Rapid modification of multi-nucleic acid probes and multi-target detection device based on electric field regulation

Abstract

The application discloses a kind of based on the rapid modification of multi-nucleic acid probe and multi-target detection device of electric field regulation and control.The instrument main body in the device integrates automatic liquid system and miniature electrochemical workstation module, for realizing programmable reagent and sample delivery and multi-channel electrochemical measurement.Detachable sensing chip is bonded by microelectrode array chip and microfluidic chip, and is connected by PCB board, to realize plug and play.The application uses electric field "positive suction-negative discharge" strategy, applies optimal positive potential to target electrode to efficiently attract probe, while applying optimal negative potential to non-target electrode to inhibit non-specific adsorption, so as to realize spatial selective rapid fixation and density accurate regulation of multi-nucleic acid probe in very short time.The device can simultaneously quantitatively detect multiple body fluid disease markers in multiple urine samples in a single operation, providing an efficient, accurate and portable multi-marker comprehensive analysis solution for early screening and instant diagnosis of tumors.

Description

Technical Field

[0001] This invention relates to the field of biosensing and analytical detection technology, and in particular to a device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field regulation. It utilizes an external electric field to achieve rapid spatial selective immobilization of multiple nucleic acid probes and simultaneously detects multiple body fluid disease biomarkers on a microelectrode array. Its applications include early screening of multiple targets in clinical body fluids, point-of-care testing (POCT), and the development of portable biosensor platforms. Background Technology

[0002] Tumor markers play a crucial role in the early screening, diagnosis, and prognostic monitoring of cancer. However, in early cancer screening and personalized treatment scenarios, there is an urgent clinical need for a portable detection platform capable of simultaneously quantifying multiple humoral molecular markers in situ within a short timeframe. On the one hand, due to the high heterogeneity of tumors, the sensitivity and specificity of single-type biomarkers are limited, making it difficult to comprehensively reflect the occurrence and development of the disease. Therefore, a multi-target combined detection strategy is urgently needed. On the other hand, current multi-target detection methods mainly rely on enzyme-linked immunosorbent assay (ELISA), mass spectrometry, or nucleic acid amplification technologies in centralized laboratories. These methods generally suffer from limitations such as long operation procedures, large instrument size, and high sample consumption. In addition, traditional probe immobilization methods (such as drop addition or whole-body immersion) are difficult to achieve precise positioning and specific modification of multiple nucleic acid probes on the surface of highly integrated and densely arranged microelectrode arrays, and are also time-consuming. In summary, current technologies are insufficient to meet the comprehensive requirements of point-of-care testing (POCT) scenarios such as bedside monitoring, community screening, and home self-testing for "minimal sample pretreatment, maximum detection throughput, and rapid result interpretation." Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field regulation.

[0004] The objective of this invention is achieved through the following technical solution: a device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field regulation, the device consisting of an instrument body and a detachable sensing chip;

[0005] The instrument body adopts an integrated design and is electrically connected to the sensor chip via a pin header and socket interface; the instrument body is divided into a reagent compartment, a detection compartment, and a liquid circuit system;

[0006] The detachable sensing chip is formed by bonding a microelectrode array chip and a microfluidic chip; the microelectrode array chip integrates multiple independent detection units, each detection unit including multiple working electrodes, a reference electrode and a counter electrode; each reaction chamber of the microfluidic chip covers a detection unit and is connected to the liquid circuit system through liquid inlet and liquid outlet channels;

[0007] During the nucleic acid probe modification stage, a positive potential is applied to the selected working electrode in the target detection unit, and a negative potential is applied to the remaining working electrodes in the same detection unit and all electrodes in other detection units. After the probe modification is completed, the liquid circuit system introduces the body fluid sample to be tested into the reaction chamber. The working electrode of each detection unit is subjected to voltammetric scanning by a potentiostat. The current data is collected and digitally processed by a microcontroller, so that quantitative detection results of multiple body fluid disease markers in multiple samples can be obtained simultaneously in a single operation.

