Method for detecting ultra-trace molecules to be tested and application

Abstract

The application provides a method for detecting ultra-trace molecules and application. The method comprises the following steps: installing a metal nano-surface enhanced Raman scattering detection chip in an in-situ electrochemical cell, wherein the in-situ electrochemical cell is provided with a working electrode and a counter electrode, the metal nano-surface enhanced Raman scattering detection chip is in a non-contact state with the working electrode, adding a to-be-detected molecule solution and an electrolyte solution into the in-situ electrochemical cell, applying a working potential on the working electrode, taking out the metal nano-surface enhanced Raman scattering detection chip, and performing surface enhanced Raman spectrum detection on the metal nano-surface enhanced Raman scattering detection chip to obtain a Raman spectrum of the to-be-detected molecule. The method has good repeatability, high detection efficiency, can obtain higher positive spectrum count under the premise of limited total amount of detection spectrum, and has reliable detection result. The application also provides application of the method for detecting ultra-trace molecules.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and in particular relates to a method and application for detecting ultra-trace molecules to be tested. Background Art

[0002] Surface-enhanced Raman spectroscopy (SERS) is a nondestructive molecular detection technique that reflects the vibrational and rotational energies of molecular bonds, creating a "fingerprint" spectrum that can specifically identify molecules. Over the past half century, SERS has seen steady growth in both fundamental research and practical applications, and has become a valuable tool for qualitative and quantitative trace detection in a wide range of fields, including surface and interface chemistry, catalysis, biology, and environmental monitoring.

[0003] Quantitative analysis has always been a huge challenge in the field of SERS, especially when detecting low concentrations (less than 1nM), where the Raman signal intensity is extremely non-repeatable. Some scholars have proposed the concept of digital surface-enhanced Raman spectroscopy, which is based on the number of single-molecule SERS event counts rather than signal intensity to overcome the quantitative inaccuracy caused by signal fluctuations. Other scholars have developed a quantitative strategy based on liquid-phase digital colloid-enhanced Raman by single-molecule counting, achieving a 1fM (10 -15 mol / L) level, and also proved that the quantitative detection error based on single-molecule counting obeys Poisson noise. However, due to the low probability of obtaining single-molecule Raman spectra by traditional SERS methods and the small number of positive spectra, scientific analysis of Poisson distribution data from a statistical perspective requires the collection of more than 10,000 sets of valid data, resulting in low detection efficiency and large errors in actual detection. At the same time, traditional SERS technology often requires the molecule solution to be tested to be immersed and incubated with the enhanced substrate for more than one hour to achieve single-molecule detection. The long detection time resolution also limits the development of this method.

[0004] Therefore, the application value of surface-enhanced Raman spectroscopy in qualitative identification and precise quantification is still greatly limited by the time resolution, efficiency, and detection limit of the detection system. Therefore, it is urgent to establish a new surface-enhanced Raman spectroscopy detection method that is fast, sensitive, efficient, and accurate. Summary of the Invention

[0005] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the present application provides a method for detecting ultra-trace molecules, which has good repeatability, high detection efficiency, and can obtain higher positive spectrum count under the premise of limited total detection spectrum amount, and the detection result is reliable, and the digital quantitative detection result can be improved by increasing the total detection times to reduce the quantitative error, and the quantitative detection error is controllable.

[0006] The present application also provides an application of the method for detecting ultra-trace molecules.

[0007] The first aspect of the present application provides a method for detecting ultra-trace molecules, comprising the following steps:

[0008] S1: installing a metal nano-surface enhanced Raman scattering detection chip in an in-situ electrochemical cell, the in-situ electrochemical cell being provided with a working electrode and a counter electrode, the metal nano-surface enhanced Raman scattering detection chip being in a non-contact state with the working electrode, adding a to-be-detected molecule solution and an electrolyte solution in the in-situ electrochemical cell, and applying a working potential on the working electrode;

[0009] S2: taking out the metal nano-surface enhanced Raman scattering detection chip, and performing surface enhanced Raman spectrum detection on the metal nano-surface enhanced Raman scattering detection chip to obtain a Raman spectrum of the to-be-detected molecule.

[0010] The present application relates to a technical solution of the method for detecting ultra-trace molecules, which has at least the following beneficial effects:

[0011] The method of the present application has good detection chip diversity, is easy to prepare, can be purchased through commercial channels, can be selected according to the properties of the target molecule to be detected, and the surface of the detection chip can be functionally modified as needed to facilitate the adsorption of the to-be-detected molecule.

[0012] In the method of the present application, the detection chip does not directly contact the working electrode during the detection process, thereby avoiding the redox reaction of the to-be-detected molecule on the working electrode.

[0013] The method of the present application is simple to operate, has fast detection response speed, and has high time resolution.

[0014] The method of the present application does not need to label the to-be-detected molecule, and can directly perform single-molecule level accurate quantitative detection on the target molecule.

[0015] The method of the present application has good repeatability and high detection efficiency, and can obtain higher positive spectrum count under the premise of limited total detection spectrum amount.

[0016] The method of the present invention has reliable detection results. The digital quantitative detection results can increase the number of positive spectra counted by increasing the total number of detection times, thereby reducing the quantitative error. The error of quantitative detection is controllable.

[0017] The present invention can realize the detection of ultra-trace analyte molecules by selecting a suitable chip and constructing it based on the electrokinetic process, and has a single-molecule level detection sensitivity for the target analyte molecules, while ensuring the precision of quantitative detection.

[0018] According to some embodiments of the invention, the method comprises qualitative detection and / or quantitative detection.

[0019] According to some embodiments of the invention, the method comprises single-molecule level quantitative detection.

