CN115753739A - Hazardous substance high-flux SERS detection method based on automatic digital microfluidic system - Google Patents

Abstract

The invention discloses a harmful substance high-flux SERS detection method based on an automatic digital microfluidic system, which comprises the following steps: designing a digital microfluidic chip to realize the mixing of trace explosive, silver sol and salt solution; respectively injecting molecules to be detected, silver sol and salt solution with different concentrations into the digital microfluidic chip and surrounding by silicone oil; firstly, distributing equivalent silver sol to be mixed with molecular drops to be detected with different concentrations respectively, and incubating for 1-6 hours to obtain mixed liquid drops; then distributing equal salt solution to be respectively mixed with the mixed liquid drops, and incubating for 5-20 minutes to obtain silver aggregates with SERS property; SERS signals of molecules to be detected with different concentrations are obtained by using a Raman microscope, and after detection, all liquid drops are transferred to a waste liquid pool for unified collection and treatment. The method can realize the automatic, high-flux and ultrasensitive SERS detection of trace explosive molecules.

Description

A high-throughput SERS detection of harmful substances based on an automated digital microfluidic system method

technical field

The invention belongs to the technical field of digital microfluidics, and specifically relates to a high-throughput SERS detection method for harmful substances based on an automated digital microfluidics system.

Background technique

With the increasing need for homeland security, explosives detection has become a subject of increasing interest in recent years. Trinitrotoluene (TNT) is a major military explosive and a major component of unexploded landmines worldwide. It should be noted that TNT is extremely toxic and is classified as a Group 1 carcinogen. Furthermore, due to the electron-absorbing properties of the three nitro groups of TNT, the aromatic ring is particularly resistant to microbial oxidative attack and ring cleavage, and exhibits persistence in the environment even at low trace concentrations. TNT pollutes millions of hectares of land around the world. In addition, 3-nitro-1,2,4-triazol-5-one (NTO) is a typical high-energy insensitive explosive with neurotoxicity and cytotoxicity. Due to the high solubility of NTO, it may enter the groundwater through the water system, thereby affecting human health in extremely low trace concentrations. The United States is gradually replacing traditional TNT explosives with NTO. With the large-scale use of NTO, it will inevitably bring pollution risks. Therefore, detecting and preventing explosives in the environment is of great significance for tracking environmental quality and protecting human health.

Over the past decade, various sensing methods including fluorescence, colorimetry, ion mobility spectroscopy, mass spectrometry, and electrochemistry have been used to detect explosives. Among them, surface-enhanced Raman spectroscopy (SERS) has attracted much attention due to its ultrasensitivity. It has been widely used in catalytic process monitoring, bioanalysis and pollution detection. With increasing requirements for safety and detection efficiency, equipment for detecting high-energy explosives should be characterized by automation, high throughput, and ultrasensitivity. In addition, traditional SERS detection is often accompanied by the problems of manual operation and low detection efficiency. Therefore, it is urgent to explore novel SERS technology with automation, high-throughput and ultra-sensitive characteristics for explosive detection.

Droplet-based microfluidics offer new opportunities for integration and automation of analytical platforms. The combination of SERS and microfluidic chips has unique advantages. Based on microdroplets, ultrasensitive, automated, and multi-target SERS detection can be realized on a chip. However, traditional microfluidic chips are complex and require external pumps and valves to work together, which is difficult to achieve. In addition, it is easy to generate channel "dead volume", causing cross-contamination. In contrast, digital microfluidics (DMF) technology does not rely on micropumps and microfluidics, and does not even require complex three-dimensional fluid channels, and has the advantages of simple structure and dynamic configuration. Furthermore, it has low sample consumption, high parallelism, and automation capabilities. However, how to implement a high-throughput process and generate SERS hotspots on DMF chips for ultrasensitive explosives detection remains two major challenges.

Contents of the invention

In order to solve the above problems, we propose a high-throughput surface-enhanced Raman scattering (SERS) detection method for harmful substances based on an automated digital microfluidic system. This method enables automated, high-throughput, and ultrasensitive SERS detection of trace explosive molecules.