[0008] Furthermore, the liquid circuit system integrates a peristaltic pump, a two-position three-way solenoid valve, and a multi-channel switching valve, enabling programmable liquid circuit switching and cleaning under the instructions of a microcontroller.

[0009] Furthermore, the detection chamber integrates a power supply module, a micro electrochemical workstation module, a microcontroller module, and a signal conversion module. The micro electrochemical workstation module integrates a programmable multi-channel potentiostat and a signal acquisition and conversion circuit.

[0010] Furthermore, the peristaltic pump and multi-channel switching valve of the liquid circuit system realize programmable switching of the liquid circuit path through a two-position three-way solenoid valve. After each nucleic acid probe modification, sealing, sample injection and detection is completed, the liquid circuit system automatically switches to the deionized water channel and discharges to the waste liquid container through a one-way valve.

[0011] Furthermore, the micro electrochemical workstation module is equipped with a multi-channel analog switch, which can switch forty electrode channels without changing the main circuit structure, and has a header interface at its front end that matches the detachable sensor chip, supporting multi-channel parallel electrochemical measurements.

[0012] Furthermore, the detachable sensor chip can achieve a pluggable electrical connection with the instrument body. Specifically, it is physically connected to the pin header interface led out from the PCB adapter board and the female header interface set at the front end of the micro electrochemical workstation module, so as to realize the electrical connection between the multi-electrode array and the potentiostat, signal conditioning and microcontroller. The pluggable electrical connection is used to quickly replace the detachable sensor chip.

[0013] Furthermore, the microcontroller module applies a positive potential to the selected working electrode in the target detection unit through the micro electrochemical workstation module, and applies a negative potential to the remaining working electrodes and other detection units in the same detection unit.

[0014] Furthermore, the microelectrode array chip integrates multiple independent detection units, the working electrodes of the detection units are arranged around the center of the detection unit, the reference electrode is located at the geometric center, and the counter electrode forms a closed ring and surrounds all the working electrodes.

[0015] Furthermore, during the rapid modification phase of the nucleic acid probe, the target working electrode is applied with a positive potential of +0.4 V, and the non-target working electrode is applied with a negative potential of -0.05 V, with the application time not exceeding 120 s.

[0016] Furthermore, the multiple humoral disease markers include interleukin-6 (IL-6), programmed death ligand (PD-L1), mucin-1 (Mucin-1), miRNA-205, and miRNA-141.

[0017] The beneficial effects of this invention are as follows: This invention proposes a device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field control, which can complete the electrochemical detection of multiple samples and multiple targets in one go within an 8-throughput reaction chamber. By applying an optimized positive potential (preferably +0.4 V) to the target electrode to attract the probe, and simultaneously applying a negative potential (preferably -0.05 V) to the non-target electrode to repel it, this "positive attraction-negative repulsion" strategy reduces the probe immobilization time from several hours of traditional static incubation to 2 minutes. This effectively overcomes the drawbacks of traditional probe immobilization methods, such as long immobilization time and difficulty in achieving precise positioning. It achieves spatially selective immobilization and density control of the probe on the surface of the microelectrode array and avoids cross-contamination between arrays. In addition, this invention adopts a detachable sensor chip design, which realizes rapid plug-and-play electrical connection through pin header and socket interface, greatly facilitating chip replacement and maintenance, reducing one-time costs, and providing convenience for mass production and rapid on-site replacement, significantly improving the portability and practicality of the device. Meanwhile, the entire detection device is highly integrated into a miniaturized instrument body, integrating automated liquid circuit control, multi-channel electrochemical measurement, and data processing functions. It achieves full automation of the entire process from probe modification, sample injection, voltammetric scanning, to result output. It can simultaneously and quantitatively detect multiple bodily fluid disease biomarkers in situ within a short time, and supports battery or external power supply. The entire device is small in size and lightweight. In summary, this invention provides an innovative solution for early tumor screening, personalized diagnosis and treatment, and home health management, offering high throughput and broad application prospects. It is particularly suitable for point-of-care diagnostic scenarios such as primary hospitals, emergency departments, and home self-testing, and can be extended to the analysis of various biological samples such as serum, saliva, and urine. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the overall design of a multi-parameter intelligent urine analyzer.

