Acetylcholinesterase detection method based on SERS (Surface Enhanced Raman Scattering) and collaborative application platform of acetylcholinesterase detection method and digital microfluidic

Abstract

The invention discloses an acetylcholin esterase SERS (Surface Enhanced Raman Scattering) detection platform and a detection method based on digital microfluidics, and designs an acetylcholin esterase SERS detection method capable of improving performance by utilizing a DMF (Dimethyl Formamide) technology on the basis of automation and accuracy of the DMF technology and aiming at the defect of poor reproducibility of the SERS technology. The SERS technology and digital microfluidics are combined in acetylcholin esterase detection for the first time, the sensitivity of acetylcholin esterase detection is further improved through combination of the SERS technology and the digital microfluidics, and the demand quantity of samples is greatly reduced. According to the detection platform and the detection method, sample treatment and SERS detection are integrated, the highly-integrated detection platform is formed, field detection is facilitated, full-process detection can be automatically completed, personal errors are reduced, multiple samples can be treated at the same time, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to biological detection, and in particular relates to a SERS-based acetylcholinesterase detection method and a collaborative application platform thereof with digital microfluidics. Background Art

[0002] The detection of acetylcholinesterase (AChE) in serum is of great significance. In the field of organophosphorus pesticide and nerve agent poisoning, it can be used for poisoning diagnosis. The degree of decrease in its activity is related to the severity of the poisoning. It can also monitor the effect during treatment and assist in adjusting the plan. In terms of liver disease, it can evaluate the synthetic function of the liver. For example, the activity is often reduced in cirrhosis, and dynamic monitoring helps to observe the progression of the disease and prognosis. In neurological diseases, it has auxiliary diagnostic value for neurodegenerative diseases such as Alzheimer's disease, and can be combined with other examinations to comprehensively judge the condition; for neuromuscular junction diseases such as myasthenia gravis, it can assist in understanding the therapeutic effect of acetylcholinesterase inhibitors and adjusting the dosage.

[0003] Surface enhanced Raman spectroscopy (SERS) refers to the process of adsorbing the analyte molecules on the rough surface of nano-metal materials, which can enhance the Raman signal of the analyte by 10 6 -10 14 times the spectral phenomenon. SERS technology has attracted widespread attention due to its simple and rapid operation, ability to detect low concentrations of substances, easy portability of detection instruments, and the ability to achieve real-time detection. Due to the advantages of fast reading and non-destructive information acquisition, SERS is considered to be an attractive detection method for chemical and biological analysis. Many strategies have been developed for indirect SERS detection of enzyme activity. When the substrate and product of the enzyme cannot provide strong SERS signals, masked or unmasked dyes (synthetic substrates containing dye groups) are used to obtain strong signals, or the product further reacts to produce changes in SERS intensity. Combined with a micro-Raman spectrometer, SERS signals can be collected from micron-scale SERS substrates.

[0004] Raman spectroscopy is label-free and highly specific for AChE detection. However, due to the inherently weak Raman activity of AChE, direct detection of AChE and neural tissue is not sensitive enough. Therefore, the present invention has developed a SERS-based acetylcholinesterase detection method with high sensitivity, low detection limit, small sample size, and short detection time.

[0005] The core principle of digital microfluidics (DMF) is to achieve precise manipulation of tiny droplets based on the electrowetting effect (EWOD). It is a special variant of chip microfluidics in which discrete droplets ranging in size from nanometers to microliters are transported on a patterned electrode array. Its basic working principle is to control the movement, splitting, merging, and other operations of the droplets by applying voltage to the electrode array to change the contact angle between the droplets and the solid surface. Specifically, the digital microfluidic system consists of a substrate, an electrode array, a hydrophobic layer, and a drive circuit. The surface of the substrate is covered with a layer of insulating hydrophobic material, and multiple independently controlled electrodes are embedded underneath. When an electrode is activated, the droplet will undergo local wetting under the action of the electric field, resulting in a decrease in the contact angle and movement of the droplet toward the activated electrode. By programming the on-off sequence and voltage changes of the electrodes, complex operations such as precise positioning, transmission, mixing, and splitting of droplets can be achieved. This pipe-free design not only avoids the clogging problem in traditional microfluidics and has significant advantages over manual operation, but also provides a high degree of flexibility and automation capabilities, making digital microfluidics an ideal technology platform for fields such as biomedicine, chemical analysis and environmental monitoring.