For reaching this purpose, the present invention adopts following technical scheme:

A high-throughput SERS detection method for harmful substances based on an automated digital microfluidic system, comprising the following steps:

(1) Design a digital microfluidic chip to realize the mixing of trace explosives, silver sol and salt solution;

(2) Inject different concentrations of the molecules to be detected, silver sol and salt solution into the digital microfluidic chip and surround them with silicone oil; first distribute equal amounts of silver sol to mix with droplets of the molecules to be detected at different concentrations, and incubate After 1-6 hours, mixed droplets were obtained; then an equal amount of salt solution was distributed to mix with the above-mentioned mixed droplets, and after incubation for 5-20 minutes, silver aggregates with SERS properties were obtained;

(3) The SERS signals of the molecules to be detected at different concentrations were obtained using a Renishaw inVia Raman microscope. After SERS detection, all droplets were transferred to the waste pool for unified collection and processing.

A further technical solution is that the digital microfluidic chip includes two parts, an upper plate and a lower plate, the lower plate includes a substrate, an electrode layer, a dielectric layer and a partially hydrophilized hydrophobic layer, and the upper plate is a hydrophobic ground The electrodes, the upper and lower plates are parallel to each other, and are separated by a gap layer; the electrode layer includes a digital microfluidic plate electrode array composed of 40 drive electrodes and 8 reserve electrodes, and also includes two sample pools, five reaction pools and A waste liquid pool and a droplet generation channel electrode array, the molecules to be detected are injected into the reaction pool, and the silver sol and salt solution are respectively injected into the sample pool.

A further technical solution is that the salt solution is selected from any one or more of sodium chloride solution, potassium chloride solution, potassium iodide solution, and sodium iodide solution.

A further technical solution is that the concentration of the salt solution is any one of 0.1M, 1M, 2M, 3M and 4M.

Its specific operation method is:

For joint system SERS performance evaluation. CV dissolved in ultrapure water was used as a probe molecule, and 1 μL of Ag sol (1 mg/mL) and 1 μL of CV aqueous solution were automatically mixed in a DMF chip and left to stand for 1-6 hours. Then, add 1 μL of salt solution (the types of salt solution include: sodium chloride, potassium chloride, potassium iodide, sodium iodide. The concentration of the salt solution is: 0.1M, 1M, 2M, 3M, 4M), and let it stand for 5-20 minutes Finally, Raman spectra were collected on a Renishaw inVia Raman microscope facility. The excitation wavelength was 532 nm, and the laser beam was focused using a 20X magnification Leica objective lens. A grating with 1800 lines/mm is used, and the spectral resolution is 1 cm ^-1 . The spectrum acquisition time was 10 s, and the laser power was kept at 0.3-15 mW in all acquisitions.

For the detection of explosive NTO. NTO dissolved in ultrapure water was automatically mixed with 1 μL of Ag sol (1 mg/mL) in a DMF chip and left to stand for 1–6 hours. Then, 1 μL of saline solution was added, and after standing for 5-20 minutes, Raman spectra were collected on RenishawinVia Raman microscope equipment. The excitation wavelength was 532 nm, and the laser beam was focused using a 20X magnification Leica objective lens. A grating with 1800 lines/mm is used, and the spectral resolution is 1 cm ^-1 . The spectrum acquisition time was 10 s, and the laser power was kept at 0.3-15 mW in all acquisitions.

For the detection of TNT. First, 4-ATP molecules were adsorbed on the surface of Ag-NPs through the formation of Ag-S bonds between sulfhydryl groups and Ag-NPs. After the 4-ATP molecule reached the adsorption equilibrium, the TNT solution was mixed with the 4-ATP-modified Ag-NP, and the TNT molecule was deprotonated by an electron-rich amine on the methyl group to form a Meissenheimer complex with 4-ATP. Then, 1 μL of saline solution was added, and after standing for 5-20 minutes, Raman spectra were collected on Renishaw inVia Raman microscope equipment. The excitation wavelength was 532 nm, and the laser beam was focused using a 20X magnification Leica objective lens. A grating with 1800 lines/mm is used, and the spectral resolution is 1 cm ^-1 . The spectrum acquisition time was 10 s, and the laser power was kept at 0.3-15 mW in all acquisitions. SERS detection of NTO, R6G, RhB was carried out with the same procedure.