[0019] Figure 2 This is a schematic diagram of the internal exploded structure of the instrument.

[0020] Figure 3 This is a schematic diagram of the overall structure of the detachable sensor chip.

[0021] Figure 4 This is a schematic diagram of the design of a micro electrochemical workstation and sensor chip.

[0022] Figure 5 This is a schematic diagram of the instrument's fluid circuit system design.

[0023] Figure 6 This is a schematic diagram of the precise immobilization of multi-target nucleic acid probes under electric field modulation.

[0024] Figure 7This is a schematic diagram of the conformational changes of the fluorescent probe on the electrode surface under electric field modulation and a comparison of confocal fluorescence images.

[0025] Figure 8 Two-dimensional potential distribution diagrams of the traditional electrode layout scheme and the optimized electrode layout scheme.

[0026] Figure 9 This is a graph showing the potential decay curves of different working electrodes along the reference electrode direction under the traditional electrode layout scheme and the optimized electrode layout scheme.

[0027] Figure 10 It is a three-dimensional electric field spatial distribution characteristic diagram under different negative voltage conditions.

[0028] Figure 11 This is a trend graph of the MB peak current signal obtained by SWV detection under different applied voltages and modification times.

[0029] Figure 12 This is a three-dimensional electric field distribution simulation diagram showing the application of a +0.4 V potential to the target working electrode and a -0.05 V potential to the other non-target working electrodes.

[0030] Figure 13 This is a schematic diagram of multi-target synchronous detection based on a competitive combination strategy. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0032] This invention proposes a device for rapid modification of multiple nucleic acid probes and detection of multiple targets based on electric field regulation. The device consists of an instrument body 1 and a detachable sensor chip 2.

[0033] like Figure 1 As shown, the main body of the instrument 1 adopts an integrated design, with overall dimensions of approximately 320 mm × 340 mm × 173 mm, and is equipped with a touch-screen host computer display 11, which can display the detection status, experimental progress, and analysis results in real time. Figure 2As shown, the instrument is divided into a reagent compartment 12, a detection compartment 13, and a liquid system 14. The reagent compartment 12 can hold eighteen 10... The mL polypropylene reagent bottle 121 can be used to store nucleic acid probe solution, blocking solution, cleaning solution, body fluid samples to be tested, and deionized water. Each reagent bottle 121 is connected to three ten-channel switching valves 141 through a Teflon tube led out of a silicone stopper. The reagent compartment door 122 facilitates sample loading and reagent storage. The liquid circuit system 14 integrates a peristaltic pump 142, a two-position three-way solenoid valve 143, and a multi-channel switching valve 141. Under the command of the microcontroller 133, the nucleic acid probe solution, blocking solution, cleaning solution, and body fluid samples to be tested are sequentially delivered to the detachable sensor chip 2, realizing programmable liquid circuit switching and cleaning. The detection compartment 13 integrates a power module 131, a micro electrochemical workstation module 132, a microcontroller 133, and a signal conversion module 134. The micro electrochemical workstation module 132 integrates a programmable multi-channel potentiostat and a signal acquisition and conversion circuit, and has a female connector 1321 at its front end that matches the sensor chip, which is conducive to the replacement and maintenance of the detachable sensor chip 2.

[0034] like Figure 3 As shown, the detachable sensor chip 2 is formed by bonding a microelectrode array chip 21 and a microfluidic chip 22, and then physically fixing and electrically connecting them via a PCB adapter board 23. The microelectrode array chip 21 integrates eight independent detection units on an insulating substrate. Each detection unit includes five working electrodes 211, one reference electrode 212, and one counter electrode 213. The microfluidic chip 22 has eight independent reaction chambers fabricated at corresponding positions, each covering one detection unit. The reaction chambers are equipped with inlet and outlet channels and have reserved steel needle connector ports 221 for communication with external liquid systems.