[0006] The prior art discloses a method for rapid detection of trace acetylcholinesterase based on SERS technology (CN113358626 A). The present invention discloses a method for rapid detection of trace acetylcholinesterase based on SERS technology. The method is based on a functionalized SERS substrate and establishes a standard curve of the enzyme content of acetylcholinesterase and the ratio of the Raman characteristic peak intensity of a heterocyclic compound containing a quaternary ammonium salt to the Raman characteristic peak intensity of an aromatic compound containing a sulfhydryl group as an internal standard. The acetylcholinesterase content in the analyte is obtained from the standard curve based on the ratio of the Raman characteristic peak intensity of the heterocyclic compound containing a quaternary ammonium salt to the Raman characteristic peak intensity of the aromatic compound containing a sulfhydryl group as an internal standard in the SERS spectrum of the analyte. The detection method can achieve rapid and ultrasensitive detection of acetylcholinesterase in microscopic biological tissues such as nerve slices. The detection method has the advantages of simple operation, low cost, and rapidity, and is suitable for research on novel biomedical problems, especially clinical microneurosurgical surgery. The SERS substrate in the above-mentioned patent is an acupuncture needle tip modified with gold nanoparticles, which requires manual operation, will produce certain errors and contamination, and uses a large number of reagents. In addition, the above-mentioned patent requires the use of a constant temperature box when incubating the reaction solution at a constant temperature, which is relatively complicated and hinders its application scenarios.

[0007] Currently, existing SERS detection methods combined with DMF mainly include: 1. Adding metal colloidal nanoparticle solutions. For colloidal nanoparticles, the respective solutions are transferred into the microchannel and mixed with the analyte. Using colloidal solutions is generally the simplest method for achieving SERS. However, quantitative SERS analysis is complicated by batch variability in nanoparticle synthesis and aging effects. In a microfluidic environment, the use of colloidal nanoparticle solutions often contaminates or even clogs the microfluidic channels. In particular, the risk of device contamination due to aging or precipitation of gold (silver) colloids is a significant issue, especially when the DMF chip is intended for multiple uses. Precipitation is another significant issue in DMF applications, as precipitated silver can lead to permanent wetting of the DMF droplet on the dielectric, hindering the EWOD process. Furthermore, these nanoparticles can sometimes negatively impact the (bio)chemical processes or other downstream processes being studied. Furthermore, such SERS substrates are often difficult or unintuitive to focus during signal acquisition. Typically, the laser spot is focused at the same depth within the droplet each time to avoid signal interference caused by varying optical pathlengths, resulting in a complex operation. 2. Integrating a fixed solid SERS substrate. To overcome the above-mentioned shortcomings of colloidal nanoparticles, immobilized SERS targets, such as immobilized nanoparticles or roughened gold (or silver) nanoparticle surfaces, can be integrated into microfluidic systems. However, in addition to the high difficulty of the manufacturing process, the integration of immobilized SERS substrates also brings a new problem, namely the irreversible adsorption of analytes, commonly known as the "memory effect." Summary of the Invention

[0008] Purpose of the Invention: To address the challenges of the prior art, the present invention provides a digital microfluidics-based SERS detection platform and method for acetylcholinesterase. By combining a digital microfluidics platform with SERS technology, the present invention designs a SERS-based acetylcholinesterase detection method that can be implemented using DMF technology. This effectively addresses the various challenges encountered in the prior art when using metal colloidal nanoparticle solutions in DMF. Furthermore, the present invention overcomes the difficulties inherent in fabricating solid SERS substrates, which place high demands on the process, and effectively mitigates the "memory effect," enabling the DMF platform to perform repeated detections and continuously provide SERS substrates within a single operation.

[0009] Technical solution: In order to achieve the above-mentioned purpose, the present invention describes an acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics, wherein the platform adopts a parallel plate form, wherein the upper electrode plate is composed of a transparent conductive material and glass used as a ground electrode, and a hydrophobic layer is spin-coated on it; the lower electrode plate is composed of a liquid storage electrode and a driving electrode array, the electrode layer of the liquid storage electrode is covered with a hydrophobic dielectric layer, and different liquid storage areas are arranged on the liquid storage electrode. When the platform is running, droplets are split out from the liquid in the liquid storage area, and the droplets are sandwiched between the two parallel plates and move in the driving electrode array area on the lower electrode plate, first entering the reaction area, and finally being moved to the waste liquid area after the reaction; the solid SERS substrate is attracted under the upper electrode plate and moved to the SERS substrate-molecule adsorption site on the upper electrode plate by magnetism. The SERS substrate-molecule adsorption site is the site on the upper electrode plate where the reaction solution and the solid SERS substrate are adsorbed.

[0010] Furthermore, the platform adopts a parallel plate structure. The DMF platform consists of two parts: an upper plate and a lower plate. The upper plate comprises a base glass substrate, an electrode layer made of a transparent conductive material (a layer of ITO transparent conductive layer), and a hydrophobic layer coated on the electrode layer. The lower plate is based on a PCB substrate and comprises an electrode layer composed of a reservoir electrode and a drive electrode array. The electrode layer is covered with a dielectric layer and a hydrophobic layer. The upper and lower plates are assembled together and separated by 300-micron thick double-sided tape. The droplet is sandwiched between the hydrophobic layers of the two parallel plates. The electrodes on the lower plate of the platform are arranged in a pattern. The lower plate is arranged with 128 electrodes, including 118 drive electrodes (2.75 mm × 2.75 mm) and 10 reservoir electrodes (8.25 mm × 10 mm), with 100-micron spacing between the electrodes. All electrodes are connected to the contact electrode via leads with a width of 40 microns. The solid-state SERS substrate is fixed or attracted to the bottom of the upper plate. The solid-state SERS substrate contains sites for adsorption of signal molecules, namely SERS active sites.