[0020] According to some embodiments of the present invention, the metal nano-surface enhanced Raman scattering detection chip is disposed between the working electrode and the counter electrode.

[0021] According to some embodiments of the present invention, the metal nano-surface enhanced Raman scattering detection chip is disposed next to the working electrode.

[0022] According to some embodiments of the present invention, the metal nano-surface enhanced Raman scattering detection chip includes at least one of a silver nano-surface enhanced Raman scattering detection chip, a copper nano-surface enhanced Raman scattering detection chip, and a zinc nano-surface enhanced Raman scattering detection chip.

[0023] According to some embodiments of the present invention, the electrolyte solution includes at least one of chloroauric acid and chloroplatinic acid.

[0024] According to some embodiments of the present invention, the concentration of the electrolyte solution is 0.01 mmol / L to 0.4 mmol / L.

[0025] According to some embodiments of the present invention, the concentration of the electrolyte solution is 0.01mmol / L, 0.02mmol / L, 0.03mmol / L, 0.04mmol / L, 0.05mmol / L, 0.06mmol / L, 0.07mmol / L, 0.08mmol / L, 0.09mmol / L, 0.1mmol / L, 0.11mmol / L, 0.12mmol / L, 0.13mmol / L, 0.14mmol / L, 0.15mmol / L, 0.16mmol / L, 0.17mmol / L, 0.18mmol / L, 0.19mmol / L, 0.2mmol The range value formed by any one of the following values ​​or any two of them is 0.1 / L, 0.21mmol / L, 0.22mmol / L, 0.23mmol / L, 0.24mmol / L, 0.25mmol / L, 0.26mmol / L, 0.27mmol / L, 0.28mmol / L, 0.29mmol / L, 0.3mmol / L, 0.31mmol / L, 0.32mmol / L, 0.33mmol / L, 0.34mmol / L, 0.35mmol / L, 0.36mmol / L, 0.37mmol / L, 0.38mmol / L, 0.39mmol / L, and 0.4mmol / L.

[0026] According to some embodiments of the present invention, when the molecule to be detected is a positively charged molecule or a neutrally charged molecule, the working potential is a negative potential; when the molecule to be detected is a negatively charged molecule, the working potential is a positive potential.

[0027] According to some embodiments of the present invention, the surface enhanced Raman spectroscopy detection includes at least one of a point scanning test, a line scanning test, and a surface scanning test.

[0028] According to some embodiments of the present invention, the detection limit of the method is 1×10 -18 M.

[0029] The second aspect of the present invention provides the use of the method of the first aspect of the present invention in environmental monitoring and biomedical testing.

[0030] The present invention provides a technical solution for the application of a method for ultra-trace detection of analyte molecules in environmental monitoring and biomedical testing, which has at least the following beneficial effects:

[0031] The application of this method in environmental monitoring and biomedical testing has the following beneficial effects:

[0032] High sensitivity: Able to detect ultra-trace amounts of analyte molecules, suitable for trace pollutants in the environment and extremely low concentrations of biomarkers in biological samples.

[0033] Strong selectivity: By functionalizing and modifying the detection chip surface, the affinity for specific target molecules can be enhanced, thereby improving the specificity of detection.

[0034] Label-free detection: No labeling is required for the molecules to be detected, which reduces the sample processing steps, avoids the impact of labeling on molecular activity, and ensures the authenticity of the detection.

[0035] Rapid response: The detection process is simple, easy to operate, and results can be obtained quickly, making it suitable for real-time monitoring and clinical diagnosis.

[0036] High reproducibility: Maintaining consistency across multiple tests ensures reliable results and helps establish standardized testing procedures.

[0037] Low error: By increasing the number of tests, the positive spectrum count is increased, effectively reducing quantitative errors and improving the credibility of the results.

[0038] Diverse applications: Suitable for the detection of a variety of molecules to be tested, and can be widely used in environmental protection, food safety, early disease screening and other fields.

[0039] Economical: The detection chip is available through commercial channels and is simple to prepare, which reduces experimental costs and increases popularity.

[0040] According to some embodiments of the present invention, the environmental monitoring includes water quality monitoring, air quality detection and soil detection.

[0041] According to some embodiments of the present invention, the biomedical tests include disease marker detection, infectious disease pathogen testing, and drug monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the scanning electron microscope image and energy spectrum of the initial pure silver sheet

[0043] Figure 2 This is a schematic diagram of the construction and detection principle of the electric power device of the present invention.

[0044] Figure 3 This is the scanning electron microscope image and energy spectrum of the silver nano-surface enhanced Raman scattering detection chip after the electric process is completed.

[0045] Figure 4 It is the Raman spectrum of the noise window and the characteristic signal window of the bimolecular crystal violet and Nile blue molecules to be measured.

[0046] Figure 5 The concentration range is 10 -18 ~10 -14 The first set of two-dimensional Raman spectra of the distribution of the mixed solution of crystal violet and Nile blue of the tested bimolecular solution M on the chip surface.

[0047] Figure 6 The concentration range is 10 -18 ~10 -14 The second set of two-dimensional Raman spectra of the distribution of the mixed solution of crystal violet and Nile blue of the tested bimolecular solution M on the chip surface.

[0048] Figure 7 The concentration range is 10 -18 ~10 -14 The third set of two-dimensional Raman spectra of the mixed solution of crystal violet and Nile blue to be tested on the chip surface.

[0049] Figure 8 The concentration range is 10 -18 ~10 -14 The first set of data for the statistics of single molecule event counts and mixed event counts in the mixed solution of crystal violet and Nile blue of the test bimolecular solution M.

[0050] Figure 9 The concentration range is 10 -18 ~10 -14 The second set of data is the statistics of single molecule event counts and mixed event counts in the mixed solution of crystal violet and Nile blue of the test bimolecular solution M.