[0035] During the nucleic acid probe modification stage, the microcontroller 133 applies a positive potential to the selected working electrode in the target detection unit through the micro electrochemical workstation module 132, and applies a negative potential to the remaining working electrodes in the same detection unit and other detection units. After probe modification is completed, the liquid circuit system 14 introduces the body fluid sample to be tested into the reaction chamber. The potentiostat performs voltammetric scanning on the working electrodes of each detection unit, and the signal acquisition and conversion circuit outputs current data in real time. After digital processing by the microcontroller, quantitative detection results of multiple body fluid disease markers, including but not limited to IL-6, PD-L1, Mucin-1, miRNA-205, and miRNA-141, in multiple samples are obtained simultaneously in a single operation.

[0036] Specifically, the liquid system 14 includes a peristaltic pump 142, a two-position three-way solenoid valve 143, and a multi-channel switching valve 141. The peristaltic pump 142 provides a stable flow rate to ensure accurate delivery of reagents and samples, and under the command of the microcontroller 133, it is responsible for the quantitative delivery of nucleic acid probe solution, blocking solution, washing solution, and the body fluid sample to be tested. The two-position three-way solenoid valve 143 and the multi-channel switching valve 141, through a programmable rotary switching mechanism, precisely distribute the liquid source to the designated detection chamber, supporting high-throughput automated experimental procedures. To avoid cross-contamination, the liquid system 14 is designed with an automated cleaning function. After each nucleic acid probe modification, sample injection, and detection, the liquid system 14 automatically switches to the deionized water channel for thorough rinsing, and then discharges the residual liquid in the flow channel to the waste liquid pool by injecting air. The overall design goal of the liquid system 14 is to achieve programmable liquid switching and cleaning. Under the command of the microcontroller 133, the pump and valve work together to automate the complex liquid transfer, distribution, and cleaning process.

[0037] Specifically, the micro electrochemical workstation module 132 is equipped with a multi-channel analog switch, which can switch forty electrode channels without changing the main circuit structure, supports multi-channel parallel electrochemical measurement, and has a female connector 1321 matching the sensor chip 2 at its front end, so as to realize a fast and convenient electrical connection between the detachable sensor chip 2 and the instrument body 1.

[0038] Specifically, the detachable sensor chip 2 is formed by bonding the microelectrode array chip 21 and the PDMS microfluidic chip 22, and then fixing them together via a PCB adapter board 23. The detachable sensor chip 2 allows for a pluggable electrical connection to the instrument body 1. Specifically, this is achieved through the pin header interface 231 on the PCB adapter board 23 and the female header interface 1321 at the front end of the micro electrochemical workstation module 132, enabling electrical connectivity between the multi-electrode array and core modules such as the potentiostat, signal conditioning, and microcontroller. This facilitates rapid replacement of the detachable sensor chip 2. Figure 4 As shown.

[0039] Specifically, the microelectrode array chip 21 integrates eight independent detection units, each comprising five working electrodes 211, one reference electrode 212, and one counter electrode 213, forming a standard three-electrode system. The working electrodes 211 have a diameter of 100 μm, and the reference electrode 212 has a diameter of 200 μm, facilitating high-density integration. The five working electrodes 211 are arranged around the center of the detection unit in a hexagonal configuration, with the reference electrode 212 located at the geometric center. This central layout was optimized using COMSOL finite element simulation to ensure consistent distances between each working electrode 211 and the reference electrode 212, thereby guaranteeing a highly uniform electric field distribution when a potential is applied, and improving the uniformity and controllability of probe modification. The reference electrode 212 is prepared with an Ag / AgCl layer on a gold seed layer via electrochemical oxidation, ensuring potential stability and long-term effectiveness. The counter electrode 213 enhances its inertness and conductivity by electrodepositing a platinum layer. It is also designed as a closed ring structure that surrounds and encloses all working electrodes 211. Its area is significantly larger than that of the working electrodes 211, which effectively reduces current density, reduces polarization effect and improves system stability.