[0011] Among them, the upper electrode plate in the platform is usually made of transparent conductive material ITO (indium tin oxide) glass, and a hydrophobic coating (such as Teflon) is spin-coated on its surface. The main function of this hydrophobic layer is to reduce the contact angle hysteresis between the droplet and the surface, thereby improving the mobility of the droplet under the action of the electric field and ensuring that the droplet can be driven and manipulated smoothly. In addition, the electrode of the upper electrode plate is an ITO conductive layer, which acts as a ground electrode. Its main function is to form a vertical electric field together with the driving electrode of the lower electrode plate. As a ground reference point, it helps to stabilize the electric field distribution, prevent charge accumulation, improve the accuracy of droplet manipulation and the stability of the digital microfluidic system operation. The lower electrode plate is usually integrated with multiple functional electrodes, among which the liquid storage electrode is used to store the reagents required during the experiment or collect the waste liquid after the reaction. The driving electrode is the core part of realizing droplet manipulation. By applying a switching voltage to control the electrowetting effect, the droplets can be accurately moved, split, merged or mixed between the electrode arrays.

[0012] Furthermore, there is a gap between the upper and lower plates for the liquid droplets to move therein.

[0013] The transparent conductive material is indium tin oxide, and the upper electrode is spin-coated with a hydrophobic layer Teflon AF1600s.

[0014] The electrode layer is covered with a hydrophobic dielectric layer - a 40-micron PTFE film.

[0015] Preferably, different liquid storage areas are provided on the liquid storage electrode, and the liquid storage areas include a mixed solution area of NBA and ATCh, a buffer area, and a sample area to be detected.

[0016] The sample area to be detected includes 1-5 samples to be detected.

[0017] Preferably, the sample area to be detected includes 3 samples to be detected.

[0018] The reaction zone is a heated constant temperature incubation zone, and the reaction solution is a sample solution in the storage zone that is split into a test solution and moves to the reaction zone to mix with the NBA and ATCh mixed solution zone to form a mixed NBA and ATCh solution.

[0019] The solid-state SERS substrate is attracted to the bottom of the upper plate by the magnet on the upper plate, and is controlled by the magnet to move into and out of the SERS substrate-molecule adsorption site.

[0020] Preferably, the steel sheet below the upper plate is attracted by the magnet on the upper plate (ie, the steel sheet modified with gold nanoparticles is the SERS substrate), and the movement of the SERS substrate is caused by the attraction of the magnet.

[0021] Among them, the preparation of the solid-state SERS substrate is to immerse a stainless steel disc in an ethanol solution containing MPTES and APTES, then wash the functionalized stainless steel disc, drop a gold nanoparticle suspension, and then wait for the water to evaporate naturally in an airless environment. After the gold nanoparticles are adsorbed on the disc, the solid-state SERS substrate is prepared.

[0022] The SERS detection method for acetylcholinesterase based on the digital microfluidics detection platform of the present invention comprises the following steps:

[0023] (1) The solutions are stored in the liquid storage area respectively. During the detection, each stored solution is split from the liquid storage area to generate small droplets of the same volume;

[0024] (2) Preparation of acetylcholinesterase standard curve: The platform splits droplets from the NBA (Nile blue) and ATCh mixed solution area in the liquid storage area and moves them to the reaction area, splits the AChE standard solution from the sample area to be tested in the liquid storage area and moves it to the reaction area and the NBA and ATCh mixed solution split from the NBA and ATCh mixed solution area to mix and react, and then moves the reaction solution to the SERS substrate-molecular adsorption site, and then the solid SERS substrate will be magnetically controlled to slowly enter the SERS substrate-molecular adsorption site to be immersed in the reaction solution. The immersion process is accompanied by competitive adsorption of NBA and TCh. After this process is completed, the SERS substrate is moved away and the solid SERS substrate is moved out of the platform to collect the Raman spectrum on the SERS substrate, collect the intensity ratio of NBA and 4-MBA (4-mercaptobenzoic acid), and make an enzyme standard curve based on the intensity ratio. Finally, the reaction solution is moved to the waste liquid area, and then PBS droplets are generated from the buffer area of the liquid storage area to clean the path where the sample to be tested has moved, thereby reducing the amount of protein in the biological sample attached to the lower plate electrode and increasing the number of uses; preventing the influence of experimental reagent residues on the next group of experimental data.

