the traditional three-electrode system of a working electrode, a reference electrode, and a counter electrode. When this device is used to excite the electrochemiluminescence of a dual reaction cell, the two reaction cells in the detection unit must share a potentiostat. This shared architecture cannot achieve independent control of the potential between the reference electrode and the working electrode of each reaction cell, resulting in an intensified electrode polarization effect and ultimately a decrease in detection sensitivity. Summary of the Invention

[0005] The purpose of the present invention is to overcome the disadvantages and deficiencies of the prior art, and provide a five-channel three-electrode electrochemiluminescence detector, which has two completely independent negative feedback loops composed of a reference electrode and a working electrode under the condition of sharing a counter electrode. The potential between the reference electrode and the working electrode constituting the loop can be independently controlled, the currents of the two reaction cells can be independently adjusted, and the two reaction cells can independently carry out electrochemiluminescence reactions.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A five-channel three-electrode electrochemiluminescence detector includes an electrochemiluminescence chip and a constant potential excitation device. The electrochemiluminescence chip includes an electrode sheet and a sample addition sheet. On the front surface of the electrode sheet, there are a shared counter electrode, a first working electrode, a first reference electrode, a second working electrode, and a second reference electrode. On the back surface of the electrode sheet, there is a driving electrode for the shared counter electrode, and the shared counter electrode is connected to the driving electrode. The sample addition sheet covers the shared counter electrode, the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode;

[0008] The bottoms of the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode and the bottom of the driving electrode serve as the electrical contact areas. The first side of the sample addition sheet corresponding to the first side of the shared counter electrode, the first working electrode, and the first reference electrode forms a first reaction cell. The second side of the sample addition sheet corresponding to the second side of the shared counter electrode, the second working electrode, and the second reference electrode forms a second reaction cell. The constant potential excitation device is connected to the electrical contact areas to stimulate the electrochemiluminescence reaction in the first reaction cell and the second reaction cell;

[0009] The constant potential excitation device includes a power supply unit, a constant potential circuit, an external interface circuit, a WiFi circuit, a control unit, a relay circuit, an OLED display screen, a reset circuit, a serial port circuit, a signal generation circuit, and a key circuit integrated on a circuit board. The constant potential circuit, the external interface circuit, the WiFi circuit, the control unit, the relay circuit, the OLED display screen, the reset circuit, the serial port circuit, the signal generation circuit, and the key circuit are respectively connected to the power supply unit. The constant potential circuit, the external interface circuit, the WiFi circuit, the relay circuit, the OLED display screen, the reset circuit, the serial port circuit, the signal generation circuit, and the key circuit are respectively connected to the control unit. The signal generation circuit is connected to the constant potential circuit. The power supply unit is connected to an external power supply. The WiFi circuit is connected to the upper computer terminal.

[0010] Further, the power supply unit includes a +5V to +3.3V step-down circuit, a +5V to +12V boost circuit, a +5V to -12V step-down circuit, a +12V to +5V step-down circuit, and a +12V to +5.21V step-down circuit. The +5V to +3.3V step-down circuit provides power for the control unit, the relay circuit, the signal generation circuit, the OLED display screen, the serial port circuit, and the WiFi circuit;

[0011] The +5V to +12V boost circuit provides power for the signal generation circuit and the potentiostat circuit, and also provides power for the +12V to +5V buck circuit and the +12V to +5.21V buck circuit; the +5V to -12V buck circuit provides power for the signal generation circuit and the potentiostat circuit, and the +12V to +5V buck circuit and the +12V to +5.21V buck circuit provide power for the signal generation circuit.

[0012] Further, the +5V to +3.3V buck circuit converts the external power supply +5V voltage into +3.3V voltage through the linear voltage regulator chip AMS1117-3.3. The +3.3V voltage is current-limited by a 4.7KΩ resistor to drive the indicator light to indicate the working state of the circuit, and the circuit is configured with 22μF and 100nF decoupling capacitors for filtering.

[0013] Further, the +5V to +12V boost circuit boosts the external power supply +5V voltage through the DC conversion chip MT3608. The boosted voltage signal is converted into a +12V output signal by the linear voltage regulator 78L12. The +12V output signal is isolated into an analog +12V voltage signal and a digital +12V voltage signal through a 0Ω resistor. The analog +12V voltage signal provides a positive power supply for the signal generation circuit and the potentiostat circuit, and the digital +12V voltage signal provides a positive power supply for the +12V to +5V buck circuit and the +12V to +5.21V buck circuit;

[0014] The +5V to -12V buck circuit converts the external power supply +5V voltage into a negative voltage signal through the asynchronous buck converter HT7463A. The negative voltage signal is converted into a -12V output signal by the linear voltage regulator 79L12. The -12V output signal is isolated into an analog -12V voltage signal and a digital -12V voltage signal through a 0Ω resistor. The analog -12V voltage signal provides a negative power supply for the signal generation circuit and the potentiostat circuit.

[0015] Further, the +12V to +5V buck circuit converts the digital +12V voltage signal into 5V voltage through the voltage reference chip REF195GSZ to provide a regulated power supply for the signal generation circuit;

[0016] The +12V to +5.21V buck circuit converts the digital +12V voltage signal into +5.21V voltage through the linear voltage regulator LM317LF. The +5.21V voltage drives the indicator light LED9 to emit light to indicate the working state of the circuit through a 10KΩ resistor for current limiting.