[0040] Specifically, in the rapid nucleic acid probe modification stage, the microcontroller 133 applies a positive potential to the selected working electrode of the target detection unit through the micro electrochemical workstation module 132, while simultaneously applying a negative potential to the remaining working electrodes within the same detection unit and other unselected detection units. Due to the negative charge of DNA molecules, when a positive potential (preferably +0.4 V) is applied to the target working electrode, the probe molecules will be electrostatically attracted, rapidly migrate to the electrode surface, and be efficiently immobilized. The +0.4 V voltage has been optimized through experiments and simulations to ensure rapid probe adsorption while avoiding structural damage. A negative potential (preferably −0.05 V) is applied to the non-target working electrodes. This negative potential forms an electrostatic repulsion field. Utilizing the positive attraction and negative repulsion effects, the spatially selective and rapid immobilization of the nucleic acid probe on the target working electrode and precise density control are achieved within a limited time, effectively suppressing non-specific adsorption of DNA probes. The −0.05 V negative potential has been verified by COMSOL simulations to form an effective repulsion region without compressing the range of the positive electric field of the target electrode, ensuring spatially selective probe modification. The application time for probe modification is preferably no more than 120 s. At this potential and duration, rapid and efficient probe immobilization can be achieved without damaging the probe structure. The liquid system 14 sequentially introduces different nucleic acid probe solutions into the corresponding detection chambers. The microcontroller 133 precisely controls the electrode potential of each detection unit, achieving spatial selectivity and sequential immobilization of multiple different nucleic acid probes on each working electrode. The entire process consumes less than 2 mL of reagents and saves two orders of magnitude of time compared to traditional static incubation, completely avoiding cross-contamination between arrays. After probe modification, the liquid system 14 automatically introduces a blocking solution to seal the electrode surface, blocking non-specific binding sites, followed by rinsing with deionized water.

[0041] Specifically, after the probes are assembled, the device automatically executes the sample detection mode. The peristaltic pump 142 mixes the body fluid sample with the detection reagent containing the MB-labeled aptamer at a 1:1 volume ratio, pushing 100 µL of the mixture into each reaction chamber and allowing it to stand for 30 min to complete target-aptamer binding. Subsequently, the micro-electrochemical workstation module 132 triggers a square-wave voltammetric scan with parameters set to an amplitude of 25 mV, a frequency of 15 Hz, 5 sampling cycles, and a single-chamber measurement duration of 9 s. The host computer 11 automatically acquires the MB peak current and matches the calibration curve, simultaneously displaying the quantitative results of IL-6, PD-L1, Mucin-1, miR-141, and miR-205 in eight samples on a single screen. The limits of detection are 28 pM, 34 pM, 27 pM, 18 pM, and 20 pM, respectively, with intra-assay RSDs all below 5%.

[0042] Example 1:

[0043] The instrument's liquid circuit system is controlled by three multi-channel switching valves (valve A, valve B, and valve C) and a two-position three-way solenoid valve, along with a peristaltic pump and check valves, to achieve precise dispensing and delivery of reagents and samples under the command of the microcontroller. A schematic diagram of the liquid circuit system is shown below. Figure 5 As shown. Valve A mainly manages various urine samples to be tested, while valve B is responsible for various reagents required for probe modification, cleaning, and sealing. Valve C, as an output valve, can be rotated to connect its channels sequentially to different detection chambers, thereby completing the sequential injection of liquid into multiple detection chambers. A two-position three-way solenoid valve is used to select the connection between valve A or valve B and valve C, allowing the instrument to flexibly switch between probe modification mode and sample detection mode.

[0044] During the probe modification stage, a two-position three-way solenoid valve connects valve B and valve C. Valve B, acting as a reagent source, manages various reagents required for probe modification, cleaning, and blocking. A microcontroller controls valve B to switch to a specific probe solution port, and a peristaltic pump drives the probe solution through valve C. Valve C, acting as an output valve, injects the probe solution sequentially into each detection chamber by rotation. After probe solution injection, the micro-electrochemical workstation applies a positive potential to the target electrode and a negative potential to the non-target electrodes to achieve rapid and precise immobilization of the nucleic acid probe on the target electrode surface. After modification, valve B switches to the deionized water channel for thorough cleaning of the liquid path and detection chambers, then switches to the air port to drain the deionized water in the liquid path to the waste tank using air pressure. This process is repeated until all probes are modified and blocked.