[0025] (3) Biological sample detection: Replace the solutions in the three sample areas to be tested on the detection platform with biological samples, and perform the biological sample detection while keeping the other steps unchanged.

[0026] The splitting is to generate droplets from the liquid storage area: first, by activating the electrodes near the liquid storage area, the liquid is pulled out of the liquid storage area using the electrowetting effect to form a connected droplet; then, the adjacent electrodes are activated to stretch the droplet to form a "liquid bridge"; then, the liquid storage area electrode is turned off and the adjacent electrodes are kept activated, so that the droplet breaks at the neck, achieving droplet splitting.

[0027] Furthermore, the present invention relates to an in vitro acetylcholinesterase SERS detection method based on digital microfluidics.

[0028] This invention combines SERS technology with digital microfluidics for the detection of acetylcholinesterase for the first time. This combination further enhances the sensitivity of acetylcholinesterase detection and significantly reduces sample requirements. The detection platform and method of this invention integrate sample processing and SERS detection, forming a highly integrated detection platform that facilitates on-site testing, automates the entire detection process, reduces human error, and can process multiple samples simultaneously, improving detection efficiency.

[0029] This invention combines enzyme detection with digital microfluidics, significantly reducing sample and reagent consumption and testing costs. Furthermore, due to its small size, it is not subject to kinetic diffusion constraints, significantly improving the effectiveness of enzyme detection. Furthermore, the invention integrates a heater plate on the back of the electrode plate, creating constant-temperature incubation conditions in the reaction and adsorption zones. This results in high integration, ease of operation, and minimal instrumentation. The invention also utilizes SERS in conjunction with DMF to expand the optimal detection range and further reduce the detection limit.

[0030] Based on the automation and accuracy of digital microfluidics, this paper combines the advantages of the digital microfluidics platform with SERS technology to achieve the effect of using DMF to improve SERS detection results. A SERS-based acetylcholinesterase detection method based on DMF technology is designed.

[0031] Digital design of enzyme activity detection process on DMF platform: Acetylthiocholine (ATCh), an acetylcholinesterase substrate with a quaternary amine group, can be adsorbed on the SERS substrate through electrostatic interaction, and after enzymatic hydrolysis, the generated thiocholine (TCh) containing a sulfhydryl group can be preferably adsorbed on the SERS substrate through covalent bonds, which is stronger than electrostatic interaction. Based on this, a SERS leverage strategy was designed to develop an ultrasensitive SERS method for AChE detection, in which the generated TCh regulates the absorbance of NBA with strong Raman activity on the SERS substrate. When there is a SERS substrate in the enzymatic hydrolysis system, a small amount of thiocholine can remove a large amount of NBA adsorbed on the SERS substrate, thereby greatly reducing the SERS signal of NBA. This is due to the difference in their inherent Raman activity and the difference in interaction with the SERS substrate. The above reactions are all carried out on the DMF platform and the DMF platform can realize the automation of the above steps. SERS technology based on SERS intensity is a semi-quantitative detection method because the reproducibility of SERS detection results is poor. To mitigate the effects of poor SERS detection reproducibility, a ratio (the ratio of the SERS intensities of two characteristic peaks), rather than the absolute intensity of a single characteristic peak, is used as a more effective indicator of molecular quantity. 4-Mercaptobenzoic acid (4-MBA) is first adsorbed onto a portion of the SERS substrate surface as an internal reference. The ratio of the characteristic peaks of NBA and 4-MBA is then used to indicate the amount of AChE. Therefore, this method can be used to detect AChE levels in small, low-concentration samples.

[0032] The present invention employs a specific substrate transfer method to open up new avenues for SERS detection in digital microfluidics, enabling seamless integration of digital microfluidics with off-chip SERS analysis. Currently, enclosed DMF chips restrict the SERS substrate from easily leaving the chip, making detection more problematic than with conventional lab-on-a-chip devices, which have sophisticated, primarily capillary-based chip interfaces. Consequently, SERS detection in DMF is rare. In the method of the present invention, high throughput can be achieved by utilizing multiple SERS active sites on the upper plate. Furthermore, the method has the following features: 1. The droplets to be analyzed remain uncontaminated (free of gold (silver) nanoparticles) in the DMF device. 2. The amount of sample consumed for each detection and analysis is minimal. 3. The movement of sample droplets through the DMF chip is not constantly subject to resistance from the SERS substrate. 4. SERS sensitivity is higher when the deposited analyte dries on the SERS substrate. 5. The reaction is automated. 6. The SERS substrate can be easily removed from the DMF platform using a magnet.