[0017] Further, the external interface circuit includes a +3.3V interface, a +12V interface, a -12V interface, a +5V interface, a +5.21V interface, a shared counter electrode interface, a first working electrode interface, a first reference electrode interface, a second working electrode interface, and a second reference electrode interface;

[0018] The +3.3V interface is connected to the output terminal of the +5V to +3.3V step-down circuit, the +12V interface is connected to the output terminal of the +5V to +12V boost circuit, the -12V interface is connected to the output terminal of the +5V to -12V step-down circuit, the +5V interface is connected to the output terminal of the +12V to +5V step-down circuit, the +5.21V interface is connected to the output terminal of the +12V to +5.21V step-down circuit, and the shared counter electrode interface, the first working electrode interface, the first reference electrode interface, the second working electrode interface, and the second reference electrode interface are respectively connected to the drive electrode, the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode.

[0019] Furthermore, the potentiostat circuit includes two voltage followers OPA604, an operational amplifier OP07, and a power amplifier BUF634. The two voltage followers OPA604 are used to maintain a constant potential between the first reference electrode and the first working electrode or between the second reference electrode and the second working electrode. The operational amplifier OP07 provides an excitation signal for the shared counter electrode, and the power amplifier BUF634 amplifies the excitation signal output by the operational amplifier OP07 again.

[0020] Furthermore, the control unit includes an STM32F103CBT6 chip, a clock circuit, a startup circuit, and a decoupling circuit. The clock circuit uses a crystal oscillator to provide an operating clock signal for the STM32F103CBT6 chip, and capacitors are connected across the crystal oscillator for filtering; the startup circuit uses header pins to be serially connected to the BOOT0 pin and the BOOT1 pin of the STM32F103CBT6 chip through resistors respectively; the decoupling circuit uses multiple capacitors to connect the 3.3V supply voltage to the ground terminal of the STM32F103CBT6 chip.

[0021] Furthermore, the serial port circuit uses a USB to TTL serial port chip, the reset circuit uses a low-level reset method, the relay circuit uses NPN and PNP transistors as switches, and the OLED display screen is used to display the status of the potentiostat excitation device and the magnitude of the excitation voltage value.

[0022] Furthermore, the signal generation circuit includes a digital-to-analog conversion chip DAC8831 and an operational amplifier OPA277. The digital signal output by the microcontroller is converted into an analog signal through the digital-to-analog conversion chip DAC8831 and the operational amplifier OPA277, and is sent to the potentiostat circuit.

[0023] Compared with the prior art, in the case of sharing a counter electrode, the present invention has a negative feedback loop composed of two completely independent reference electrodes and working electrodes. The potential between the reference electrode and the working electrode forming the loop can be independently controlled, and the currents of the two reaction cells can be independently adjusted, avoiding the mutual interference between the electrochemical reactions in the two reaction cells, enabling the two reaction cells to independently carry out electrochemiluminescence reactions. The present invention can achieve the simultaneous excitation of reactants in the same system or different systems, expanding the application scope of the three-electrode electrochemiluminescence detector in the field of electrochemiluminescence detection and improving the detection efficiency.

[0024] The constant potential excitation device of the present invention adopts a triggering mechanism that first connects to the reference electrode and the working electrode on the electrochemiluminescence chip and then connects to the shared counter electrode, overcoming the situation where a two-electrode electrochemiluminescence is formed first between the working electrode and the counter electrode when the excitation device connects the three electrodes simultaneously, significantly improving the controllability of the electrochemiluminescence reaction trigger and providing more stable and accurate constant potential control for electrochemiluminescence detection.

[0025] The constant potential excitation device of the present invention sets the parameters of the amperometric chronoamperometry method through the upper computer terminal, with simple operation and low cost. The present invention realizes dual control through the key terminal of the key circuit and the upper computer terminal, supports two control methods of wired and wireless, and provides a flexible and diversified control scheme. The present invention adopts a low-power circuit design and a simplified operation process, reducing the maintenance cost and complexity, meeting the requirements of long-term continuous operation, and is especially suitable for the detection application scenarios that require continuous electrochemiluminescence. Description of the Drawings

[0026] Figure 1 It is a schematic structural diagram of an electrochemiluminescence chip.

[0027] Figure 2 It is a schematic structural diagram of an electrode sheet and a sample adding sheet.

[0028] Figure 3 It is a schematic structural diagram of an upper cover.

[0029] Figure 4 It is a schematic structural diagram of a lower cover.

[0030] Figure 5 It is a connection block diagram of each module of the circuit board.

[0031] Figure 6 It is a schematic diagram of the +5V to +3.3V circuit of the power supply unit.

[0032] Figure 7 It is a schematic diagram of the +5V to +12V circuit of the power supply unit.

[0033] Figure 8 It is a schematic diagram of the +5V to -12V circuit of the power supply unit.

[0034] Figure 9 Schematic diagram of the +12V to +5V circuit for the power supply unit.

[0035] Figure 10 Schematic diagram of the +12V to +5.21V circuit for the power supply unit.

[0036] Figure 11 Schematic diagram of the potentiostatic circuit.

[0037] Figure 12 Schematic diagram of the external interface circuit.

[0038] Figure 13 Schematic diagram of the WiFi circuit.

[0039] Figure 14 Schematic diagram of the control unit circuit.

[0040] Figure 15 Schematic diagram of the relay circuit.

[0041] Figure 16 Schematic diagram of the OLED display circuit.