[0045] During the sample detection stage, a two-position three-way solenoid valve connects valve A and valve C. Valve A, acting as the sample source, manages the urine sample to be tested. The microcontroller controls valve A to sequentially select the sample to be tested, and, in conjunction with the rotating channel of valve C, achieves orderly matching and injection of the sample into the detection chamber. Before matching the next sample, the system automatically switches to the deionized water channel to flush the liquid path to avoid cross-contamination between samples. To prevent backflow or backflow of the solution during drainage, one-way valves are installed between the waste liquid tank and each detection chamber. Under the coordinated control of the host and slave computers, the instrument can flexibly switch between the two major processes of probe modification and sample detection, achieving efficient and automated multi-channel fluid control and management.

[0046] Example 2:

[0047] The core of the rapid nucleic acid probe assembly method based on electric field modulation lies in utilizing the overall negative charge of DNA molecules. By precisely encoding the potential of a multi-electrode array, the "attraction-repulsion" distribution control of the probes is achieved. Specifically, this method applies a positive potential to the target electrode, using electrostatic attraction to drive the rapid migration and efficient immobilization of negatively charged probe molecules. Simultaneously, a negative potential is applied to the non-target electrode, suppressing non-specific adsorption of the probes through electrostatic repulsion. This enables spatially selective modification of the probes on the electrode surface, such as... Figure 6 As shown. By switching the potential polarity of different electrodes ( Figure 6 The method (AE) sequentially performs spatial selective immobilization of five DNA probes—IL-6, PD-L1, Mucin-1, miR-205, and miR-141—on different electrodes in the array. Compared to traditional immobilization methods, this method can modify multiple nucleic acid probes in a single channel, and by adjusting the voltage and reaction time, the assembly rate and density of the probes can be precisely controlled, significantly improving modification efficiency and controllability. The effectiveness of this method has been verified by in-situ observation of the fluorescence signal changes of the probes on the electrode surface using confocal fluorescence microscopy. Under a positive potential, the negatively charged phosphate groups on the DNA backbone are electrostatically attracted and tilt towards the gold surface, reducing the distance between the fluorophore and the gold surface, resulting in quenching and a significant weakening of the fluorescence signal. Under a negative potential, they are electrostatically repelled and tend to stand upright, causing the fluorophore to move away from the gold surface, weakening the quenching effect, and thus enhancing the fluorescence signal. Figure 7 This demonstrates that an electric field can reversibly modulate the conformation of the probe on the electrode surface, thereby affecting the fluorescence signal, providing key technical support for high-throughput multi-target detection.

[0048] Example 3:

[0049] The layout design of the microelectrode array directly determines the uniformity of the electric field distribution on the electrode surface, thus affecting the precise controllability of the nucleic acid probe during electric field-induced modification. The microelectrode array consists of five independent working electrodes, one reference electrode, and one counter electrode. The counter electrode is designed as a semi-circular ring structure surrounding the working electrodes, with a significantly larger area than the working electrodes, which reduces the current density on the electrode surface, minimizes polarization effects, and improves system stability. To improve the symmetry and uniformity of the electric field distribution, the reference electrode is placed at the geometric center of the five working electrode array, ensuring that the distance between each working electrode and the reference electrode is completely consistent, thus eliminating potential inhomogeneity caused by distance differences. The optimized layout is compared with the traditional layout using finite element simulation analysis (COMSOL). In the traditional scheme, the reference electrode is usually placed outside the counter electrode, in an arc shape (…). Figure 8 (A in the original text); while the design of this study places the reference electrode in the center, and the five working electrodes are symmetrically arranged around the reference electrode in the form of regular hexagonal vertices (A in the original text). Figure 8 In step B), a positive potential is applied to each working electrode relative to the reference electrode, while a negative potential is applied to the other electrodes, thus obtaining... Figure 8 The two-dimensional electric potential field distribution is shown. Simulation results show that the electric field distribution under the optimized layout is more symmetrical and uniform.