[0033] The SERS substrate in the present invention is a new form, that is, a steel sheet modified with gold nanoparticles. The advantages are: the SERS substrate is relatively large, it is easier to collect signals, and the designed SERS substrate can be manipulated with a magnet. DMF is used to manipulate droplets instead of manual operation. Less reagent is used, the speed is faster, the error is smaller, the impact of the diffusion effect of droplets on the results can be improved, human errors and contamination can be reduced, and the operator can be prevented from contacting chemical reagents, which is safer. At the same time, multiple samples can be processed on DMF. The present invention combines the DMF platform and the method for detecting acetylcholinesterase, which can be used to adsorb SERS active substances in the DMF platform, and the DMF platform can be easily removed for SERS signal collection.

[0034] The digital microfluidic system of the present invention consists of a digital microfluidic chip, a microcontroller, a droplet drive module, and a temperature control module. The microcontroller serves as the control core of the system; the droplet drive module is connected to the electrode array on the lower plate of the digital microfluidic chip, is used to receive corresponding instructions from the microcontroller and provide driving voltage for droplet movement; the temperature control module is used to adjust and feedback the liquid reaction temperature information of the electrode array module, realizing closed-loop control of the reaction temperature. Droplet control is achieved by driving HV507, which is used to generate high-voltage signals to drive the electrodes in the digital microfluidic chip to control the movement, splitting, merging, and mixing of droplets. HV507 is a high-voltage serial-to-parallel converter for electrostatic applications, supporting an output of up to 300V, and is mainly used in precision electronic systems requiring high voltage and multi-channel control. Its core function is to convert low-voltage digital signals into high-voltage parallel outputs, which is suitable for industrial and scientific research scenarios with special requirements for voltage and current. By changing the contact angle of the droplets through the 300V high voltage, the precise movement of droplets on the hydrophobic medium is achieved, which is used for the automation of biochemical experiments.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0036] 1. The detection platform and detection method provided by the present invention ensure that the droplets to be analyzed in the DMF device remain uncontaminated (free of gold (silver) nanoparticles), while simultaneously minimizing the amount of sample droplets consumed for analysis. The movement of sample droplets through the DMF chip is unaffected by the SERS substrate, and when the deposited analyte dries on the SERS substrate, SERS sensitivity is high. The method of the present invention can be de-automated, and the SERS substrate can be easily removed from the DMF platform using a magnetic field.

[0037] 2. Compared to other detection methods, the method of the present invention is not only simple to operate but also requires less sample for detection. Furthermore, during implementation, the method of the present invention shortens the reaction time of acetylcholinesterase and the acquisition time of Raman spectra. For the detection of AChE content in biological samples, the SERS substrate adsorption treatment time can be shortened to as little as 8 minutes. The entire detection time can be controlled within 10 minutes.

[0038] 3. The method of the present invention is an ultrasensitive method for detecting acetylcholinesterase. Even for trace amounts of biological tissue such as serum, its detection limit is 1-2 orders of magnitude lower than that of other methods (Anal Chem., 2008, 80, 3769-3776; Sens. Acta. B, 2018, 259, 75-82).

[0039] 4. The instrument for collecting Raman signals in the method of the present invention is mature. The portable Raman spectrometer is cheap, easy to operate, and only about the size of a smart phone, and can be easily integrated with existing clinical surgical platforms. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the scanning electron microscope image of the SERS substrate;

[0041] Figure 2 Comparison of the results of automated experiments using DMF and manual experiments: (a) NBA Raman intensity signals from 5 repeated experiments performed manually, (b) NBA Raman intensity signals from 5 repeated experiments using DMF;

[0042] Figure 3 Design for the partition of DMF;

[0043] Figure 4 The overall process of processing the SERS substrate for detecting acetylcholinesterase content on DMF;

[0044] Figure 5 is the enzyme standard curve detected by this method;

[0045] Figure 6 The enzyme standard curve was fitted for the optimal detection range of this method.

[0046] Figure 7 The Raman signal of 4-MBA adsorbed on the SERS substrate changes with the number of days;

[0047] Figure 8 Figure 1: (a) Polyimide film electric heating film; (b) Temperature controller; (c) The back of the electric heating film adhered to the lower plate of the digital microfluidic chip;

[0048] Figure 9 To control the droplets on DMF by turning the electrodes on and off;

[0049] Figure 10 The overall physical image of DMF: (a) splitting from the liquid reservoir to generate small droplets, (b) droplet migration, (c) droplet merging, and (d) droplet mixing;

[0050] Figure 11 Schematic diagram of the cross section of the adsorption process between the SERS substrate and the molecular adsorption site;

[0051] Figure 12 Schematic diagram of the upper and lower parallel plates in the acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to the accompanying drawings and examples.

[0053] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0054] The gold nanoparticles (GNSs) in the present invention were prepared according to the method of Minimally Invasive Surface-EnhancedRaman Scattering Detection with Depth Profiles Based on a Surface-EnhancedRaman Scattering-Active Acupuncture Needle. Anal Chem, 2011, 83: 6191-6195, and the concentration parameters were: OD 712 The absorbance is 8 nm.