[0042] Figure 17 Schematic diagram of the reset circuit.

[0043] Figure 18 Schematic diagram of the serial port circuit.

[0044] Figure 19 Schematic diagram of the signal generation circuit.

[0045] Figure 20 Schematic diagram of the key circuit.

[0046] Figure 21 Graph of the linear fitting relationship between hydrogen peroxide concentration and ECL luminescence intensity.

[0047] Figure 22 Schematic diagram of simultaneous excitation and separate excitation of the first reaction cell and the second reaction cell.

[0048] Figure 23 Graph comparing the ECL signal intensities in the first reaction cell and the second reaction cell under simultaneous excitation and separate excitation.

[0049] Figure 24 Relationship between hydrogen peroxide concentration and ECL luminescence intensity in the first reaction cell under different excitation devices.

[0050] Figure 25 Relationship between hydrogen peroxide concentration and ECL luminescence intensity in the second reaction cell under different excitation devices.

[0051] Figure 26 Effect of the driving voltage provided by the potentiostatic excitation device on the ECL luminescence intensity.

[0052] Figure 27 For Ru(bpy)3 2+ Linear fitting relationship diagram between the concentration and the ECL luminescence intensity.

[0053] Figure 28 Schematic diagram of the presence / absence of physical separation between the first reaction cell and the second reaction cell.

[0054] Figure 29 Comparison of the ECL luminescence intensity when the potentiostatic excitation device simultaneously excites the first reaction cell and the second reaction cell with / without physical separation under two different reaction systems.

[0055] Explanation of the reference numerals in the attached drawings:

[0056] 1 - Electrochemiluminescence chip; 11 - Electrode sheet; 111 - Shared counter electrode; 112 - First working electrode; 113 - First reference electrode; 114 - Second working electrode; 115 - Second reference electrode; 116 - Driving electrode; 117 - Central through hole; 118 - Electrical contact area; 12 - Sampling sheet; 121 - First reaction cell; 122 - Second reaction cell; 13 - Upper cover; 131 - First observation area; 132 - Second observation area; 133 - Upper opening groove; 14 - Lower cover; 141 - Groove; 142 - Lower opening groove. Detailed implementation manners

[0057] The five - path three - electrode electrochemiluminescence detector of the present invention will be further described below in conjunction with the attached drawings and specific embodiments.

[0058] Please refer to Figure 1 and Figure 2 The present invention discloses a five - path three - electrode electrochemiluminescence detector, including an electrochemiluminescence chip 1 and a five - path three - electrode electrochemiluminescence potentiostatic excitation device. The electrochemiluminescence chip 1 includes an electrode sheet 11, a sampling sheet 12, an upper cover 13 and a lower cover 14. The electrode sheet 11 and the sampling sheet 12 are arranged between the upper cover 13 and the lower cover 14. On the front side of the electrode sheet 11, there are a shared counter electrode 111, a first working electrode 112, a first reference electrode 113, a second working electrode 114 and a second reference electrode 115. On the back side of the electrode sheet 11, there is a driving electrode 116 of the shared counter electrode 111. The shared counter electrode 111 and the driving electrode 116 are physically connected through the central through hole 117. The sampling sheet 12 covers the shared counter electrode 111, the first working electrode 112, the first reference electrode 113, the second working electrode 114 and the second reference electrode 115.

[0059] Please refer to Figure 1 andFigure 2 The bottom ends of the first working electrode 112, the first reference electrode 113, the second working electrode 114, and the second reference electrode 115 and the bottom end of the driving electrode 116 serve as the electrical contact area 118, sharing the first side of the counter electrode 111, the first side of the sampling piece 12 corresponding to the first working electrode 112 and the first reference electrode 113 to form the first reaction cell 121, and sharing the second side of the counter electrode 111, the second side of the sampling piece 12 corresponding to the second working electrode 114 and the second reference electrode 115 to form the second reaction cell 122. The potentiostatic excitation device is connected to the electrical contact area 118 to excite the first reaction cell 121 and the second reaction cell 122 to generate an electrochemiluminescence reaction.

[0060] Please refer to Figure 2 、 Figure 3 and Figure 4 ,the electrode sheet 11 is a transparent PET plastic plate constructed by screen printing conductive carbon ink. The central through hole 117 on the electrode sheet 11 is cut by a laser cutting machine. When screen printing the electrode sheet 11, the carbon ink will penetrate through the central through hole 117, thereby physically connecting the shared counter electrode 111 and the driving electrode 116. The sampling piece 12 is prepared by cutting non-woven fabric with a laser cutting machine. The upper cover 13 and the lower cover 14 are both made by 3D printing technology. The upper cover 13 is provided with a first observation area 131, a second observation area 132, and an upper opening groove 133, and the lower cover 14 is provided with a groove 141 and a lower opening groove 142. The upper opening groove 133 of the upper cover 13 and the lower opening groove 142 of the lower cover 14 are used for the electrical connection between the electrical contact area 118 and the potentiostatic excitation device.

[0061] The potentiostatic excitation device includes a power supply unit, a potentiostatic circuit, an external interface circuit, a WiFi circuit, a control unit, a relay circuit, an OLED display screen, a reset circuit, a serial port circuit, a signal generation circuit, and a key circuit integrated on a circuit board. The potentiostatic circuit, the external interface circuit, the WiFi circuit, the control unit, the relay circuit, the OLED display screen, the reset circuit, the serial port circuit, the signal generation circuit, and the key circuit are respectively connected to the power supply unit. The potentiostatic circuit, the external interface circuit, the WiFi circuit, the relay circuit, the OLED display screen, the reset circuit, the serial port circuit, the signal generation circuit, and the key circuit are respectively connected to the control unit. The signal generation circuit is connected to the potentiostatic circuit. The power supply unit is connected to an external power supply. The WiFi circuit is connected to the upper computer terminal.