[0050] Example 4:

[0051] To more accurately and quantitatively evaluate the electric field distribution of the two layout schemes, the potential distribution variation within a 500 μm range extending from the center of each working electrode towards the reference electrode was further analyzed. Traditional layout ( Figure 9 The A and B sections exhibit significant inhomogeneity, with large differences in the potential decay rate of the working electrode towards the reference electrode at different locations; while the optimized layout ( Figure 9 The C and D parameters significantly improve the non-uniformity of potential decay, resulting in uniform potential decay across all electrodes. Therefore, by optimizing the position of the reference electrode and the symmetrical layout of the working electrodes, the spatial distribution of the electric field is significantly improved. In the actual electric field-induced probe modification process, more precise and uniform electric field control can be achieved, thereby effectively ensuring the consistency of probe modification density across different electrodes and improving the reliability of multi-target detection.

[0052] Example 5:

[0053] In the process of electric field-induced modification of multi-target nucleic acid probes, it is necessary not only to apply an appropriate positive potential to the target electrode to attract the probe, but also to apply a negative potential to adjacent electrodes to suppress non-specific adsorption of the probe, so as to ensure the controllability of nucleic acid probe modification density and efficiency. Through simulation of the three-dimensional electric field distribution, the influence of the combination of positive and negative potentials on the electric field distribution was systematically studied. Figure 10With a fixed voltage of +0.8 V applied to five target working electrodes (#1 to #5) relative to the reference electrode, the remaining four working electrodes were subjected to the same negative voltages (-0.05 V, -0.10 V, -0.15 V, and -0.20 V). The three-dimensional electric field spatial distribution characteristics under different negative voltage conditions were systematically analyzed. The results show that the magnitude of the applied negative potential directly determines the electric field boundary and the effective modification region. When the negative potential of the non-target electrode is controlled within −0.15 V, the influence range of the negative electric field is small and does not significantly limit the positive potential region generated by the target electrode. Probe molecules can effectively migrate to the surface of the target electrode, thereby achieving efficient modification. However, when the negative potential increases to −0.20 V, the combined effect of multiple negative potential electrodes compresses the positive electric field region into a confined spherical region, significantly reducing the effective region of electric field-induced modification, thereby reducing the adsorption efficiency of the probe and the controllability of the modification density.

[0054] Example 6:

[0055] Based on simulation analysis of the potential application strategy, the optimal modification parameters for the nucleic acid probe were further determined through experiments. Figure 11 The variation trend of probe signal with induction time under different voltages is shown. It can be observed that at voltages of 0.1 V and 0.4 V, the probe current signal increases linearly with time, with 0.4 V exhibiting higher fixation efficiency. However, when the voltage increases to 0.8 V, the current signal continues to rise for the first 120 seconds, but decreases after this point, suggesting that while high voltage promotes probe enrichment, it may also trigger the destruction of gold-sulfur bonds, leading to partial desorption of the fixed probe. This phenomenon is more pronounced at 1.2 V. Considering both modification efficiency and stability, 0.4 V was ultimately selected as the optimal induction voltage. Furthermore, COMSOL simulations verified that applying +0.4 V to the target electrode while simultaneously applying −0.05 V to the non-target electrode can create a moderately negative electric field repulsion region. Figure 12 This voltage combination effectively suppresses non-specific adsorption of the probe to non-target electrodes without compressing the positive electric field range of the target electrode, thus providing good selective modification conditions between electrodes and reliable technical support for high-throughput detection of multiple targets.

[0056] Example 7:

[0057] This invention discloses the simultaneous detection of urinary system tumor markers (IL-6, PD-L1, Mucin-1, miRNA-205, and miRNA-141) on the same platform for bladder cancer and other related tumors. Five working electrodes deployed on each channel of the sensor chip are modified with specific probes corresponding to the five targets. Figure 13The 3' end of the aptamers or antichains of the five targets is modified with MB molecules. If the target is present in the urine sample, it will bind to the corresponding aptamer or antichain. Unbound free aptamers / antichains flow across the electrode surface and bind to complementary probes immobilized on the electrode, thus bringing MB molecules to the electrode interface. Through parallel measurements using a multi-electrode array, the system can detect the electrochemical signal intensity of MB molecules on each electrode surface. This signal intensity is negatively correlated with the number of free aptamers / antichains, allowing for the deduction of the actual content of the target biomarker in the urine. Utilizing parallel measurements of the multi-electrode array and chip-select control of an analog switch, this device can simultaneously and sensitively detect five targets in a single experiment, providing a feasible multi-biomarker integrated detection platform for clinical urine testing and early tumor screening.