[0055] Example 1

[0056] Preparation of a solid-state SERS substrate that can be used for a DMF-SERS integrated detection platform:

[0057] A stainless steel disc with a diameter of 0.2 cm and a thickness of 50 μm was cut with a laser. After washing with ethanol, the disc was immersed in an ethanol solution containing 3% (v / v) MPTES and 1% (v / v) APTES for 12 hours. The functionalized stainless steel disc was then washed with ethanol three times, 2.5 μL of gold nanoparticle (GNSs) suspension was dripped on each disc, and then stored in a box with no air circulation, and the air humidity and airflow were controlled to be relatively stable without too much change. After the water evaporated naturally and the GNSs were adsorbed on the disc, the preparation of the solid-state SERS substrate that can be used for DMF-SERS was completed. Figure 1 This is an electron microscope image of the solid-state SERS substrate.

[0058] Example 2

[0059] Structural design of DMF-SERS integrated platform:

[0060] Figure 10 This is a physical picture of the entire DMF system. Figure 10 The left is the DMF drive system. Figure 10 The right is the DMF platform. Figure 12 As shown, the DMF platform adopts a parallel plate format (also known as a closed type). The DMF platform consists of two parts: an upper plate and a lower plate. In the parallel plate format, the droplet is sandwiched between the hydrophobic layers of the two parallel plates and moves in the driving electrode array area on the lower plate. The lower plate uses a PCB substrate as the base, and an electrode layer consisting of a liquid storage electrode and a driving electrode array is provided on the base. The electrode material is mainly a copper electrode, and the electrode layer is covered with a hydrophobic dielectric layer (using a 40-micron PTFE film layer). The liquid storage electrode and the driving electrode are in the same plane. The liquid storage electrode is used to store the reagents required during the experiment or collect the waste liquid after the reaction. The driving electrode is the core part of realizing droplet manipulation. By applying a switching voltage to control the electrowetting effect, the droplets can be precisely moved, split, merged, or mixed between the electrode arrays. The upper plate, which serves as a ground electrode, is composed of a transparent conductive material (indium tin oxide (ITO)) and glass. The upper plate is based on a glass substrate, on which an electrode layer of the transparent conductive material, indium tin oxide, is applied. A hydrophobic layer (Teflon AF1600s) is spin-coated on the electrode layer. The upper and lower plates are assembled together and separated by 300-micron thick double-sided tape. The droplet is sandwiched between two parallel plates. The electrodes on the lower and middle plates of the platform are arranged in a pattern. The lower plate has 128 electrodes, including 118 drive electrodes (2.75 mm × 2.75 mm) and 10 large electrodes (8.25 mm × 10 mm), with 100-micron spacing between them. All electrodes are connected to the contact electrode via 40-micron wide wires. The SERS substrate-molecule adsorption site is the final site of adsorption of the signal molecule on the solid-state SERS substrate constructed in Example 1. This site actually occurs at a point on the ITO glass. This site of adsorption between the SERS substrate and the molecule is known as the "SERS substrate-molecule" adsorption site. The solid-state SERS substrate is attracted to the bottom of the upper plate by the magnet on the upper plate, and is controlled by the magnet to move into and out of the SERS substrate-molecule adsorption site.

[0061] Different liquid storage areas are set on the liquid storage electrode. When the platform is running, droplets are split from the liquid in the liquid storage area. The droplets are sandwiched between two parallel plates and move in the driving electrode array area on the lower electrode plate. They react in the heated constant temperature incubation area in the reaction area and are finally moved to the waste liquid area after the reaction. Five different liquid storage areas are set on the liquid storage electrode, and the liquid storage areas include a mixed solution of NBA and ATCh, sample 3 to be tested, PBS, sample 2 to be tested, and sample 1 to be tested.

[0062] Example 3

[0063] Droplet manipulation steps on the DMF platform, such as Figure 9 As shown:

[0064] Generate droplets from the liquid reservoir: First, by activating the electrodes near the liquid reservoir, the electrowetting effect is used to pull the liquid out of the reservoir to form a connected droplet; then, the adjacent electrodes are activated to stretch the droplet to form a "liquid bridge"; then, the liquid reservoir electrode is turned off and the adjacent electrodes are kept activated, causing the droplet to break at the neck, achieving droplet splitting.

[0065] Droplet merging: First, two or more droplets to be merged are transferred to adjacent positions through an electrode array to bring them close to each other; then, the electrodes between the droplets are activated, and the electrowetting effect is used to reduce the contact angle between the droplets and the substrate surface, prompting the droplets to contact each other and form a connection; then, by adjusting the electric field strength or turning off the middle electrode, the droplets naturally merge under the action of surface tension to form a larger droplet; finally, the merged droplets are mixed (such as by activating the electrodes to make them move repeatedly) to ensure that the liquid is fully mixed and evenly distributed.