[0051] Figure 10 The concentration range is 10 -18 ~10 -14 The third set of data is the statistics of single molecule event counts and mixed event counts in the mixed solution of crystal violet and Nile blue of the test bimolecular solution M.

[0052] Figure 11 is 10 -18 ~10 -14 Crystal violet quantitative detection data of M.

[0053] Figure 12 is 10 -18 ~10 -14 Nile blue quantitative detection data of M.

[0054] Figure 13 is 10 -15 ~10 -13 Quantitative detection data of perfluorohexanoic acid of M.

[0055] Figure 14 is 10 -15 ~10 -13 Enrofloxacin quantitative detection data of M. DETAILED DESCRIPTION

[0056] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0057] In a first aspect, in some embodiments of the present invention, the first aspect of the present invention provides a method for detecting an ultra-trace amount of a molecule to be detected, comprising the following steps:

[0058] S1: Installing a metal nano-surface enhanced Raman scattering detection chip in an in-situ electrochemical cell, wherein the in-situ electrochemical cell is provided with a working electrode and a counter electrode, wherein the metal nano-surface enhanced Raman scattering detection chip and the working electrode are in a non-contact state, adding a solution of molecules to be tested and an electrolyte solution to the in-situ electrochemical cell, and applying a working potential to the working electrode;

[0059] S2: taking out the metal nano-surface enhanced Raman scattering detection chip, performing surface enhanced Raman spectroscopy detection on the metal nano-surface enhanced Raman scattering detection chip, and obtaining a Raman spectrum of the molecule to be detected.

[0060] A technical solution in the method for ultra-trace detection of analyte molecules of the present invention has at least the following beneficial effects:

[0061] The method of the present invention has good detection chip diversity, is easy to prepare, and can also be purchased through commercial channels. It can be selected according to the properties of the target molecule to be measured. The surface of the detection chip can also be functionally modified as needed to facilitate the adsorption of the molecule to be measured.

[0062] In the method of the present invention, during the detection process, the detection chip does not directly contact the working electrode, thereby avoiding the redox reaction of the molecule to be detected on the working electrode.

[0063] The method of the present invention is simple to operate, has a fast detection response speed, and has a high detection time resolution.

[0064] The method of the present invention does not require labeling of the target molecule and can directly perform precise quantitative detection of the target molecule at the single molecule level.

[0065] The method of the present invention has good repeatability and high detection efficiency, and can obtain a higher positive spectrum count under the premise of a limited total amount of detected spectra.

[0066] The method of the present invention has reliable detection results. The digital quantitative detection results can increase the number of positive spectra counted by increasing the total number of detection times, thereby reducing the quantitative error. The error of quantitative detection is controllable.

[0067] The present invention can realize the detection of ultra-trace analyte molecules by selecting a suitable chip and constructing it based on the electrokinetic process, and has a single-molecule level detection sensitivity for the target analyte molecules, while ensuring the precision of quantitative detection.

[0068] In combination with the first aspect, in some embodiments of the present invention, the method includes qualitative detection and / or quantitative detection.

[0069] In conjunction with the first aspect, in some embodiments of the present invention, the method includes single-molecule level quantitative detection.

[0070] Single-molecule quantitative detection means this method can precisely identify and measure the concentration and properties of individual molecules. This highly sensitive assay can effectively capture target molecules at extremely low concentrations, making it suitable for applications such as early disease detection, environmental monitoring, and basic scientific research. It ensures the reliability and accuracy of test results and helps reveal the dynamic changes and interactions of trace substances.

[0071] In combination with the first aspect, in some embodiments of the present invention, a metal nano-surface enhanced Raman scattering detection chip is disposed between the working electrode and the counter electrode.

[0072] The metal nano-surface enhanced Raman scattering detection chip is arranged between the working electrode and the counter electrode. The surface enhancement effect of the metal nanomaterial increases the Raman signal intensity and improves the detection sensitivity. Furthermore, the non-contact configuration between the chip and the electrode avoids the reaction of the electrode surface, reduces interference, and improves data reliability. In addition, this layout simplifies the experimental process, facilitates sample processing and data acquisition. Furthermore, the chip position optimizes the electrochemical reaction conditions, which helps to improve detection efficiency. Furthermore, the chip can be functionally modified according to different experimental requirements to adapt to a variety of molecular detection. Ultimately, this design improves the sensitivity and accuracy of detection and expands the scope of application.

[0073] In combination with the first aspect, in some embodiments of the present invention, a metal nano-surface enhanced Raman scattering detection chip is disposed next to the working electrode.

[0074] Positioning the chip close to the electrodes enhances the intensity of the Raman signal and increases sensitivity to the target molecules. This layout helps more effectively capture target molecules near the electrodes, improving detection efficiency. Avoiding direct contact with the electrodes reduces interference from electrochemical reactions on the Raman signal. This allows experimenters to easily adjust the chip position for optimization under different experimental conditions. Furthermore, the chip can be functionalized as needed to adapt to the detection of different types of molecules.

[0075] It needs to be particularly pointed out that the traditional surface-enhanced Raman spectrum detection means is to immerse the roughened silver substrate in the solution to be detected, or to modify the silver substrate to better adsorb the molecules to be detected, and then test the Raman spectrum of the silver substrate to obtain the information of the detected substance, which is the ordinary and conventional way. The similarity between the present application and the prior art is that the silver sheet is roughened by an electrochemical method, and it needs to be pointed out that the present application uses the electrochemical method to roughen the silver sheet only to prepare a rough silver enhancement substrate, and there is no special requirement, and the preparation method of the silver substrate does not need to be specific, and any commercially available gold silver enhancement substrate can be used.

