High-sensitivity recyclable microfluidic Raman detection substrate

Abstract

The invention relates to the technical field of nano photon fluidics, and discloses a high-sensitivity recyclable microfluidic Raman detection substrate, which comprises: a substrate; the metal rough film enhancement layer is arranged on the surface of the substrate, and the enhancement layer comprises a copper deposition layer and a gold deposition layer and is used for providing surface enhanced Raman scattering sites for target molecules; the micro-fluidic channel is arranged on the surface of the metal rough film enhancement layer, and the micro-fluidic channel is provided with at least one inlet and one outlet and is used for guiding a liquid sample to flow through the surface of the enhancement layer. According to the invention, the metal rough film enhancement layer formed by depositing the copper layer and the gold layer on the substrate and the nanoscale rough structure on the surface of the metal rough film enhancement layer are utilized to generate high-density localized surface plasma resonance hot spots, so that Raman scattering signals of target molecules adsorbed on the metal rough film enhancement layer are greatly enhanced; according to the invention, the detection sensitivity can be obviously improved, and the effective detection requirement on ultra-trace molecules can be met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanophotonic fluidics, in particular to a high-sensitivity recyclable microfluidic Raman detection substrate. BACKGROUND

[0002] Raman spectroscopy is a kind of scattering spectrum based on the interaction between light and molecules, which realizes high specificity, label-free and non-destructive identification and analysis of substances by recording the characteristic spectrum of molecular vibration ("molecular fingerprint"). Raman spectroscopy has been widely used in biomedical, chemical analysis, environmental monitoring and food safety fields. In order to overcome the low signal intensity of traditional Raman, surface-enhanced Raman scattering (SERS) technology has emerged. Through the local surface plasmon resonance effect generated by metal nanostructures, SERS can greatly enhance the Raman signal and improve the detection sensitivity to the level of trace or even single molecule, promoting the wide application of Raman technology in high-sensitivity molecular detection field.

[0003] With the continuous expansion of application scenarios, higher requirements are put forward for the performance of SERS substrate. On the one hand, the substrate needs to have high enhancement ability and excellent signal consistency to support high sensitivity and high reliability detection; on the other hand, the recyclable use ability, cost-effectiveness and adaptability of the substrate in complex environment also become the bottleneck that needs to be broken through in practical application. However, Raman sensing often encounters many difficulties in practical application: (1) Difficult to recycle: Most SERS substrates are disposable materials, which are easily contaminated or irreversibly damaged after use, and the strong adsorption of target molecules on the metal surface ("memory effect") makes it difficult to completely remove the residual molecules, affecting the consistency of subsequent detection, limiting the recycling of the substrate, resulting in high detection cost, and difficult to meet the needs of high-throughput or long-term online monitoring.

[0004] (2) The lower limit of detection concentration is high: Many traditional SERS substrates have insufficient enhancement factor, which makes it difficult to realize reliable detection of ultra-trace or even single molecule level, limiting the application value of the technology in the detection of extremely low concentration samples.

[0005] (3) Poor signal stability: Some SERS substrates have problems such as uneven structure, insufficient chemical stability or limited service life, which affect the performance consistency and data reliability in long-term use.

[0006] (4) Background signal interference: In complex sample background, fluorescence interference, stray light and non-target substance signal are easy to affect the quality of Raman detection, reduce the sensitivity and specificity of detection.

[0007] (5) It is difficult to realize real-time detection: most traditional SERS substrates cannot be effectively integrated into microfluidic systems, and it is difficult to support high-throughput, automation, real-time dynamic detection, which limits its promotion in modern online monitoring and microfluidic chip integration applications. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a high-sensitivity recyclable microfluidic Raman detection substrate, which solves the problems of the conventional SERS substrate caused by memory effect, such as difficult recycling, insufficient detection sensitivity, poor signal stability, and difficult real-time monitoring.

[0009] To achieve the above object, the present application is realized by the following technical scheme: The present application provides a high-sensitivity recyclable microfluidic Raman detection substrate. The substrate comprises: a substrate; a metal rough film enhancement layer disposed on the surface of the substrate; and a microfluidic channel disposed on the surface of the metal rough film enhancement layer.

[0010] The metal rough film enhancement layer comprises a copper deposition layer and a gold deposition layer. The structure of the enhancement layer is designed to form a surface with nanoscale roughness, and the rough structure can produce a localized surface plasmon resonance (LSPR) effect, providing high-intensity Raman signal enhancement sites for target molecules adsorbed thereon.

[0011] The microfluidic channel has at least one inlet and one outlet, which functions to limit and guide the flow of liquid sample on the surface of the metal rough film enhancement layer.

[0012] In a specific embodiment, the substrate of the present application is an optically transparent substrate in the visible light band, and the optically transparent substrate in the visible light band is a glass sheet. The transparent property allows the collection of Raman signals to be performed from one side of the substrate.