[0062] As Figure 5As shown in the figure, the power supply unit includes a +5V to +3.3V buck circuit, a +5V to +12V boost circuit, a +5V to -12V buck circuit, a +12V to +5V buck circuit, and a +12V to +5.21V buck circuit. The +5V to +3.3V buck circuit supplies power to the control unit, relay circuit, signal generation circuit, OLED display, serial port circuit, and WiFi circuit. The +5V to +12V boost circuit supplies power to the signal generation circuit and potentiostat circuit, and also supplies power to the +12V to +5V buck circuit and the +12V to +5.21V buck circuit; the +5V to -12V buck circuit supplies power to the signal generation circuit and potentiostat circuit, and the +12V to +5V buck circuit and the +12V to +5.21V buck circuit supply power to the signal generation circuit.

[0063] As Figure 6 shown, the +5V to +3.3V buck circuit converts the external power supply +5V voltage into +3.3V voltage through the linear voltage regulator chip AMS1117-3.3. The +3.3V voltage is current-limited by a 4.7KΩ resistor to drive the indicator light to indicate the working state of the circuit. The circuit is configured with 22μF and 100nF decoupling capacitors for filtering and stabilizing the power supply, reducing voltage fluctuations and high-frequency noise, and ensuring the stable operation of the circuit.

[0064] As Figure 7 shown, the +5V to +12V boost circuit boosts the external power supply +5V voltage through the DC-DC converter chip MT3608. The boosted voltage signal is converted into a high-precision, low-ripple +12V output signal by the linear voltage regulator 78L12. Further, the +12V output signal is isolated into an analog +12V voltage signal and a digital +12V voltage signal through a 0Ω resistor. The analog +12V voltage signal supplies a low-noise positive voltage power supply to the signal generation circuit and potentiostat circuit, and the digital +12V voltage signal supplies a positive voltage power supply to the +12V to +5V buck circuit and the +12V to +5.21V buck circuit. The circuit is configured with an inductor to store energy in the boost converter and release energy during the switching cycle to increase the output voltage. The configured Schottky diode is used to prevent reverse current flow and provide a current flow path for the circuit during the switching cycle.

[0065] As Figure 8 shown, the +5V to -12V buck circuit converts the external power supply +5V voltage into a negative voltage signal through the asynchronous buck converter HT7463A. The negative voltage signal is converted into a high-precision, low-ripple -12V output signal by the linear voltage regulator 79L12. Further, the -12V output signal is isolated into an analog -12V voltage signal and a digital -12V voltage signal through a 0Ω resistor. The analog -12V voltage signal supplies a stable negative voltage power supply to the signal generation circuit and potentiostat circuit.

[0066] AsFigure 9 As shown, the +12V to +5V step-down circuit converts the digital +12V voltage signal into a low-noise and highly stable 5V voltage through the voltage reference chip REF195GSZ, providing a precise regulated power supply for the signal generation circuit. As Figure 10 shown, the +12V to +5.21V step-down circuit converts the digital +12V voltage signal into a +5.21V voltage through the linear voltage regulator LM317LF. The +5.21V voltage drives the indicator LED9 to emit light through a 10KΩ resistor to limit the current and indicate the working state of the circuit.

[0067] As Figure 11 shown, the potentiostat circuit includes the first voltage follower OPA604, the first voltage follower OPA604, the operational amplifier OP07, and the power amplifier BUF634. The first voltage follower OPA604 and the first voltage follower OPA604 can enhance the potential driving ability. The first voltage follower OPA604 is used to maintain the potential between the first reference electrode and the first working electrode constant. The second voltage follower OPA604 is used to maintain the potential between the second reference electrode and the second working electrode constant. The operational amplifier OP07 provides an excitation signal for the shared counter electrode, and the power amplifier BUF634 amplifies the excitation signal output by the operational amplifier OP07 again to solve the problem of insufficient excitation current caused by sharing a single shared counter electrode between the first reaction cell and the second reaction cell. To effectively overcome interference in the circuit and ensure impedance matching of the linear circuit, the potentiostat circuit uses parallel reverse diodes to prevent components from being damaged due to overvoltage and overcurrent.

[0068] As Figure 12 shown, the external interface circuit includes a +3.3V interface, a +12V interface, a -12V interface, a +5V interface, a +5.21V interface, a shared counter electrode interface, a first working electrode interface, a first reference electrode interface, a second working electrode interface, and a second reference electrode interface. The +3.3V interface is connected to the output terminal of the +5V to +3.3V step-down circuit, the +12V interface is connected to the output terminal of the +5V to +12V boost circuit, the -12V interface is connected to the output terminal of the +5V to -12V step-down circuit, the +5V interface is connected to the output terminal of the +12V to +5V step-down circuit, the +5.21V interface is connected to the output terminal of the +12V to +5.21V step-down circuit, and the shared counter electrode interface, the first working electrode interface, the first reference electrode interface, the second working electrode interface, and the second reference electrode interface are respectively connected to the drive electrode, the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode.

