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

Abstract

The invention provides a method for detecting ultra-trace molecules to be detected and application. According to the method, a metal nano surface enhanced Raman scattering detection chip is installed in an in-situ electrochemical cell, a working electrode and a counter electrode are arranged in the in-situ electrochemical cell, and the metal nano surface enhanced Raman scattering detection chip and the working electrode are in a non-contact state; adding a to-be-detected molecular solution and an electrolyte solution into the in-situ electrochemical cell, applying a working potential to the working electrode, then taking out the metal nano surface enhanced Raman scattering detection chip, and carrying out surface enhanced Raman spectrum detection on the metal nano surface enhanced Raman scattering detection chip to obtain the metal nano surface enhanced Raman scattering detection chip. And obtaining the Raman spectrum of the molecule to be detected. The method is good in repeatability and high in detection efficiency, a higher positive spectrum count can be obtained on the premise that the total spectrum amount is detected in a limited mode, and the detection result is reliable. The invention further provides application of the detection method for the ultra-trace molecules to be detected.

Description

Technical Field

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

[0002] Surface-enhanced Raman spectroscopy (SERS) is a non-destructive molecular detection technology that reflects the information of molecular bond vibration energy and rotation energy. It is a "fingerprint" spectrum that can specifically identify molecules. In the past half century, the basic research and practical application of SERS have grown steadily, and it has developed into a means of qualitative and quantitative analysis of trace detection in a wide range of fields such as surface and interface chemistry, catalysis, biology and environmental monitoring.

[0003] Quantitative analysis has always been a huge challenge in the SERS field, especially when detecting low concentrations (less than 1nM), 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 to achieve 1fM (10 -15 mol / L) level, and 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 point of view requires the collection of more than 10,000 sets of valid data, which makes the detection efficiency low and the error large in the actual detection process. At the same time, traditional SERS technology often needs to immerse the molecule solution to be tested and the enhanced substrate for more than 1 hour to achieve single-molecule level detection. The long detection time resolution also limits the development of this method.

[0004] It can be seen that the application value of surface-enhanced Raman spectroscopy in qualitative identification, precise quantification and other application scenarios is still greatly limited due to the time resolution, efficiency and detection limit problems 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 invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention provides a method for detecting ultra-trace molecules to be tested, which has good repeatability and high detection efficiency, can obtain higher positive spectrum counts under the premise of a limited total amount of detected spectra, and the detection results are reliable. The digital quantitative detection results can increase the count amount of positive spectra by increasing the total number of detections, thereby reducing the quantitative error, and the error of quantitative detection is controllable.

[0006] The invention also provides an application for detecting ultra-trace molecules to be detected.

[0007] The first aspect of the present invention provides a method for ultra-trace detection of a molecule to be detected, comprising the following steps:

[0008] S1: installing a metal nano-surface enhanced Raman scattering detection chip in an in-situ electrochemical cell, wherein a working electrode and a counter electrode are provided in the in-situ electrochemical cell, wherein 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 detected and an electrolyte solution into the in-situ electrochemical cell, and applying a working potential to the working electrode;

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

[0010] A technical solution in the method for ultra-trace detection of molecules to be tested of the present invention has at least the following beneficial effects:

[0011] The method of the present invention has good detection chip diversity, is easy to prepare, can also 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 molecule to be detected.

[0012] 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.

[0013] The method of the invention has the advantages of simple operation, fast detection response speed and high detection time resolution.

[0014] The method of the present invention does not require labeling of the molecules to be detected and can directly perform accurate quantitative detection of the target molecules at the single molecule level.

[0015] 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.

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

[0017] The present invention can realize the detection of ultra-trace molecules to be detected based on the electrokinetic process by selecting a suitable chip, and has a single-molecule level detection sensitivity for the target molecule to be detected, 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, 0.2mmo The range value formed by any one of the values ​​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, or any two of them.

[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 relates to a technical solution for the application of the method for ultra-trace detection of molecules to be tested 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 functionally modifying the detection chip surface, the affinity for specific target molecules can be enhanced and the specificity of detection can be improved.

[0034] Label-free detection: There is no need to label the molecules to be tested, 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, which effectively reduces the quantitative error and improves the credibility of the results.

[0038] Diverse applications: It is 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 can be obtained through commercial channels and is simple to prepare, which reduces experimental costs and improves 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 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 It 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 electrokinetic 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 distribution 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 is the statistics of single molecule event counts and mixed event counts in the mixed solution of crystal violet and nile blue of the tested bimolecular solution M.

[0050] Fig. 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 tested bimolecular solution M.

[0051] Fig.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 tested bimolecular solution M.