[0013] In a specific embodiment, the copper deposition layer is located between the substrate and the gold deposition layer. This structure helps to form an effective nanoscale rough morphology on the surface of the gold deposition layer.

[0014] In a specific embodiment, the thickness of the copper deposition layer is 0.9-1.1 nm (preferably 1 nm), and the thickness of the gold deposition layer is 9.5-10.5 nm (preferably 10 nm).

[0015] In a specific embodiment, the metal rough film enhancement layer is formed by a thermal evaporation process.

[0016] In a specific embodiment, the shape of the microfluidic channel is set as a curved channel.

[0017] In one embodiment, the substrate further comprises a transparent cover plate. The transparent cover plate covers the microfluidic channel and seals the microfluidic channel, so that the surface of the metal rough film enhancement layer and the transparent cover plate together form a closed flow detection cavity.

[0018] The second aspect of the present application provides an online detection and regeneration method using the microfluidic Raman detection substrate. The method comprises a detection step and a regeneration step.

[0019] The detection step specifically comprises: inputting a solution to be detected containing target molecules from the inlet of the microfluidic channel, and in the process of the solution flowing through the metal rough film enhancement layer, the target molecules are adsorbed on the surface of the enhancement layer; at the same time, the adsorbed target molecules are collected and detected in real time by using a Raman spectrometer.

[0020] The regeneration step specifically comprises: after the detection step is completed, inputting a sodium borohydride (NaBH4) solution as a cleaning solution from the inlet of the microfluidic channel.

[0021] The technical mechanism of the regeneration step is that the sodium borohydride solution can react with the surface of the gold deposition layer to break the chemical adsorption bond formed between the target molecules and the surface of the gold deposition layer. Thus, the previously adsorbed target molecules are separated from the surface of the enhancement layer and flow out with the liquid, thereby removing the surface residues, restoring the detection activity of the substrate, and achieving online regeneration of the substrate.

[0022] In one embodiment, after the regeneration step is completed, the detection step can be repeatedly performed to use the regenerated substrate for the next round of Raman spectrum detection, thereby achieving multiple cycles of the substrate.

[0023] The present application provides a high-sensitivity recyclable microfluidic Raman detection substrate. The present application has the following advantages: 1. The metal rough film enhancement layer formed by depositing a copper layer and a gold layer on the substrate uses the nanoscale rough structure on its surface to generate a high-density local surface plasmon resonance hot spot, thereby greatly enhancing the Raman scattering signal of the target molecules adsorbed thereon. Therefore, the present application significantly improves the detection sensitivity and can meet the effective detection requirements of ultra-trace molecules.

[0024] 2. The present application uses sodium borohydride solution as a cleaning medium to initiate a substitution reaction on the surface of the gold deposition layer and break the chemical bond formed between the adsorbed target molecules and the surface. This process can effectively remove the surface residues to inhibit the memory effect commonly seen on conventional SERS substrates. Therefore, the present substrate can support multiple cycles of use and maintain signal consistency and stability in continuous detection, thereby reducing the analysis cost and improving the long-term operation ability of the system.

[0025] 3、The application combines the metal rough film enhancement layer with the microfluidic channel, thereby constructing a dynamic flow detection environment, and can also promote the target molecules in the flowing sample to fully contact the enhancement sites on the surface of the enhancement layer, compared with the static detection mode, the efficiency and sensitivity of real-time detection are improved, and the flowing sample can be collected on-line by Raman spectrum, the technical requirements of high-throughput and automatic detection are met, and the application range of the surface enhanced Raman scattering technology in continuous monitoring field is expanded.

[0026] 4、The whole system structure of the application is simple, the manufacturing process is easy to realize, and the design of the microfluidic channel has flexibility, in addition, the prepared metal rough film enhancement layer has high light transmittance, can make the substrate compatible with high numerical aperture detection mode, therefore the collection efficiency and imaging quality of Raman signal can be further improved, and the application potential of the system in the fields of high-resolution detection and integrated microfluidic chip detection is expanded. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a schematic diagram of the traditional Raman scattering process and the surface enhanced Raman scattering process of the application, in which (a) is the traditional Raman scattering process, and (b) is the surface enhanced Raman scattering process; Figure 2 It is an experimental detection schematic diagram of the application, in which In represents an inlet, Out represents an inlet, and Molecular represents a solution to be measured; Figure 3 It is a schematic diagram of the change of Raman signal intensity in the detection-regeneration cycle test of multiple rounds of the application, in which the horizontal coordinate Cycle number represents the cycle number, the vertical coordinate Intensity (a.u.) represents the Raman signal intensity, BPT represents the Raman signal intensity measured when the biphenyl-4-thiol solution to be measured is detected, Mean (Intensity) represents the average signal intensity, and NaBH4 represents the signal intensity obtained when the substrate is washed and regenerated by sodium borohydride solution and then detected by Raman spectrum again. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be described in detail below with reference to the drawings in the specification of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0029] In order to better understand the application, the above content will be described in detail in combination with specific embodiments.