[0069] As Figure 13As shown, the WiFi circuit communicates wirelessly with the host computer via the TCP protocol. The WiFi circuit acquires the data transmitted from the host computer and transfers the data to the control unit via USART2 to control the voltage of the potentiostat circuit.

[0070] As Figure 14 shown, the control unit includes an STM32F103CBT6 chip, a clock circuit, a startup circuit, and a decoupling circuit. The STM32F103CBT6 chip is connected to the clock circuit, the startup circuit, and the decoupling circuit. The clock circuit uses a crystal oscillator to provide a running clock signal for the STM32F103CBT6 chip, and capacitors are connected across the crystal oscillator to filter and eliminate inductive interference. The startup circuit uses header pins to be connected in series with the BOOT0 pin and the BOOT1 pin of the STM32F103CBT6 chip through resistors respectively. The decoupling circuit uses multiple capacitors to connect the 3.3V supply voltage to the ground terminal of the STM32F103CBT6 chip, and the multiple capacitors are close to the STM32F103CBT6 chip to reduce noise and interference on the power line.

[0071] As Figure 15 shown, the relay circuit uses an NPN transistor and a PNP transistor as switches; the control unit releases a voltage signal to control the turn-off of the switches, and then manipulates the attraction and separation of the armature in the relay, thereby separately controlling the conduction and turn-off of the excitation signal of the shared counter electrode, so that the potentiostatic excitation device is first connected to the first reference electrode, the second reference electrode, the first working electrode, and the second working electrode of the electrochemiluminescence chip, and then connected to the shared counter electrode.

[0072] As Figure 16 shown, the OLED display communicates with the control unit via the I2C protocol, and the OLED display is used to display the status of the potentiostatic excitation device and the magnitude of the excitation voltage value. As Figure 17 shown, the reset circuit includes a reset button, a resistor, and a capacitor, and adopts a low-level reset method. During the operation of the control unit, after pressing the reset button, the reset pin in the reset circuit is directly conducted to GND, and the capacitor discharges to reset the control unit. After releasing the reset button, the capacitor continues to charge, and after a few milliseconds, the charging is completed and the reset circuit is open, and the control unit enters the working state.

[0073] As Figure 18 shown, the serial port circuit uses a USB-to-TTL serial port chip as the core, configures a crystal oscillator and filter capacitors. The CH340 D+ and CH340 D- in the USB interface are connected to the UD+ and UD- of the serial port chip, and the serial port chip then sends data to the STM32F103CBT6 chip through RXT and TXD, so as to ensure the safe burning of the external program into the control unit.

[0074] As Figure 19As shown in the figure, the signal generation circuit includes a 16-bit digital-to-analog conversion chip DAC8831 and a high-precision operational amplifier OPA277 with low noise and low temperature drift. The digital signal output by the microcontroller is converted into an analog signal through the digital-to-analog conversion chip DAC8831 and the operational amplifier OPA277, and then sent to the potentiostatic circuit. The 16-bit digital-to-analog conversion chip DAC8831 communicates with the control unit through the SPI protocol. Cooperating with the operational amplifier OPA277 can improve the output driving ability of the digital-to-analog conversion chip DAC8831, thereby providing an accurate voltage signal for the potentiostatic circuit.

[0075] As Figure 20 shown in the figure, the key circuit is connected to the control unit through DuPont wires. It can not only select different excitation voltages, but also control the on / off of the excitation signal. The upper computer communicates with the control unit remotely through the WiFi circuit to achieve wireless control of the circuit board. By setting the parameters of the chronoamperometry method on the upper computer, the operation is simple.

[0076] Application Example 1

[0077] This application example uses a five-channel three-electrode electrochemiluminescence detector to quantitatively detect hydrogen peroxide in the luminol / hydrogen peroxide system.

[0078] (1) Use NaOH solution (0.1M) to adjust the pH value of distilled water to 10 for preparing 5 mM luminol solution. Use distilled water to prepare hydrogen peroxide solutions with concentrations of 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM. The hydrogen peroxide solutions with different concentrations are mixed with 5 mM luminol solution at a volume ratio of 1:1 to obtain the test solution.

[0079] (2) Use a pipette to drop 35 μL of the test solution onto the first reaction cell and the second reaction cell, and press the upper cover and the lower cover tightly to assemble an electrochemiluminescence chip.

[0080] (3) Adopt the chronoamperometry method, and apply a 2.5V excitation voltage to the electrochemiluminescence chip through the potentiostatic excitation device of the five-channel three-electrode electrochemiluminescence detector. After the five-channel three-electrode electrochemiluminescence detector finishes the detection, further analyze and process the electrochemiluminescence imaging data through Origin software.

[0081] (4) The results are as Figure 21 shown in the figure. It can be seen from the figure that: by using the potentiostatic excitation device of the present invention, as the hydrogen peroxide concentration increases, the electrochemiluminescence intensity in the first reaction cell and the second reaction cell also increases accordingly. There is a good linear relationship between the hydrogen peroxide concentration and the electrochemiluminescence intensity. For the first reaction cell, the linear fitting equation is Y = 7.3574X + 1.8585 (R A 2= 0.9907, n = 5). For the second reaction cell, the linear fitting equation is Y = 7.3919X + 1.8434 (R B 2 = 0.9841, n = 5). Therefore, the five-channel three-electrode electrochemiluminescence detector of the present invention has the potential for quantitative detection of hydrogen peroxide.