[0066] Droplet mixing: First, the droplets to be mixed are transferred to the same location through an electrode array, so that they come into contact and merge into a larger droplet. Next, by alternately activating multiple sets of electrodes around the droplet, the electrowetting effect is used to drive the droplet to move back and forth or rotate on the substrate surface, forming internal convection. Then, through repeated movement or rotation operations, the liquid inside the droplet is fully mixed to ensure uniformity. Finally, the electrode activation is stopped to stabilize the droplet at the target location.

[0067] Example 4

[0068] Establishment of acetylcholinesterase detection method based on DMF-SERS detection platform:

[0069] Solution preparation: Dissolve 2 mg (500 u) of acetylcholinesterase (ATCh) in 1 ml of 0.1 M phosphate buffered saline (PBS) at pH 8.0 as a stock solution and store at -20°C. Dissolve acetylthiocholine in 0.1 M PBS at pH 8.0 and prepare immediately before use. Prepare a concentration of 1 × 10-2 NBA was dissolved in 0.1M PBS (pH 8.0) and 1×10 -4 The concentration of M was prepared as a stock solution and stored at 4°C.

[0070] First, the SERS substrate prepared in Example 1 was immersed in a 1×10 -4 M of 4-MBA for 10 min and set aside to 1×10 -4 M's 4-MBA is used as a reference. Figure 7 The Raman signal of 4-MBA adsorbed on the SERS substrate changes with the number of days, indicating that 4-MBA can be adsorbed on the SERS substrate first. -4 mol / L ATCh and 1×10 -6 mol / L NBA solution was mixed and set aside.

[0071] Detection method: The solutions were stored in Figure 3 During the test, the solution generates small droplets of the same volume from the reservoir. Figure 4 Various steps in .

[0072] Preparation of acetylcholinesterase standard curve: The detection platform splits three droplets from the ATCh and NBA (Nile Blue A) mixed solution area in the liquid storage area and moves them to the three reaction areas respectively. AChE standard solutions with different dilution ratios are split from the three test sample areas in the liquid storage area and moved to the three reaction areas respectively to mix with the NBA and ATCh mixed solution for 3 minutes. The reaction solution is then moved to the three SERS substrate-molecule adsorption sites. Subsequently, the three solid-state SERS substrates will be magnetically controlled to slowly enter the three SERS substrate-molecule adsorption sites to be immersed in the reaction solution for 5 minutes ( Figure 11 The immersion process is accompanied by competitive adsorption of NBA and TCh. After this process, the SERS substrate is removed and the solid-state SERS substrate is moved off the platform to collect the Raman spectrum on the SERS substrate. The intensity ratio of NBA and 4-MBA (4-mercaptobenzoic acid) is collected, and an enzyme standard curve is generated based on the intensity ratio. Finally, the reaction solution is moved to the waste liquid area. Subsequently, three PBS solutions are generated from the buffer zone in the liquid storage area to clean the path of the sample to be tested. This reduces the amount of protein in the biological sample attached to the lower plate electrode, increases the number of uses, and prevents the impact of experimental reagent residue on the next set of experimental data. Figure 5 The enzyme standard curve detected by this method shows that the detection limit of this method is 1×10 -5 U / mL, the optimal detection range is 1-0.0001U / mL. Figure 6As shown, a linear fit was performed within the optimal detection range, and the linear equation was y = -1.1268x - 0.0745 (R 2 =0.986).

[0073] Biological sample testing: Replace the solutions in the three test sample areas of the detection platform with biological samples. All other steps remain unchanged and the biological sample is tested. The obtained data is compared with the standard curve to determine the concentration of acetylcholinesterase. Rabbit serum diluted 6400-fold was used as the biological sample for testing. The ratio of the Raman characteristic peak intensity of NBA to that of 4-MBA, the internal standard, was calculated to be 3.934. The acetylcholinesterase content in rabbit serum was calculated based on the linear equation and multiplied by the dilution factor to obtain 1.773 U / mL.

[0074] Further, if Figure 8 As shown, the optimal reaction temperature for the enzyme in this invention is 37°C. To achieve this optimal reaction temperature, a temperature control module was designed. It primarily consists of a heating film, a temperature sensor, and a temperature controller. The heating film is a polyimide film, which offers excellent properties for fast heat transfer and stable internal resistance. The heating film is attached to the back of the heated, constant-temperature incubation area of the digital microfluidic chip baseplate using double-sided tape. The temperature is collected by the temperature sensor, and controlled by the temperature controller to maintain the incubation area at a constant 37°C.

[0075] Example 5

[0076] To compare the results of automated experiments using the DMF platform with those of manual experiments, the SERS substrate was adsorbed with a mixed solution of NBA and ATCh using the DMF platform without adding the sample to be tested, consuming approximately 2.25 μL of reagent. Alternatively, the SERS substrate was placed directly into a centrifuge tube, and the mixed solution of NBA and ATCh was manually added using a pipette, consuming approximately 10 μL of reagent. Each experiment was repeated five times.