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

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

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

[0055] Fig.14 For 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 a working electrode and a counter electrode are provided in the in-situ electrochemical cell, wherein 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 detected and an electrolyte solution into 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 spectrum detection on the metal nano-surface enhanced Raman scattering detection chip, and obtaining the Raman spectrum of the molecule to be detected.

[0060] A technical solution in the method for ultra-trace detection of molecules to be tested 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, can also 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 molecule to be detected.

[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 invention has the advantages of simple operation, fast detection response speed and high detection time resolution.

[0064] The method of the present invention does not require labeling of the molecules to be detected and can directly perform accurate quantitative detection of the target molecules 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, and the digital quantitative detection results can increase the counting amount of positive spectra by increasing the total number of detection times, thereby reducing the quantitative error, and the error of quantitative detection is controllable.

[0067] The present invention can realize the detection of ultra-trace molecules to be detected based on the electrokinetic process by selecting a suitable chip, and has a single-molecule level detection sensitivity for the target molecule to be detected, 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 combination with the first aspect, in some embodiments of the present invention, the method includes single molecule level quantitative detection.

[0070] Single-molecule level quantitative detection means that the method can accurately identify and measure the concentration and characteristics of individual molecules. This highly sensitive detection can effectively capture target molecules at extremely low concentrations and is suitable for fields such as early disease detection, environmental monitoring, and basic scientific research. It ensures the reliability and accuracy of the test results and helps to 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 nano-material 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 and helps to improve the 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] The position close to the electrode increases the intensity of the Raman signal and enhances the detection sensitivity of the molecules to be tested. This layout helps to more effectively capture the molecules to be tested near the electrode and improve the detection efficiency. Avoiding direct contact with the electrode reduces the interference of the electrochemical reaction on the Raman signal. It is convenient for experimenters to adjust the chip position and achieve optimization under different experimental conditions. In addition, the chip can be functionalized as needed to adapt to the detection of different types of molecules.

[0075] It should be noted that the traditional means of surface enhanced Raman spectroscopy detection is to immerse the roughened silver substrate in the test solution, or to perform some functional modification on the silver substrate to better adsorb the test molecules, and then test the Raman spectrum of the silver substrate to obtain the information of the test object. This is a common and conventional method. The similarity between the present invention and the prior art is that the silver sheet is roughened by an electrochemical method. It should be noted that the present invention uses an electrochemical method to roughen the silver sheet only to prepare a rough silver enhanced substrate, and there is no special requirement. The preparation method of the silver substrate does not need to be specific, and it can also be any commercially available gold and silver enhanced substrate.

[0076] The specificity of the present invention lies in the construction method of electrodynamic-enhanced Raman spectroscopy. Specifically, in the method of the present invention, the detection chip is not used 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 tested is placed near the working electrode, but also not in contact with the working electrode. That is, in the present invention, the chip to be tested is not electrically connected.

[0077] In traditional electrochemical surface enhanced Raman testing methods, the chip used in the test is used as a working electrode and needs to be connected to electricity. The chip to be tested is directly used as a working electrode and voltage needs to be applied. One of the disadvantages of this is that after the test object is adsorbed onto the working electrode, that is, the chip to be tested, a certain degree of redox reaction will occur, thus affecting its detection.

[0078] In combination with the first aspect, in 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.

[0079] In combination with the first aspect, in some embodiments of the present invention, the solution of the molecule to be detected has a series of concentration gradients, and the concentration of the solution of the molecule to be detected decreases 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 it 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, which is convenient for subsequent quantitative analysis. By covering multiple concentration gradients, the target molecule can be detected in 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 and improve the accuracy and reliability of the test results. By covering multiple concentration levels, it can be ensured that the changes in the molecule to be tested are more comprehensively reflected in practical 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 the prior art uses an electrochemical reduction process to produce a gold-silver composite substrate on a silver substrate using, for example, a HAuCl4 solution at a constant potential. The feature of the present invention is that the silver substrate chip is immersed in a HAuCl4 solution and does not require an electro-reduction effect, because the direct replacement reaction between the silver nanoparticles and the gold solution can produce an Ag-Au nanoshell structure with a hollow structure. The enhancement performance of this hollow shell structure is better than the structural enhancement effect produced by directly using the silver chip as a working electrode to electro-deposit gold.

[0083] Therefore, the feature 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, so that the effect of electrodynamics can be utilized to drive the molecules to be tested to the chip to be tested by means of electromigration, and the chip is not directly powered, so the oxidation-reduction reaction of the molecules to be tested will not occur. 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 it is not feasible to simply place the detection chip between the working electrode and the counter electrode to energize it, and the effect is not good. In the scheme of the present invention, the key point is to use chloroauric acid 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 generating an Ag-Au shell structure to confine the molecules to be tested and improve the detection effect. Therefore, the scheme 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 detection effect.