[0082] Application Example 2

[0083] In this application example, a five-channel three-electrode electrochemiluminescence detector is used to verify whether the first reaction cell and the second reaction cell can independently quantitatively detect hydrogen peroxide in the luminol / hydrogen peroxide system.

[0084] (1) The test solution used is similar to that in Example 2.

[0085] (2) As Figure 22 (A) shows, use a pipette to add 35 μL of the test solution to the first reaction cell and the second reaction cell, press the upper cover and the lower cover tightly to close, and assemble into an electrochemiluminescence chip; as Figure 22 (B) shows, cover only the first reaction cell or the second reaction cell with the sample addition piece, where 17 μL of the test solution is added, press the upper cover and the lower cover tightly to close, and assemble into an electrochemiluminescence chip, and the first reaction cell or the second reaction cell is separately excited.

[0086] (3) The detection process is similar to that in Application Example 1, and the results are as Figure 23 shown.

[0087] It can be seen from the figure that for each hydrogen peroxide concentration, there is no significant difference in the electrochemiluminescence intensity caused by the first reaction cell under simultaneous excitation and the first reaction cell under separate excitation; there is also no significant difference in the electrochemiluminescence intensity caused by the second reaction cell under simultaneous excitation and the second reaction cell under separate excitation. These results indicate that under the condition of simultaneous excitation, the two reaction cells on the electrochemiluminescence chip are independent of each other and do not interfere with each other.

[0088] Application Example 3

[0089] On the basis of Application Example 2, in this application example, a potentiostatic excitation device and a traditional potentiostat are used for an electrochemiluminescence comparative experiment.

[0090] (1) Use NaOH solution (0.1 M) to adjust the pH value of distilled water to 10 to prepare a 5 mM luminol solution, and use distilled water to prepare hydrogen peroxide solutions with concentrations of 1 mM, 2 mM, 3 mM, and 4 mM. The hydrogen peroxide solutions with different concentrations are mixed with the 5 mM luminol solution at a volume ratio of 1:1 to obtain the test solution.

[0091] (2) The assembly method and detection process of the electrochemiluminescence chip are similar to those in Application Example 1, and the results are as Figure 24 and Figure 25 shown. It can be seen from the figure that as the hydrogen peroxide concentration in the test solution increases, the electrochemiluminescence intensities triggered by the potentiostatic excitation device and the traditional potentiostat have the same changing trend and detection effect, indicating that the potentiostatic excitation device of the present invention has good application potential in electrochemiluminescence detection.

[0092] Application Example 4

[0093] In this application example, a five-channel three-electrode electrochemiluminescence detector is used to study the influence of different driving voltages provided by the potentiostatic excitation device on the electrochemiluminescence intensity under the luminol / hydrogen peroxide system.

[0094] (1) The pH value of distilled water is adjusted to 10 with NaOH solution (0.1 M) to prepare a 5 mM luminol solution, and a 1 mM hydrogen peroxide solution is prepared with distilled water. The 1 mM hydrogen peroxide solution and the 5 mM luminol solution are mixed at a volume ratio of 1:1 to obtain the test solution.

[0095] (2) The driving voltages provided by the electrochemiluminescence potentiostatic excitation device of the five-channel three-electrode electrochemiluminescence detector are set to 2.5 V, 2.75 V, 3.0 V, 3.5 V, 4.0 V, and 4.5 V.

[0096] (3) The chip assembly method and detection process are similar to those in Example 2, and the results are as Figure 26 shown. It can be seen from the figure that as the driving voltage increases from 2.5 V to 4.5 V, the electrochemiluminescence intensity in the first reaction cell increases from 0.70×10 5 to 35.13×10 5 , and the electrochemiluminescence intensity in the second reaction cell increases from 0.72×10 5 to 35.80×10 5 . Therefore, the electrochemiluminescence chip of the present invention can be well triggered by different driving voltages provided by the potentiostatic excitation device.

[0097] Application Example 5

[0098] In this application example, a five-channel three-electrode electrochemiluminescence detector is used to detect different concentrations of Ru(bpy)3 2+ / TPA) system. 2+ under the tris(bipyridine)ruthenium(III) / tripropylamine (Ru(bpy)3

[0099] (1) PBS solution (10×) is used to prepare Ru(bpy)3 solutions with concentrations of 0.1 mM, 0.5 mM, 2.5 mM, and 5 mM 2+Solutions, Ru(bpy)3 with different concentrations 2+ The solution and the TPA solution (obtained by diluting 98% TPA with PBS (pH 7.4)) were mixed in a volume ratio of 1:1 to obtain the test solution; the excitation voltage provided by the potentiostatic excitation device was 3V.

[0100] (2) The assembly method and detection process of the electrochemiluminescence chip are similar to those in Application Example 1, and the results are as Figure 27 shown. It can be seen from the figure that: using the five-channel three-electrode electrochemiluminescence detector of the present invention, as the concentration of Ru(bpy)3 2+ increases, the electrochemiluminescence intensities in the first reaction cell and the second reaction cell also increase correspondingly. There is a good linear relationship between the concentration of Ru(bpy)3 2+ and the electrochemiluminescence intensity. For the first reaction cell, the linear fitting equation is Y = 9.8559X + 0.3548 (R A 2 = 0.9878, n = 5). For the second reaction cell, the linear fitting equation is Y = 9.5298X + 0.2767 (R B 2 = 0.9863, n = 5). Therefore, the five-channel three-electrode electrochemiluminescence detector of the present invention has the potential for quantitative detection of Ru(bpy)3 2+ .