[0076] The specificity of the present application lies in the construction method of electrodynamic-enhanced Raman spectrum, specifically, in the method of the present application, the detection chip does not serve as the working electrode in the electrochemical system, but is placed between the working electrode and the counter electrode (similar to a sandwich structure, but not in contact with the working electrode and the counter electrode), or the chip to be detected is placed near the working electrode, also without contact with the working electrode, that is, in the present application, the chip to be detected is not connected to electricity.

[0077] The traditional electrochemical surface-enhanced Raman test means uses the chip as the working electrode, which needs to be connected to electricity, and the chip to be detected is directly used as the working electrode, which needs to be applied with voltage, one of the disadvantages of which is that after the molecules to be detected are adsorbed on the working electrode, that is, the chip to be detected, a certain degree of redox reaction will occur, thereby affecting the detection.

[0078] In combination with the first aspect, in some embodiments of the present application, the metal nano-surface-enhanced Raman scattering detection chip includes at least one of a silver nano-surface-enhanced Raman scattering detection chip, a copper nano-surface-enhanced Raman scattering detection chip, and a zinc nano-surface-enhanced Raman scattering detection chip.

[0079] In combination with the first aspect, in some embodiments of the present application, the solution of the molecules to be detected has a series of concentration gradients, and the concentration of the solution of the molecules to be detected is reduced by at least 0.1 order of magnitude each time.

[0080] The solution of the molecule to be tested has a series of concentration gradients, and each time the concentration decreases by at least 0.1 orders of magnitude, which can effectively evaluate the sensitivity of the detection method at different concentrations, help determine the lowest detectable concentration, and help establish a standard curve between concentration and signal intensity to facilitate subsequent quantitative analysis. By covering multiple concentration gradients, the target molecule can be detected over a wider range to meet the needs of different samples. Testing at different concentrations helps to optimize experimental conditions and find the best detection parameters. Smaller concentration changes can provide more refined quantitative data, improve the accuracy and reliability of the test results, and by covering multiple concentration levels, it can ensure that the changes in the molecule to be tested are more comprehensively reflected in actual applications. As a result, these effects improve the overall performance of the detection method and enhance its applicability in various applications.

[0081] In combination with the first aspect, in some embodiments of the present invention, the electrolyte solution includes at least one of chloroauric acid and chloroplatinic acid.

[0082] It should be noted that existing technologies utilize an electrochemical reduction process, using constant-potential electrodeposition of, for example, HAuCl4 solution onto a silver substrate to produce a gold-silver composite substrate. However, the present invention is unique in that the silver substrate chip is immersed in the HAuCl4 solution, eliminating the need for electroreduction. Instead, a direct substitution reaction between the silver nanoparticles and the gold solution produces a hollow Ag-Au nanoshell structure. This hollow shell structure exhibits superior structural reinforcement compared to the structural reinforcement achieved by directly electrodepositing gold on a silver chip as the working electrode.

[0083] Therefore, the characteristic of the present invention is that the metal nano-surface enhanced Raman scattering detection chip is in a non-contact state with the working electrode, that is, the detection chip is constructed between the working electrode and the counter electrode. In this way, the effect of electrokinetic force can be utilized to drive the molecules to be tested to the chip by means of electromigration. Moreover, the chip is not directly energized, and no redox reaction of the molecules to be tested occurs. At the same time, the Au-Ag structure generated during the immersion of the silver substrate in HAuCl4 as an electrolyte anchors and confines the molecules to be tested at the chip interface, producing a stronger enhancement effect. Therefore, the detection limit of the method of the present invention is higher, which can reach -1×10 -18 M, which is much higher than the detection limit of existing technologies.

[0084] It should also be noted that simply placing the detection chip between the working electrode and the counter electrode to energize it is not feasible, and the effect is not good. In the solution of the present invention, the key point is that chloroauric acid is used as an electrolyte. After the molecules to be tested are driven to the chip interface by electric power, a replacement reaction is required between the nanoparticle structure on the surface of the silver chip and chloroauric acid (or chloroplatinic acid), thereby producing an Ag-Au shell structure to confine the molecules to be tested and improve the detection effect. Therefore, the solution of the present invention can be understood as a two-step strategy. First, the molecules are driven by electric power to the chip interface, and second, the shell structure generated by the replacement reaction of the silver substrate and chloroauric acid is required to confine the enhanced detection effect.

[0085] Experiments have found that if the detection chip is used as a working electrode, as in the prior art, to attract the target molecules, the effect of electrodepositing chloroauric acid is actually worse. This is because the dense gold layer produced by the rapid electrodeposition of chloroauric acid on the silver interface does not enhance the detection effect. Furthermore, because the detection target of the present invention can be a single molecule at very low concentrations, excessive electrodeposition of gold on the silver chip substrate can cover the target molecules. During detection, the Raman background of the matrix is ​​too high, thus drowning out the Raman signal of the target molecules.

[0086] It should also be noted that the single-molecule SERS phenomenon occurs on molecules adsorbed on special areas of the nanostructured metal surface, called SERS "hotspots". Only molecules adsorbed on efficient hotspots will contribute to the overall Raman signal. Most importantly, efficient hotspots are rare, and at ultra-low concentrations, the amount of adsorbed analyte is expected to be very small. This makes the probability of obtaining single-molecule Raman spectra by traditional SERS methods low, and the number of positive spectra is small. It is often necessary to test more than 10,000 sets of spectra to obtain enough positive spectra for statistical analysis. At the same time, traditional SERS technology often requires the solution of the molecule to be tested to be immersed and incubated with the enhanced substrate for more than 1 hour to achieve single-molecule level detection, and the detection time is relatively slow. Using the strategy in the present invention, the response time of the detection can be shortened to 20 minutes. At the same time, due to the principle of electrokinetic binding surface confinement, the positive spectrum rate of the detection is greatly improved, which can reduce the total number of detections and improve the detection efficiency. As shown in the data in the present invention, only 900 counts (n=3) are required to achieve very accurate quantitative detection.