Compared with the prior art, the present invention has the following beneficial effects: the present invention designs a high-throughput detection process for trace explosive NTO based on an electrode array composed of 40 driving electrodes and 8 reserve electrodes. Through the screening of the type and concentration of the salt solution, the SERS hot spot in the microfluidic chip was regulated, and the sensitive detection of trace explosive NTO was realized. Based on automated digital microfluidics, it reduces the contact between people and harmful substances and ensures the safety of testing personnel.

Description of drawings

Fig. 1 is the DMF-SERS high-throughput detection process of the present invention;

Fig. 2 is the aggregation situation of silver nanoparticles in the DMF chip before and after the present invention adds salt;

Fig. 3 is the Raman response of DMF-SERS to the CV of 10 ^-7 M under the presence of 1M different kinds of salts of the present invention;

Figure 4 is the Raman response of DMF-SERS to the CV of 10 ^-7 M in the presence of different concentrations of KI in the present invention;

Figure 5 is the Raman response of DMF-SERS in the presence of different concentrations (10 ^-4 -10 ^-8 M) of NTO of the present invention;

Fig. 6 is the Raman response of DMF-SERS in the presence of different concentrations (10 ^-4 -10 ^-7 M) of TNT of the present invention.

Detailed ways

In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through formal channels.

Example 1

The detection effect of the DMF-SERS combined system under different types of salt solutions:

In order to realize the aggregation of Ag-NPs in Ag sol, generate a localized electric field, and achieve SERS enhancement, an inorganic salt-induced nanoparticle aggregation method was designed. Inorganic salts act as coagulants, destroying the electrical double layer of Ag-NPs and causing Ag-NPs to aggregate. Figure 2 shows the digital photographs of the Ag-NPs aggregation state before and after salt addition on the DMF chip. After adding salt, the Ag-NPs aggregated to generate hot spots, which was beneficial for the expression of SERS signal. CV dissolved in ultrapure water was used as a probe molecule, and 1 μL of Ag sol (1 mg/mL) and 1 μL of CV aqueous solution were automatically mixed in a DMF chip and left to stand for 2 hours. Then, 1 μL of saline solution was added (1M, the types of saline solution include: sodium chloride, potassium chloride, potassium iodide, sodium iodide.), and after standing for 20 minutes, Raman spectra were collected on RenishawinVia Raman microscope equipment. The excitation wavelength was 532 nm, and the laser beam was focused using a 20X magnification Leica objective lens. A grating with 1800 lines/mm is used, and the spectral resolution is 1 cm ^-1 . The spectral acquisition time was 10 s, and the laser power was kept at 15 mW during all acquisitions. Figure 3 shows the SERS spectra of the probe molecule CV (10 ^-7 M) collected after adding different kinds of salts.

Example 2

The detection effect of the DMF-SERS combined system under different salt solution concentrations:

CV dissolved in ultrapure water was used as a probe molecule, and 1 μL of Ag sol (1 mg/mL) and 1 μL of CV aqueous solution were automatically mixed in a DMF chip and left to stand for 2 hours. Then, 1 μL of salt solution was added (KI, concentration of salt solution: 0.1M, 1M, 2M, 3M, 4M.), and after standing for 20 minutes, Raman spectra were collected on Renishaw inVia Raman microscope equipment. The excitation wavelength was 532 nm, and the laser beam was focused using a 20X magnification Leica objective lens. A grating with 1800 lines/mm is used, and the spectral resolution is 1 cm ^-1 . The spectral acquisition time was 10 s, and the laser power was kept at 15 mW during all acquisitions. Figure 4 shows the SERS spectra of the probe molecule CV (10 ⁻⁷ M) collected after adding different concentrations of salt, and a significant SERS signal of CV can be observed.