[0101] Application Example 6

[0102] In this application example, a five-channel three-electrode electrochemiluminescence detector was used to verify the feasibility of electrochemiluminescence reactions of different systems in different reaction cells on the electrochemiluminescence chip under different systems of luminol / hydrogen peroxide and Ru(bpy)3 2+ / TPA, and further verify the potential of simultaneous quantitative detection of analytes in different reaction systems on a single electrochemiluminescence chip.

[0103] (1) A 5 mM Ru(bpy)3 2+ solution was prepared using a PBS solution (10×), and was mixed with the TPA solution in a volume ratio of 1:1 to obtain the test solution for the Ru(bpy)3 2+ / TPA system; the pH value of distilled water was adjusted to 10 using a NaOH solution (0.1 M) to prepare a 5 mM luminol solution, and a 5 mM hydrogen peroxide solution was prepared using distilled water. The 5 mM hydrogen peroxide solution and the 7 mM luminol solution were mixed in a volume ratio of 1:1 to obtain the test solution for the luminol system. The excitation voltage provided by the potentiostatic excitation device was 3V.

[0104] (2) As shown in Figure 28 (A), 35 μL of the test solution for the luminol / hydrogen peroxide system or Ru(bpy)32+ The test solution of the / TPA system was added dropwise to the first reaction cell and the second reaction cell, and the upper cover and the lower cover were pressed tightly and closed to assemble an electrochemiluminescence chip; as Figure 28 (B) shows, the first reaction cell and the second reaction cell are physically separated. 17 μL of the test solution of the luminol / hydrogen peroxide system was added dropwise to the first reaction cell, and 17 μL of Ru(bpy)3 2+ / TPA system test solution was added dropwise to the second reaction cell. The upper cover and the lower cover were pressed tightly and closed to assemble an electrochemiluminescence chip.

[0105] (3) The detection process of the electrochemiluminescence chip is similar to that of Application Example 1, and the results are as Figure 29 shown. Group 1 is the luminol / hydrogen peroxide system when the two reaction cells are not physically separated; Group 2 is the luminol / hydrogen peroxide system in the first reaction cell and the Ru(bpy)3 2+ / TPA system in the second reaction cell when the two reaction cells are physically separated; Group 3 is the Ru(bpy)3 2+ / TPA system when the two reaction cells are not physically separated.

[0106] It can be seen from the figure that: under the luminol / hydrogen peroxide system, there is no significant difference in the electrochemiluminescence intensity caused by the first reaction cell when the two reaction cells are not physically separated and the first reaction cell when the two reaction cells are physically separated; under the Ru(bpy)3 2+ / TPA system, there is no significant difference in the electrochemiluminescence intensity caused by the second reaction cell when the two reaction cells are not physically separated and the second reaction cell when the two reaction cells are physically separated. These results indicate that the five-channel three-electrode electrochemiluminescence detector of the present invention has good application potential when simultaneously detecting different electrochemiluminescence systems.

[0107] The above description is a detailed description of the preferred feasible embodiment, but the embodiment is not used to limit the scope of the patent application. Any equivalent changes or modifications made under the disclosed technical spirit shall fall within the scope of the covered patent.

Claims

1. A five-channel three-electrode electrochemiluminescence detector, characterized in that It includes an electrochemiluminescence chip and a potentiostatic excitation device. The electrochemiluminescence chip includes an electrode sheet and a sample addition sheet. On the front side of the electrode sheet, there are a shared counter electrode, a first working electrode, a first reference electrode, a second working electrode, and a second reference electrode. On the back side of the electrode sheet, there is a driving electrode for the shared counter electrode. The shared counter electrode is connected to the driving electrode. The sample addition sheet covers the shared counter electrode, the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode. The bottom ends of the first working electrode, the first reference electrode, the second working electrode, the second reference electrode, and the bottom end of the driving electrode serve as the electrical contact area. The first side of the sample addition sheet corresponding to the first side of the shared counter electrode, the first working electrode, and the first reference electrode constitutes the first reaction cell. The second side of the sample addition sheet corresponding to the second side of the shared counter electrode, the second working electrode, and the second reference electrode constitutes the second reaction cell. The potentiostatic excitation device is connected to the electrical contact area to stimulate the electrochemiluminescence reaction in the first reaction cell and the second reaction cell. The potentiostatic excitation device includes a power supply unit, a potentiostatic circuit, an external interface circuit, a WiFi circuit, a control unit, a relay circuit, an OLED display screen, a reset circuit, a serial port circuit, a signal generation circuit, and a key circuit integrated on a circuit board. The potentiostatic circuit, the external interface circuit, the WiFi circuit, the control unit, the relay circuit, the OLED display screen, the reset circuit, the serial port circuit, the signal generation circuit, and the key circuit are respectively connected to the power supply unit. The potentiostatic circuit, the external interface circuit, the WiFi circuit, the relay circuit, the OLED display screen, the reset circuit, the serial port circuit, the signal generation circuit, and the key circuit are respectively connected to the control unit. The signal generation circuit is connected to the potentiostatic circuit. The power supply unit is connected to an external power supply. The WiFi circuit is connected to the upper computer terminal.