[0087] In combination with the first aspect, in some embodiments of the present invention, the concentration of the electrolyte solution is 0.01 mmol / L to 0.4 mmol / L.

[0088] In combination with the first aspect, in some embodiments of the present invention, the concentration of the electrolyte solution is 0.01mmol / L, 0.02mmol / L, 0.03mmol / L, 0.04mmol / L, 0.05mmol / L, 0.06mmol / L, 0.07mmol / L, 0.08mmol / L, 0.09mmol / L, 0.1mmol / L, 0.11mmol / L, 0.12mmol / L, 0.13mmol / L, 0.14mmol / L, 0.15mmol / L, 0.16mmol / L, 0.17mmol / L, 0.18mmol / L, 0.19mmol / L, 0.2mmol / L The range value formed by any one of the following values, or any two of them, is 0.21mmol / L, 0.22mmol / L, 0.23mmol / L, 0.24mmol / L, 0.25mmol / L, 0.26mmol / L, 0.27mmol / L, 0.28mmol / L, 0.29mmol / L, 0.3mmol / L, 0.31mmol / L, 0.32mmol / L, 0.33mmol / L, 0.34mmol / L, 0.35mmol / L, 0.36mmol / L, 0.37mmol / L, 0.38mmol / L, 0.39mmol / L, and 0.4mmol / L.

[0089] In combination with the first aspect, in some embodiments of the present invention, when the molecule to be detected is a positively charged molecule or a neutrally charged molecule, the working potential is a negative potential; when the molecule to be detected is a negatively charged molecule, the working potential is a positive potential.

[0090] In combination with the first aspect, in some embodiments of the present invention, a working potential is applied to the working electrode for a period of 5 to 60 minutes.

[0091] In combination with the first aspect, in some embodiments of the present invention, surface enhanced Raman spectroscopy detection includes at least one of a point scanning test, a line scanning test, and a surface scanning test.

[0092] In combination with the first aspect, in some embodiments of the present invention, the detection limit of the method is 1×10 -18 M.

[0093] In combination with the first aspect, in some embodiments of the present invention, surface-enhanced Raman spectroscopy detection includes performing a surface scanning test on the detection chip to obtain a two-dimensional Raman spectrum of the distribution of the molecules to be detected on the chip surface.

[0094] In combination with the first aspect, in some embodiments of the present invention, obtaining the Raman spectrum of the molecule to be detected includes selecting a noise region away from any peak belonging to the molecule to be detected and a blank sample as a noise window, and determining a noise threshold for judging the presence of the molecule to be detected.

[0095] In combination with the first aspect, in some embodiments of the present invention, obtaining the Raman spectrum of the molecule to be tested includes selecting a signal region belonging to the characteristic peak of the molecule to be tested as a signal window, and using whether the maximum intensity (Is) of the characteristic peak in the signal window containing the characteristic Raman peak of the molecule to be tested is greater than the noise threshold to determine whether the molecule to be tested is detected in the spectrum. If the molecule to be tested is detected, it is a positive spectrum, and if the molecule to be tested is not detected, it is a negative spectrum.

[0096] In combination with the first aspect, in some embodiments of the present invention, the method for detecting ultratrace test molecules includes the following steps: determining the concentration of the test molecules in the test molecule solution based on the number of times and / or probability that the Raman spectrum of the test molecules in the test molecule solution is judged as a positive spectrum.

[0097] In combination with the first aspect, in some embodiments of the present invention, the method for ultra-trace detection of the molecule to be tested includes the following steps: determining the concentration of the molecule to be tested in the molecule to be tested solution based on a mathematical correlation of the number of times and / or probability that the Raman spectrum of the molecule to be tested in the molecule to be tested solution is judged as a positive spectrum.

[0098] In combination with the first aspect, in some embodiments of the present invention, the method for detecting ultra-trace target molecules includes the following steps: adjusting the properties of the metal nano-surface enhanced Raman scattering detection chip, or functionally modifying the detection chip to better bind to the target molecule to be detected.

[0099] In combination with the first aspect, in some embodiments of the present invention, the method for detecting ultra-trace amounts of analyte molecules includes the following steps: adjusting the time for applying the working potential on the working electrode.

[0100] In combination with the first aspect, in some embodiments of the present invention, the method for detecting ultra-trace test molecules includes the following steps: adjusting the test parameters of the surface enhanced Raman spectroscopy detection process, including but not limited to scanning mode, exposure time, incident laser wavelength, and laser power.

[0101] In combination with the first aspect, in some embodiments of the present invention, the method for detecting ultra-trace test molecules includes the following steps: adjusting the total number of sampling times of the surface-enhanced Raman spectroscopy detection; and\or the total number of positive spectra detected as the test molecules.

[0102] In a second aspect, some embodiments of the present invention provide applications of the method of the first aspect of the present invention in environmental monitoring and biomedical testing.

[0103] The present invention provides a technical solution for the application of a method for ultra-trace detection of analyte molecules in environmental monitoring and biomedical testing, which has at least the following beneficial effects:

[0104] The application of this method in environmental monitoring and biomedical testing has the following beneficial effects:

[0105] High sensitivity: Able to detect ultra-trace amounts of analyte molecules, suitable for trace pollutants in the environment and extremely low concentrations of biomarkers in biological samples.

[0106] Strong selectivity: By functionalizing and modifying the detection chip surface, the affinity for specific target molecules can be enhanced, thereby improving the specificity of detection.