Example 3

High-throughput detection of 5 different concentrations of NTO explosive samples:

Different concentrations of NTO molecules (at positions 16, 24, ^{40, 39, 28, 10 -4} -10 ^-8 M), Ag sol (at position 5) and saline solution (KI, 1M, at position 2) were injected into DMF chip and surrounded by silicone oil. First, 5 units (1 μL) of Ag sol were isolated from position 5, mixed with NTO droplets of different concentrations (positions 16, 24, 40, 39, 28), and incubated for 2 h. Then, saline solution (position 2) was mixed with the above-mentioned droplets separately. After 20 min of incubation, Ag-NPs aggregates with SERS properties were observed. Finally, the SERS signals of different concentrations of analyte molecules were obtained using a Renishaw inVia Raman microscope (Fig. 5). After SERS detection, all droplets can be transferred to the waste pool (position 12) for unified collection and processing.

Example 4

High-throughput detection of 5 different concentrations of TNT explosive samples:

Mix 100 μL of Ag sol (1 mg/mL) and 500 μL of 4-ATP ethanol solution (10-5 M) and incubate for 2 hours, then wash with ultrapure water by centrifugation, add 100 μl of ultrapure water, and ultrasonically disperse to obtain 4- ATP-grafted Ag-NPs. Different concentrations of TNT molecules (at positions 16, 24, 40, 39, 28, ^{10 -4} -0M), 4-ATP-grafted Ag-NPs (at position 5) and saline solution (KI, 1M, at position 2) respectively injected into the DMF chip and surrounded by silicone oil. First, one unit (1 μL) of 4-ATP-grafted Ag-NPs was detached from position 5, mixed with different concentrations (positions 16, 24, 40, 39, 28) of TNT droplets, and incubated for 2 h. Then, saline solution (position 2) was mixed with the above-mentioned droplets separately. After 20 minutes of incubation, the SERS signals of different concentrations of analyte molecules were obtained using a RenishawinVia Raman microscope (Fig. 6). After SERS detection, all droplets can be transferred to the waste pool (position 12) for unified collection and processing.

Although the present invention has been described here with reference to the illustrative examples of the present invention, the above-mentioned examples are only preferred implementations of the present invention, and the implementation of the present invention is not limited by the above-mentioned examples. It should be understood that those skilled in the art Many other modifications and embodiments can be devised which will fall within the scope and spirit of the principles disclosed in this application.

Claims (4)

1. A high-throughput SERS detection method for harmful substances based on an automated digital microfluidic system, characterized in that it comprises the following steps: (1) Design a digital microfluidic chip to realize the mixing of trace explosives, silver sol and salt solution; (2) Inject different concentrations of the molecules to be detected, silver sol and salt solution into the digital microfluidic chip and surround them with silicone oil; first distribute equal amounts of silver sol to mix with droplets of the molecules to be detected at different concentrations, and incubate After 1-6 hours, mixed droplets were obtained; then an equal amount of salt solution was distributed to mix with the above-mentioned mixed droplets, and after incubation for 5-20 minutes, silver aggregates with SERS properties were obtained; (3) The SERS signals of the molecules to be detected at different concentrations were obtained using a Renishaw inVia Raman microscope. After SERS detection, all droplets were transferred to the waste pool for unified collection and processing. 2. The high-throughput SERS detection method for harmful substances based on an automated digital microfluidic system according to claim 1, wherein the digital microfluidic chip includes two parts, an upper and a lower plate, and the lower plate includes Substrate, electrode layer, medium layer and partially hydrophilized hydrophobic layer. The upper plate is a hydrophobic ground electrode. The upper and lower plates are parallel and separated by a gap layer; the electrode layer includes 40 drive electrodes and 8 reserve electrodes. The digital microfluidic plate electrode array composed of electrodes also includes two sample pools, five reaction pools and a waste liquid pool and droplet generation channel electrode array, the molecules to be detected are injected into the reaction pools, the Silver sol and saline solution were injected into the sample cell respectively. 3. the harmful substance high-throughput SERS detection method based on automated digital microfluidic system according to claim 1, is characterized in that, described saline solution is selected from sodium chloride solution, potassium chloride solution, potassium iodide solution, iodine Any one or more of the sodium chloride solution. 4. The hazardous substance high-throughput SERS detection method based on the automated digital microfluidic system according to claim 3, wherein the concentration of the salt solution is any of 0.1M, 1M, 2M, 3M, 4M A sort of.

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