2. The five-channel three-electrode electrochemiluminescence detector according to claim 1, wherein, The power supply unit includes a +5V to +3.3V buck circuit, a +5V to +12V boost circuit, a +5V to -12V buck circuit, a +12V to +5V buck circuit, and a +12V to +5.21V buck circuit. The +5V to +3.3V buck circuit provides power for the control unit, the relay circuit, the signal generation circuit, the OLED display screen, the serial port circuit, and the WiFi circuit. The +5V to +12V boost circuit provides power for the signal generation circuit and the potentiostatic circuit, and also provides power for the +12V to +5V buck circuit and the +12V to +5.21V buck circuit. The +5V to -12V buck circuit provides power for the signal generation circuit and the potentiostatic circuit. The +12V to +5V buck circuit and the +12V to +5.21V buck circuit provide power for the signal generation circuit.

3. The five-channel three-electrode electrochemiluminescence detector according to claim 2, wherein The +5V to +3.3V buck circuit converts the external power supply +5V voltage into +3.3V voltage through the linear voltage regulator chip AMS1117-3.

3. The +3.3V voltage is current-limited through a 4.7KΩ resistor to drive the indicator light to indicate the working state of the circuit. The circuit is configured with 22μF and 100nF decoupling capacitors for filtering.

4. The five-channel three-electrode electrochemiluminescence detector according to claim 2, wherein The +5V to +12V boost circuit boosts the external power supply +5V voltage through the DC conversion chip MT3608. The boosted voltage signal is converted into a +12V output signal by the linear voltage regulator 78L12. The +12V output signal is isolated into an analog +12V voltage signal and a digital +12V voltage signal through a 0Ω resistor. The analog +12V voltage signal provides a positive power supply for the signal generation circuit and the potentiostat circuit, and the digital +12V voltage signal provides a positive power supply for the +12V to +5V buck circuit and the +12V to +5.21V buck circuit. The +5V to -12V buck circuit converts the external power supply +5V voltage into a negative voltage signal through the asynchronous buck converter HT7463A. The negative voltage signal is converted into a -12V output signal by the linear voltage regulator 79L12. The -12V output signal is isolated into an analog -12V voltage signal and a digital -12V voltage signal through a 0Ω resistor. The analog -12V voltage signal provides a negative power supply for the signal generation circuit and the potentiostat circuit.

5. The five-channel three-electrode electrochemiluminescence detector according to claim 4, wherein The +12V to +5V buck circuit converts the digital +12V voltage signal into 5V voltage through the voltage reference chip REF195GSZ to provide a regulated power supply for the signal generation circuit. The +12V to +5.21V buck circuit converts the digital +12V voltage signal into +5.21V voltage through the linear voltage regulator LM317LF. The +5.21V voltage drives the indicator LED9 to emit light through a 10KΩ resistor to indicate the working state of the circuit.

6. The five-channel three-electrode electrochemiluminescence detector according to claim 2, wherein The external interface circuit includes a +3.3V interface, a +12V interface, a -12V interface, a +5V interface, a +5.21V interface, a shared counter electrode interface, a first working electrode interface, a first reference electrode interface, a second working electrode interface, and a second reference electrode interface. The +3.3V interface is connected to the output terminal of the +5V to +3.3V buck circuit, the +12V interface is connected to the output terminal of the +5V to +12V boost circuit, the -12V interface is connected to the output terminal of the +5V to -12V buck circuit, the +5V interface is connected to the output terminal of the +12V to +5V buck circuit, the +5.21V interface is connected to the output terminal of the +12V to +5.21V buck circuit. The shared counter electrode interface, the first working electrode interface, the first reference electrode interface, the second working electrode interface, and the second reference electrode interface are respectively connected to the drive electrode, the first working electrode, the first reference electrode, the second working electrode, and the second reference electrode.

7. The five-channel three-electrode electrochemiluminescence detector according to claim 1, wherein The potentiostat circuit includes two voltage followers OPA604, an operational amplifier OP07, and a power amplifier BUF634. The two voltage followers OPA604 are used to maintain the potential constant between the first reference electrode and the first working electrode or between the second reference electrode and the second working electrode. The operational amplifier OP07 provides an excitation signal for the shared counter electrode, and the power amplifier BUF634 amplifies the excitation signal output by the operational amplifier OP07 again.

8. The five-channel three-electrode electrochemiluminescence detector according to claim 1, characterized in that The control unit includes an STM32F103CBT6 chip, a clock circuit, a startup circuit, and a decoupling circuit. The clock circuit uses a crystal oscillator to provide an operating clock signal for the STM32F103CBT6 chip, and capacitors are connected across the crystal oscillator for filtering. The startup circuit uses header pins to be serially connected to the BOOT0 pin and the BOOT1 pin of the STM32F103CBT6 chip through resistors respectively. The decoupling circuit uses multiple capacitors to connect the 3.3V supply voltage to the ground terminal of the STM32F103CBT6 chip.

9. The five-channel three-electrode electrochemiluminescence detector according to claim 1, wherein The serial port circuit uses a USB-to-TTL serial port chip. The reset circuit uses a low-level reset method. The relay circuit uses NPN and PNP transistors as switches. The OLED display screen is used to display the status of the potentiostatic excitation device and the magnitude of the excitation voltage value.

10. The five-channel three-electrode electrochemiluminescence detector according to claim 1, characterized in that, The signal generation circuit includes a digital-to-analog conversion chip DAC8831 and an operational amplifier OPA277. The digital signal output by the microcontroller is converted into an analog signal through the digital-to-analog conversion chip DAC8831 and the operational amplifier OPA277, and is sent to the potentiostatic circuit.

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