[0107] Label-free detection: No labeling is required for the molecules to be detected, which reduces the sample processing steps, avoids the impact of labeling on molecular activity, and ensures the authenticity of the detection.

[0108] Rapid response: The detection process is simple, easy to operate, and results can be obtained quickly, making it suitable for real-time monitoring and clinical diagnosis.

[0109] High reproducibility: Maintaining consistency across multiple tests ensures reliable results and helps establish standardized testing procedures.

[0110] Low error: By increasing the number of tests, the positive spectrum count is increased, effectively reducing quantitative errors and improving the credibility of the results.

[0111] Diverse applications: Suitable for the detection of a variety of molecules to be tested, and can be widely used in environmental protection, food safety, early disease screening and other fields.

[0112] Economical: The detection chip is available through commercial channels and is simple to prepare, which reduces experimental costs and increases popularity.

[0113] In combination with the second aspect, in some embodiments of the present invention, environmental monitoring includes water quality monitoring, air quality detection and soil detection.

[0114] Water quality monitoring primarily involves detecting pollutants (such as heavy metals and organic pollutants) and microorganisms in water. Air quality monitoring primarily involves monitoring harmful gases (such as VOCs and PM2.5) and pollutants in the air. Soil testing primarily involves assessing the levels of pesticides, heavy metals, and other harmful substances in the soil.

[0115] In conjunction with the second aspect, in some embodiments of the present invention, biomedical testing includes disease marker detection, infectious disease pathogen testing, and drug monitoring.

[0116] Disease marker testing includes detecting tumor markers or viral load. Infectious disease pathogen testing includes rapid identification of pathogens (such as bacteria and viruses) to assist in clinical trials. Drug monitoring involves monitoring drug concentrations in patients to optimize medication regimens and safety.

[0117] These applications contribute to improving the effectiveness of public health and environmental protection.

[0118] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0119] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0120] Unless otherwise specified, "room temperature" in the present invention means 25°C±5°C.

[0121] Unless otherwise specified, “about” in the present invention means that the allowable error is within ±2%.

[0122] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0123] Example

[0124] Using a silver nano-surface enhanced Raman scattering detection chip as the surface-enhanced Raman substrate, the ultra-trace mixed bimolecular solution of crystal violet and Nile blue was quantitatively detected by electrodynamic enrichment.

[0125] 1. Preparation of silver nano-surface enhanced Raman scattering detection chip

[0126] The smooth pure silver sheet was washed with ethanol and acetone three times respectively, and the washed and dried pure silver sheet was polished with 1000 mesh and 3000 mesh sandpaper respectively, and then washed and dried with ultrapure water for use. The scanning electron microscope and energy spectrum of the smooth Ag sheet were as follows: Figure 1As shown. The silver sheet was electrochemically treated on a three-electrode electrochemical workstation with 0.1M KOH as the electrolyte. The silver sheet was the working electrode, the platinum electrode was the counter electrode, and the Ag / AgCl electrode was the reference electrode. Cyclic voltammetry was first used to roughen the silver sheet. The cyclic voltammetry curve was scanned over a potential range of +0.5 to -0.5 V (vs Ag / AgCl) at a scan rate of 10 mV / s for 5 cycles. After the cyclic voltammetry scan, the silver sheet was reduced at a constant potential of -1.2 V (vs Ag / AgCl) for 800 s to stabilize the roughened silver sheet substrate. The roughened silver sheet was taken out, rinsed repeatedly with ultrapure water and dried. The roughened silver sheet was placed in an embedding box, and 1 mL of 0.93 mM silver colloid solution (the solvent of the silver colloid solution was water and the particle size was 15 nm) was added thereto. After soaking for 3 hours, the silver sheet substrate was taken out, rinsed with ultrapure water and dried to prepare a silver nano-surface enhanced Raman scattering detection chip.

[0127] 2. Preparation of samples to be tested

[0128] Prepare a 10mM chloroauric acid (Aladdin, 99%) solution and place it in a glass sample bottle protected from light. Prepare a 0.1mM crystal violet (Macklin, 95%) ethanol solution and a Nile blue (Sigma-Aldrich) water solution, mix them in equal proportions, and then dilute them in a gradient of 10 to 100 μg / mL. -18 ~10 -14 A bimolecular mixed solution of crystal violet and Nile blue.

[0129] 3. Construction of electric power device

[0130] The construction and experimental principle of the electrokinetic-surface enhanced Raman detection device used in the present invention are as follows: Figure 2 As shown, first a 1.5×1cm 2 The silver sheet working electrode was encapsulated in the in-situ electrochemical cell, and the silver nano-surface enhanced Raman scattering detection chip (area 0.5×0.5cm 2 ) is fixed above the working electrode, 3 mm away from the working electrode (the chip to be tested and the working electrode are not in contact). Then, 20 mL of the crystal violet and Nile blue bimolecular mixed solution to be tested and 0.5 mL of 10 mM chloroauric acid solution are added to the electrochemical cell and shaken well. 2 A titanium sheet was used as the counter electrode in the electrochemical cell, above the counter electrode and the silver nano-surface enhanced Raman scattering detection chip, and ~8 mm away from the working electrode. Finally, a potential of -1.2 V vs Ag / AgCl was applied to the working electrode, and a current of I~1.4×10 -4A. After 20 minutes of power-on, the silver nano-surface enhanced Raman scattering detection chip to be tested is taken out for Raman spectrum detection. Under the action of the electric field, the cationic dyes crystal violet and Nile blue are enriched on the interface of the silver nano-surface enhanced Raman scattering detection chip to be tested due to electromigration. At the same time, due to the replacement reaction between the silver nanostructure on the chip and the aqueous solution of chloroauric acid, an Au-Ag shell with a hollow nanostructure is produced, which not only confines and anchors the target molecules to be tested, but also has a stronger Raman enhancement effect, thereby improving the detection performance. The scanning electron microscope and energy spectrum of the silver nano-surface enhanced Raman scattering detection chip after the electric process are as follows: Figure 3 As shown, it can be seen that the chip interface has a uniform and dense nanostructure, which brings ubiquitous Raman enhancement "hot spots". At the same time, the quantitative data of Au elements in the energy spectrum show that Au elements already exist on the chip surface, proving the formation of Au-Ag shells in hollow nanostructures.