[0085] The experiment found that if the detection chip is used as a working electrode to attract the molecules to be detected, the effect of electro-depositing chloroauric acid will be worse, because the dense gold layer produced by the rapid electro-deposition process of chloroauric acid on the silver interface is not well enhanced. In addition, since the detection object of the present invention can be a single molecule detection with a very low concentration, excessive electro-deposition of gold on the silver chip substrate will cover the molecules to be detected. During the detection, it will be found that the Raman background of the matrix is ​​too high, thereby drowning the Raman signal of the molecules to be detected.

[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 "hot spots". Only molecules adsorbed on efficient hot spots will contribute to the overall Raman signal. Most importantly, efficient hot spots are rare, and at ultra-low concentrations, the amount of adsorbed analyte is expected to be small, which 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 groups 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 20min. At the same time, due to the principle of electrokinetic binding surface confinement, the positive spectrum rate of the detection is greatly improved, so that the total number of detections can be reduced and the detection efficiency can be improved. 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 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, or any two of the values.

[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 min to 60 min.

[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 diagram 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 a 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 a 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 ultra-trace 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 detecting ultra-trace test molecules includes the following steps: determining the concentration of the test molecules in the test molecule solution based on a mathematical correlation of the number of times and / or probability that the Raman spectrum of the test molecule in the test molecule 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 test 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 relates to a technical solution for the application of the method for ultra-trace detection of molecules to be tested 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 functionally modifying the detection chip surface, the affinity for specific target molecules can be enhanced and the specificity of detection can be improved.

[0107] Label-free detection: There is no need to label the molecules to be tested, 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, which effectively reduces the quantitative error and improves the credibility of the results.

[0111] Diverse applications: It is 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 can be obtained through commercial channels and is simple to prepare, which reduces experimental costs and improves 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] Among them, water quality monitoring mainly refers to the detection of pollutants (such as heavy metals, organic pollutants) and microorganisms in water. Air quality testing mainly refers to the monitoring of harmful gases (such as VOCs, PM2.5) and pollutants in the air. Soil testing mainly refers to the assessment of the content 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, the biomedical test includes disease marker detection, infectious disease pathogen testing, and drug monitoring.

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

[0117] The above applications help to improve the effectiveness of public health and environmental protection.

[0118] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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, the description with reference to the terms "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 schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[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 conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0123] Example

[0124] Using silver nano-surface enhanced Raman scattering detection chip as surface enhanced Raman substrate, 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 for three times, 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 later use. The scanning electron microscope and energy spectrum of the smooth Ag sheet are shown in Figure 2. Figure 1As shown. The silver sheet was electrochemically treated on an electrochemical workstation with a three-electrode system. The electrolyte was 0.1M KOH, the working electrode was a silver sheet, the counter electrode was a platinum sheet, and the reference electrode was an Ag / AgCl electrode. First, the silver sheet was roughened by cyclic voltammetry. The cyclic voltammetry curve was scanned in the potential range of +0.5 to -0.5V (vs Ag / AgCl), the scanning rate was 10mV / s, and the number of scanning circles was 5. After the cyclic voltammetry scan, the silver sheet was reduced at a constant potential of -1.2V (vs Ag / AgCl) for 800s to stabilize the prepared roughened silver sheet substrate. The roughened silver sheet was taken out, rinsed repeatedly with ultrapure water and dried, and 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 away from light for later use. Prepare a 0.1mM crystal violet (Macklin, 95%) ethanol solution and a Nile blue (Sigma-Aldrich) aqueous solution, mix them in equal proportions, and then dilute them in a gradient of 1 order of magnitude to prepare a concentration gradient of 10 -18 ~10 -14 M is 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 1.5×1 cm 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 ) was fixed above the working electrode, 3 mm away from the working electrode (the chip to be tested and the working electrode were not in contact). Then, 20 mL of the bimolecular mixed solution of crystal violet and Nile blue to be tested and 0.5 mL of 10 mM chloroauric acid solution were added to the electrochemical cell and shaken well. 2 The titanium sheet was used as the counter electrode and placed in the electrochemical cell, above the counter electrode and the silver nano-surface enhanced Raman scattering detection chip, and ~8mm away from the working electrode. Finally, a potential of -1.2V vs Ag / AgCl was applied to the working electrode, and the current I was ~1.4×10 -4A. After 20 minutes of power-on, take out the silver nano-surface enhanced Raman scattering detection chip to be tested 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 limits 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 taking out the silver nano-surface enhanced Raman scattering detection chip to be tested from the electrochemical cell, rinse it repeatedly with ultrapure water for 3 times, then put the chip into a culture dish filled with ultrapure water and place it in the Raman confocal spectrometer for Raman spectral surface scanning imaging, so that the chip is always kept wet, and the liquid surface is about 800μm away from the chip interface. Use 633nm as the laser wavelength, laser power 1.7mW, 50x objective lens, static scanning mode for surface scanning, exposure time 1s, X-axis scanning width -1000μm~+1030μm, Y-axis scanning width -1000μm~+1030μm, detection step length 70μm, and a total of 900 points for collection.