[0030] Please see the attached Figure 1 , an embodiment of the present invention provides a highly sensitive and recyclable microfluidic Raman detection substrate.

[0031] Example 1 This embodiment provides a method for preparing a highly sensitive and reusable microfluidic Raman detection substrate, comprising: Substrate pretreatment Take a glass slide (glass substrate, size 2 cm x 2 cm) and ultrasonically clean it in beakers containing acetone, anhydrous ethanol, and deionized water for 15 minutes each. After cleaning, dry the slide in a stream of nitrogen to obtain a clean, dry substrate. Store in a clean environment until ready for use.

[0032] Preparation of metal rough film reinforcement layer Fix the clean substrate treated in step 1 on the sample stage of the thermal evaporation coating equipment. Start the vacuum system and pump the background vacuum of the vacuum chamber to 5.0×10 -4 First, the evaporation source parameters were set to deposit a 1.0 nm thick copper layer on the substrate surface at a deposition rate of 0.4 Å / s. After the copper layer was deposited, the evaporation source parameters were adjusted to deposit a 10.0 nm thick gold layer on the copper layer at a deposition rate of 0.2 Å / s without disrupting the vacuum environment. After the deposition process was completed, the evaporation source was turned off, and the sample was removed from the vacuum chamber after naturally cooling to room temperature. At this point, a substrate with a metal roughness film reinforcement layer was obtained.

[0033] Microfluidic system assembly Take the microfluidic channel structure and place it on the surface of the metal rough film reinforcement layer prepared in step 2. Then, take a transparent acrylic plate of matching size as a cover plate and cover it on the microfluidic channel structure. Apply uniform pressure to the overall structure through an external clamp so that the cover plate, microfluidic channel structure and substrate fit tightly together to form a closed flow detection cavity with a fluid inlet and outlet. At this point, the preparation of the highly sensitive and recyclable microfluidic Raman detection substrate described in this embodiment is completed. At the same time, an oil immersion lens is used for detection. The specific experimental schematic diagram is shown as follows. Figure 2 As shown; Comparative Example 1: Compared with Example 1, the difference is that in step 2, during the preparation of the metal roughness film reinforcement layer, no copper layer is deposited. Instead, a 10.0 nm thick gold layer is directly deposited on the clean and dry substrate surface. The remaining preparation steps and conditions are the same as in Example 1.

[0034] Comparative Example 2: The detection substrate used in this comparative example is exactly the same as the substrate prepared in Example 1. The difference between this example and the regeneration method described in the present application is that deionized water is used as the cleaning solution instead of sodium borohydride solution in the subsequent online regeneration step.

[0035] Test Example 1 Substrate detection sensitivity performance test To verify the performance of the technical solution of the present application in terms of detection sensitivity, the detection substrate prepared in Example 1 is compared with the detection substrate prepared in Comparative Example 1.

[0036] The experimental steps are as follows: first, biphenyl-4-thiol is prepared into a low concentration test solution using anhydrous ethanol. Then, using a microsyringe pump, the test solution is injected into the microfluidic channel of the substrate of Example 1 and Comparative Example 1 at a constant flow rate, respectively.

[0037] During the solution flow, the same Raman spectrometer is used to collect spectra from the same area on the metal rough film enhancement layer of the two substrates under exactly the same instrument parameters, including laser wavelength, power, integration time and objective magnification. The signal intensity of the Raman characteristic peak of biphenyl-4-thiol at 1288 cm -1 is recorded, and each sample is measured three times. The results are recorded in Table 1.

[0038] Table 1: Comparison of Raman signal intensity of biphenyl-4-thiol characteristic peak between Example 1 and Comparative Example 1 substrates Experimental summary: From the test data in Table 1, it can be seen that under the same detection conditions, the Raman signal intensity obtained from the detection substrate prepared in Example 1 is significantly higher than that obtained from the detection substrate prepared in Comparative Example 1. This result shows that the introduction of a copper deposition layer under the gold deposition layer plays a decisive role in improving the signal enhancement effect of the substrate. The technical mechanism lies in that the pre-deposited copper layer as a topography inducing layer changes the growth mode of the subsequent gold layer, thereby constructing a nano-scale rough structure with higher density and better enhancement effect on the surface of the final metal rough film enhancement layer. These structures can produce more intense localized surface plasmon resonance, thereby greatly amplifying the Raman scattering signal of biphenyl-4-thiol molecules adsorbed thereon, achieving higher detection sensitivity.