[0131] 4. Surface enhanced Raman spectroscopy test

[0132] After removing the silver nanoparticle surface-enhanced Raman scattering detection chip from the electrochemical cell, rinse it three times with ultrapure water. The chip was then placed in a petri dish filled with ultrapure water and placed on a Raman confocal spectrometer for Raman spectral scanning imaging. The chip was kept moist, with the liquid level approximately 800 μm from the chip interface. The laser wavelength was 633 nm, the laser power was 1.7 mW, the objective lens was 50x, and the surface scan was set to static scanning mode with an exposure time of 1 s, an X-axis scan width of -1000 μm to +1030 μm, a Y-axis scan width of -1000 μm to +1030 μm, a detection step size of 70 μm, and a total of 900 acquisition points.

[0133] 5. Quantitative calculation and statistical analysis

[0134] All the Raman spectra were baseline-removed using WiRE5.2 software (Renishaw). Figure 4 As shown, select 340~370cm -1 As the noise window, select 400~440cm -1 As the signal window of crystal violet, 570-610 cm -1 As the signal window of Nile Blue. For the calculation of noise threshold, 340-370 cm -1 Data within the range is every 10cm -1 Import a column into Excel, remove negative spectral intensity values ​​and outliers with strong signals and obvious peaks, and retain spectral intensity data of more than 800 different points after screening. Calculate the average value and standard deviation of this group of data, and use the average value + 10 times the standard deviation as the threshold for determining whether crystal violet and Nile blue are detected.

[0135] like Figure 5 As shown in Figure 2, the threshold values ​​of the five concentration gradients in the first experimental group were 155.3, 153.9, 158.2, 160.0, and 154.7, respectively. Figure 6 As shown in Figure 2, the threshold values ​​of the five concentration gradients in the second experimental group were 154.9, 148.3, 154.8, 156.1, and 162.0, respectively. Figure 7 As shown in Figure 2, the threshold values ​​of the five concentration gradients in the third experimental group were 153.1, 174.2, 179.5, 173.9, and 160.0, respectively. Figures 5-7 As shown, the points above the threshold are white, that is, the positive spectrum counting points where crystal violet or Nile blue signals are detected, and the points below the threshold are black, that is, the negative spectrum.

[0136] The number of positive spectra in each experimental group was then counted. For the calculation of crystal violet spectra, the 400-440 cm -1 Data within the range is every 5cm -1 Import a column into Excel, filter the positive spectral points greater than or equal to the threshold and remove the duplicate points. The retained data are the crystal violet counts. For the calculation of Nile Blue spectrum, 570-610 cm -1 Data within the range is every 5cm -1 Import a column into Excel, filter the positive spectral points greater than or equal to the threshold and remove the duplicate points, and the retained data is the Nile blue count. Among them, by filtering the duplicate points with both crystal violet count and Nile blue count, they are recorded as mixed counts. The statistical data of each experimental group are as follows Figures 8-10 shown.

[0137] Figure 11 (a) shows the dependence of the average number of crystal violet single molecule counts (gray bars, the statistical data have subtracted the number of mixed events, and only the number of single molecule crystal violet events is counted) of the three experimental groups on the crystal violet concentration, and the error bars represent the standard deviation of the number of crystal violet single molecule counts at each concentration (n=3).

[0138] The expected error value based on Poisson statistics was calculated as 1 / √N, where N is the total number of positive single-molecule events (black curve) with 900 total counts per measurement, as Figure 11 Panel (a) shows that the relative standard deviation (black circles) of single-molecule events detected three times for each concentration is consistent with the expected error of a Poisson distribution. Figure 11 (b) shows that the positive rate of single-molecule events of each concentration of crystal violet is positively correlated with the crystal violet concentration on a logarithmic coordinate, where R 2 =0.995.

[0139] like Figure 11As shown in the figure, through the electrokinetic-surface enhanced Raman detection strategy, the chip and the molecular solution to be tested only need 20 minutes of contact time to achieve 10 -18 The quantitative detection of M level reflects the fast time resolution and high sensitivity of the method of the present invention. Only three sets of parallel experiments, each with 900 tests, can reach 10 -18 The relative standard deviation of the quantitative detection of crystal violet was 25.0% for 10 -15 The relative standard deviation of the crystal violet quantitative detection of M was 12.4%, which reflects the convenience, efficiency and high accuracy of the method of the present invention.

[0140] Figure 12 (a) shows the dependence of the average number of Nile blue single molecule counts (gray bars, the statistical data have subtracted the number of mixed events, and only the number of single molecule Nile blue events is counted) of the three experimental groups on the Nile blue concentration, and the error bars represent the standard deviation of the number of Nile blue single molecule counts at each concentration (n=3).

[0141] The expected error value based on Poisson statistics was calculated as 1 / √N, where N is the total number of positive single-molecule events (black curve) with 900 total counts per measurement, as Figure 12 Panel (a) shows that the relative standard deviation (black circles) of single-molecule events detected three times for each concentration is consistent with the expected error of a Poisson distribution. Figure 12 (b) shows that the positive rate of single-molecule events of each concentration of Nile Blue is positively correlated with the concentration of Nile Blue on a logarithmic scale, where R 2 =0.999.