[0133] 5. Quantitative calculation and statistical analysis

[0134] All the Raman spectra measured were baseline-removed by 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 the negative values ​​of spectral intensity, strong signals, and outliers with obvious peaks, and retain more than 800 spectral intensity data at 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 1, 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 to 7 As shown, the points above the threshold are white points, 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, 400-440 cm -1 Data within the range is every 5cm -1 Import a column into Excel, filter the positive spectral points that are greater than or equal to the threshold and remove the duplicate points. The retained data are the crystal violet counts. For the calculation of the 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 that are 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 counts and Nile blue counts, they are recorded as mixed counts. The statistical data of each experimental group is as follows Figures 8 to 10 shown.

[0137] Fig.11 In the figure, (a) shows the dependence of the average number of crystal violet single molecule counts (gray bars, the statistical data has 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 with 900 total counts per measurement (black curve), as Fig.11 (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 the Poisson distribution. Fig.11 (b) shows that the positive rate of single-molecule events of each concentration of crystal violet is positively correlated with the concentration of crystal violet on a logarithmic scale, where R 2 =0.995.

[0139] like Fig.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, 900 tests per set, can reach 10 -18 The relative standard deviation of the quantitative detection of crystal violet was 25.0%, 10 -15 The relative standard deviation of the crystal violet quantitative detection of M is 12.4%, which reflects the convenience, high efficiency and high accuracy of the method of the present invention.

[0140] Fig.12 In the figure, (a) shows the dependence of the average number of Nile blue single molecule counts (gray bars, the statistical data has 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 with 900 total counts per measurement (black curve), as Fig.12 (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 the Poisson distribution. Fig.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 Fig.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, 900 tests per set, can reach 10 -18 The relative standard deviation of the quantitative detection of Nile Blue was 17.6%, 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 actual 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 substrates below ng·L. –1Enrofloxacin, as the most widely used animal antibiotic, has been detected in large quantities in the environment, usually at a concentration of less than 10 ng·L in groundwater. –1 .

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

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

[0147] Based on the strategy developed by the present invention, the quantitative detection limit of perfluorohexanoic acid and enrofloxacin can reach 1fM (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 invention also provides application of the method of the 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 functionally modifying the detection chip surface, the affinity for specific target molecules can be enhanced and the specificity of detection can be improved.

[0152] Label-free detection: There is no need to label the molecules to be tested, 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, which effectively reduces the quantitative error and improves the credibility of the results.

[0156] Diverse applications: It is 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 can be obtained through commercial channels and is simple to prepare, which reduces experimental costs and improves popularity.

[0158] The present invention has been described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for detecting ultra-trace amounts of molecules to be tested, 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 a working electrode and a counter electrode are provided in the in-situ electrochemical cell, wherein 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 detected and an electrolyte solution into the in-situ electrochemical cell, and applying a working potential to the working electrode; S2: taking out the metal nano-surface enhanced Raman scattering detection chip, performing surface enhanced Raman spectrum detection on the metal nano-surface enhanced Raman scattering detection chip, and obtaining the Raman spectrum of the molecule to be detected.

2. The method according to claim 1, characterized in that: The metal nano-surface enhanced Raman scattering detection chip is arranged between the working electrode and the counter electrode.

3. The method according to claim 1, characterized in that: The metal nano-surface enhanced Raman scattering detection chip is arranged beside the working electrode.

4. The method according to any one of claims 1 to 3, characterized in that 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.

5. The method according to any one of claims 1 to 3, characterized in that The electrolyte solution includes at least one of chloroauric acid and chloroplatinic acid.

6. The method according to claim 5, characterized in that The concentration of the electrolyte solution is 0.01 mmol / L to 0.4 mmol / L.

7. The method according to any one of claims 1 to 3, characterized in that When the molecule to be detected is a positively charged molecule or an electrically neutral 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.

8. The method according to any one of claims 1 to 3, 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.

9. The method according to any one of claims 1 to 3, characterized in that The detection limit of the method was 1×10 - 18 M.

10. Use of the method according to any one of claims 1 to 9 in environmental monitoring and biomedical testing.

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