[0058] It should be stated that the content and specific embodiments of this invention are intended to demonstrate the practical application of the technical solutions provided by this invention, and should not be construed as limiting the scope of protection of this invention. Any modifications and changes made to this invention within the spirit and scope of the claims fall within the protection scope of this invention.

Claims

1. A device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation, characterized in that, The device consists of a main instrument body and a detachable sensor chip; The instrument body adopts an integrated design and is electrically connected to the sensor chip via a pin header interface. The instrument body is divided into a reagent compartment, a detection compartment, and a liquid system. The liquid system integrates a peristaltic pump, a two-position three-way solenoid valve, and a multi-channel switching valve, enabling programmable liquid path switching and cleaning under the command of a microcontroller. The peristaltic pump and the multi-channel switching valve of the liquid system achieve programmable switching of the liquid path through the two-position three-way solenoid valve. After each nucleic acid probe modification, sealing, sample injection, and detection, the liquid system automatically switches to the deionized water channel and discharges to the waste container via a one-way valve. The detachable sensor chip is formed by bonding a microelectrode array chip and a microfluidic chip. The microelectrode array chip integrates multiple independent detection units, each of which includes multiple working electrodes, a reference electrode, and a counter electrode. The working electrodes of the detection unit are arranged around the center of the detection unit, the reference electrode is located at the geometric center, and the counter electrode forms a closed ring and surrounds all the working electrodes. Each reaction chamber of the microfluidic chip covers a detection unit and is connected to the liquid path system through inlet and outlet channels. During the nucleic acid probe modification stage, a positive potential is applied to the selected working electrode in the target detection unit, and a negative potential is applied to the remaining working electrodes in the same detection unit and all electrodes in other detection units. After probe modification, the liquid circuit system introduces the body fluid sample to be tested into the reaction chamber. The working electrode of each detection unit is scanned by a potentiostat, and the current data is collected and digitally processed by a microcontroller. In a single operation, quantitative detection results of multiple body fluid disease markers in multiple samples are obtained simultaneously.

2. The device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation according to claim 1, characterized in that, The detection chamber integrates a power supply module, a micro electrochemical workstation module, a microcontroller module, and a signal conversion module. The micro electrochemical workstation module integrates a programmable multi-channel potentiostat and a signal acquisition and conversion circuit.

3. The device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation according to claim 2, characterized in that, The micro electrochemical workstation module is equipped with a multi-channel analog switch, which can switch forty electrode channels without changing the main circuit structure. It also has a header interface at its front end that matches the detachable sensor chip, supporting multi-channel parallel electrochemical measurements.

4. The device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation according to claim 3, characterized in that, The detachable sensor chip can be plugged into the instrument body for electrical connection. Specifically, it is physically connected to the pin header interface on the PCB adapter board and the female header interface on the front end of the micro electrochemical workstation module, so as to realize the electrical connection between the multi-electrode array and the potentiostat, signal conditioning and microcontroller. The pluggable electrical connection is used to quickly replace the detachable sensor chip.

5. The device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation according to claim 2, characterized in that, The microcontroller module applies a positive potential to the selected working electrode in the target detection unit through the micro electrochemical workstation module, and applies a negative potential to the remaining working electrodes and other detection units in the same detection unit.

6. The device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation according to claim 1, characterized in that, During the rapid modification phase of the nucleic acid probe, the positive potential applied to the target working electrode is +0.4 V, and the negative potential applied to the non-target working electrode is -0.05 V, with the application time not exceeding 120 s.

7. The device for rapid modification and multi-target detection of multiple nucleic acid probes based on electric field modulation according to claim 1, characterized in that, The various humoral disease markers include interleukin-6, programmed death ligand, mucin-1, miRNA-205, and miRNA-141.

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