[0142] like Figure 12 As shown in the figure, through the electrokinetic-surface enhanced Raman detection strategy, the chip and the molecular solution to be tested only need 20 minutes of contact time to achieve 10 -18 The quantitative detection of M level reflects the fast time resolution and high sensitivity of the method of the present invention. Only three sets of parallel experiments, each with 900 tests, can reach 10 -18 The relative standard deviation of the quantitative detection of Nile Blue was 17.6% for 10 -15 The relative standard deviation of the Nile Blue quantitative detection of M was 8.4%, which reflects the convenience, high efficiency and high accuracy of the method of the present invention.

[0143] 6. Actual pollutant detection

[0144] In order to establish the potential of the present invention in practical measurement, the present invention selected perfluorohexanoic acid and enrofloxacin as demonstration examples. Due to their unique structure and surface activity, perfluoro compounds have weak binding ability with Raman enhanced substrates, and traditional SERS methods cannot detect the Raman enhanced substrate below ng·L. –1Enrofloxacin, the most widely used animal antibiotic, has been detected in large quantities in the environment, usually at concentrations below 10 ng·L in groundwater. –1 .

[0145] Figure 13 The dependence of the average number of positive counts (grey bars) on the perfluorohexanoic acid concentration is shown. Error bars represent standard deviation (n = 3). The relative standard deviation (black dots) and the expected error value based on Poisson statistics (black curve) are also shown. The positive rate at each concentration is positively correlated with the perfluorohexanoic acid concentration on a logarithmic scale.

[0146] Figure 14 The dependence of the average number of positive counts (grey bars) on enrofloxacin concentration is shown. Error bars represent standard deviation (n=3). The relative standard deviation (black dots) and the expected error value based on Poisson statistics (black curve) are also shown. The positive rate at each concentration is also shown to be positively correlated with enrofloxacin concentration on a logarithmic scale.

[0147] Based on the strategy developed by the present invention, the quantitative detection limit of perfluorohexanoic acid and enrofloxacin can reach 1 fM (pg·L) under the premise of a relative error of 20%. –1 The ultra-high sensitivity and reliable statistical distribution of this strategy are the key foundations for achieving these quantitative measurements.

[0148] The present invention also provides applications of the method of the present invention in environmental monitoring and biomedical testing.

[0149] It can be understood that the application of this method in environmental monitoring and biomedical testing has the following beneficial effects:

[0150] High sensitivity: Able to detect ultra-trace amounts of analyte molecules, suitable for trace pollutants in the environment and extremely low concentrations of biomarkers in biological samples.

[0151] Strong selectivity: By functionalizing and modifying the detection chip surface, the affinity for specific target molecules can be enhanced, thereby improving the specificity of detection.

[0152] Label-free detection: No labeling is required for the molecules to be detected, which reduces the sample processing steps, avoids the impact of labeling on molecular activity, and ensures the authenticity of the detection.

[0153] Rapid response: The detection process is simple, easy to operate, and results can be obtained quickly, making it suitable for real-time monitoring and clinical diagnosis.

[0154] High reproducibility: Maintaining consistency across multiple tests ensures reliable results and helps establish standardized testing procedures.

[0155] Low error: By increasing the number of tests, the positive spectrum count is increased, effectively reducing quantitative errors and improving the credibility of the results.

[0156] Diverse applications: Suitable for the detection of a variety of molecules to be tested, and can be widely used in environmental protection, food safety, early disease screening and other fields.

[0157] Economical: The detection chip is available through commercial channels and is simple to prepare, which reduces experimental costs and increases popularity.

[0158] The present invention has been described in detail above with reference to the embodiments. However, the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the spirit of the present invention.

Claims

1. A method for detecting ultra-trace amounts of a molecule to be detected, characterized in that: The following steps are involved: S1: installing a metal nano-surface enhanced Raman scattering detection chip in an in-situ electrochemical cell, wherein the in-situ electrochemical cell is provided with a working electrode and a counter electrode, the metal nano-surface enhanced Raman scattering detection chip and the working electrode are in a non-contact state, adding a molecular solution to be tested and an electrolyte solution to the in-situ electrochemical cell, applying a working potential to the working electrode, the metal nano-surface enhanced Raman scattering detection chip is arranged between the working electrode and the counter electrode, the metal nano-surface enhanced Raman scattering detection chip comprises at least one of a silver nano-surface enhanced Raman scattering detection chip, a copper nano-surface enhanced Raman scattering detection chip, and a zinc nano-surface enhanced Raman scattering detection chip, and the electrolyte solution comprises at least one of chloroauric acid and chloroplatinic acid; S2: taking out the metal nano-surface enhanced Raman scattering detection chip, performing surface enhanced Raman spectroscopy detection on the metal nano-surface enhanced Raman scattering detection chip, and obtaining a Raman spectrum of the molecule to be detected; When the molecule to be measured is a positively charged molecule or a neutrally charged molecule, the working potential is a negative potential; When the molecule to be measured is a negatively charged molecule, the working potential is a positive potential; The detection limit of the method was 1×10 -18 M, The method determines the concentration of the molecule to be detected in the molecule to be detected solution according to the number or probability of the Raman spectrum of the molecule to be detected in the molecule to be detected solution being judged as a positive spectrum.

2. The method according to claim 1, characterized in that The concentration of the electrolyte solution is 0.01 mmol / L~0.4 mmol / L.

3. The method according to claim 1, characterized in that The surface enhanced Raman spectroscopy detection includes at least one of a point scanning test, a line scanning test and a surface scanning test.

4. Use of the method according to any one of claims 1 to 3 in environmental monitoring and biomedical testing.

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