[0077] Figure 2 Comparison of DMF automated and manual results. Manual errors, both between different substrates and between different sites on the same substrate, are greater than those in automated DMF experiments. This demonstrates that automated DMF experiments offer advantages over manual operations in terms of labor, time, efficiency, and accuracy.

Claims

1. A digital microfluidics-based acetylcholinesterase SERS substrate molecular adsorption platform, characterized in that: The platform adopts the form of parallel plates, wherein the upper plate is composed of a transparent conductive material and glass used as a ground electrode, and a hydrophobic layer is spin-coated on it; the lower plate is composed of a liquid storage electrode and a driving electrode array, the electrode layer of the liquid storage electrode is covered with a hydrophobic dielectric layer, and different liquid storage areas are set on the liquid storage electrode. When the platform is in operation, droplets are split from the liquid in the liquid storage area, and the droplets are sandwiched between the two parallel plates and move in the driving electrode array area on the lower plate, first entering the reaction area, and finally being moved to the waste liquid area after the reaction; the solid SERS substrate is attracted under the upper plate and moved to the SERS substrate-molecule adsorption site on the upper plate through magnetism. The SERS substrate-molecule adsorption site is the site where molecules in the reaction solution on the upper plate adsorb to the solid SERS substrate.

2. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 1, characterized in that: The transparent conductive material is preferably indium tin oxide, and a hydrophobic layer Teflon AF1600s is spin-coated on the upper electrode.

3. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 1, characterized in that: The electrode layer of the lower electrode plate is covered with a PTFE film serving as a dielectric layer and a hydrophobic layer.

4. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 1, characterized in that: Different liquid storage areas are provided on the liquid storage electrode, and the liquid storage areas include a mixed solution area of NBA and ATCh, a buffer area, and a sample area to be detected.

5. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 4, characterized in that: The sample area to be detected includes 1-5 samples to be detected.

6. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 1, characterized in that: The solid-state SERS substrate is attracted to the bottom of the upper plate by a magnet provided on the upper plate, and moves under the control of the magnet to enter and leave the SERS substrate-molecule adsorption site.

7. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 1, characterized in that: The reaction zone is a heated constant temperature incubation zone, and the reaction solution is a sample zone in the storage zone. The sample zone is split into a test solution and moved to the reaction zone to mix with the NBA and ATCh mixed solution zone to form a mixed NBA and ATCh solution.

8. The acetylcholinesterase SERS substrate molecular adsorption platform based on digital microfluidics according to claim 1, characterized in that: The solid-state SERS substrate is prepared by immersing a stainless steel disc in an ethanol solution containing MPTES and APTES, then washing the functionalized stainless steel disc with ethanol, dripping a gold nanoparticle suspension onto it, and then allowing the water to evaporate naturally in an airless environment. After the gold nanoparticles are adsorbed on the disc, the solid-state SERS substrate is prepared.

9. A digital microfluidics-based SERS detection method for acetylcholinesterase based on the platform of claim 1, characterized in that: The steps include: (1) The solutions are stored in the liquid storage area respectively. During the detection, each stored solution is split from the liquid storage area to generate small droplets of the same volume; (2) Preparation of acetylcholinesterase standard curve: the platform splits droplets from the NBA (Nile blue) and ATCh mixed solution area in the liquid storage area and moves them to the reaction area, splits the AChE standard solution from the sample area to be tested in the liquid storage area and moves it to the reaction area and the NBA and ATCh mixed solution area to be mixed and reacted, and then moves the reaction solution to the SERS substrate-molecule adsorption site, and then the solid-state SERS substrate will be magnetically controlled to slowly enter the SERS substrate-molecule adsorption site to be immersed in the reaction solution, and the immersion process is accompanied by competitive adsorption of NBA and TCh. After this process is completed, the SERS substrate is moved away and the solid-state SERS substrate is moved out of the platform to collect the Raman spectrum on the SERS substrate, collect the intensity ratio of NBA and 4-MBA (4-mercaptobenzoic acid), and make an enzyme standard curve based on the intensity ratio. Finally, the reaction solution is moved to the waste liquid area, and then PBS droplets are generated from the buffer area of the liquid storage area to clean the path where the sample to be tested has moved; (3) Biological sample detection: The solution in the sample area to be tested on the platform is replaced with the biological sample, and the other steps remain unchanged to perform the biological sample detection.

10. The detection method according to claim 9, characterized in that: The splitting is achieved by activating the electrodes near the liquid storage area and using the electrowetting effect to pull the liquid out of the liquid storage area to form a connected droplet; then, activating the adjacent electrodes to stretch the droplet to form a "liquid bridge"; then, closing the liquid storage area electrode and keeping the adjacent electrodes activated, so that the droplet breaks at the neck, achieving droplet splitting.

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