[0039] Test Example 2 Substrate recycling performance and stability test To verify the performance of the technical solution described in the present application in terms of recycling performance, the detection substrate prepared in Example 1 is subjected to multiple rounds of detection-regeneration cycle tests.

[0040] The experimental procedure is as follows: first, inject the biphenyl-4-thiol test solution into the microfluidic channel, collect the Raman spectrum under the same spectral parameters as in Test Example 1, and record the signal intensity of the biphenyl-4-thiol characteristic peak at 1288 cm -1 Subsequently, inject the sodium borohydride solution into the channel as a cleaning solution, and continuously flush at a constant flow rate for a certain period of time to complete the online regeneration of the substrate.

[0041] After the regeneration step is completed, inject deionized water to flush away the residual cleaning solution. After completing the above steps, inject the biphenyl-4-thiol test solution again for the second detection. This "detection-regeneration-flushing" process is repeated 10 times, and the signal intensity detected in each cycle is recorded. The results are shown in Table 2.

[0042] Table 2: Substrate cycle performance test data Experimental summary: As can be seen from the test data in Table 2 (as shown in Figure 3 After 10 detection-regeneration cycles, the Raman signal intensity remains above 95% of the initial intensity, without significant attenuation, showing high signal consistency and detection stability of the system. This result confirms the high efficiency of the regeneration method. The technical mechanism is that the sodium borohydride solution can effectively break the chemical bonds formed by the biphenyl-4-thiol molecules adsorbed on the surface of the gold deposition layer through a chemical substitution reaction, thereby completely removing the target molecules from the surface of the enhancement layer. This regeneration process can restore the detection activity of the surface of the enhancement layer without damaging the nanostructure of the metal rough film, thus ensuring that the substrate can still provide stable and reliable Raman signal enhancement effect in multiple continuous uses, solving the technical problem of the difficulty of recycling of traditional substrates.

[0043] Test Example 3 Memory effect inhibition effect test To verify the effectiveness of the regeneration method described in the present application in inhibiting signal residues, this test was performed. Two completely identical probe substrates prepared according to Example 1 were taken, and were labeled as Group A and Group B, respectively.

[0044] The experimental steps are as follows: First, the test solution of biphenyl-4-thiol of the same concentration is injected into the microfluidic channels of the substrates of Group A and Group B respectively, and the spectrum is collected. Subsequently, the two groups of substrates are cleaned and regenerated: Group A corresponds to Example 1 (using sodium borohydride solution as the cleaning liquid), and Group B corresponds to Example 2 (using deionized water as the cleaning liquid). After the cleaning and regeneration steps are completed, a pure solvent (anhydrous ethanol) that does not contain biphenyl-4-thiol is injected into the microfluidic channels of the two substrates. Under the same instrument parameters as the initial detection, the Raman spectrum of the enhanced layer surface of the two groups of substrates is collected again, and the 1288cm -1 The residual signal intensity of the characteristic peak of biphenyl-4-thiol at 400 nm is recorded in Table 3.

[0045] Table 3: Memory effect suppression performance test data Experimental summary: As shown in the test data in Table 3, the signal intensity of the biphenyl-4-thiol characteristic peak in the subsequent spectra of Group A substrates cleaned with sodium borohydride solution has been reduced to the background noise level, and no effective signal is observed. In contrast, the signal intensity of the biphenyl-4-thiol characteristic peak in the subsequent spectra of Group B substrates cleaned only with deionized water is still significantly detected. This result clearly demonstrates that the regeneration method adopted by the present invention can effectively remove surface-adsorbed molecules, thereby completely suppressing the memory effect.

[0046] The technical mechanism of this phenomenon lies in the fact that biphenyl-4-thiol molecules form a strong chemical adsorption bond with the surface of the gold deposit through their sulfur atoms. Conventional solvent rinses (such as those in Group B) do not provide sufficient energy to break this chemical bond, resulting in residual molecules. The sodium borohydride solution employed in this invention, however, triggers a chemical substitution reaction on the surface of the gold deposit. This reaction actively destroys the chemical bond between the target molecule and the gold surface, completely removing the target molecule from the enhancement layer surface, restoring the substrate surface to a clean state and providing interference-free surface conditions for subsequent precise detection.

[0047] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A highly sensitive and recyclable microfluidic Raman detection substrate, characterized in that: include: substrate; A metal rough film enhancement layer is provided on the surface of the substrate, wherein the enhancement layer comprises a copper deposition layer and a gold deposition layer, and is used to provide surface enhanced Raman scattering sites for target molecules; a microfluidic channel disposed on the surface of the metal rough film reinforcement layer, the microfluidic channel having at least one inlet and one outlet for guiding a liquid sample to flow through the surface of the reinforcement layer; The copper deposition layer is located between the substrate and the gold deposition layer, and the metal rough film reinforcement layer has light transmittance; The thickness of the copper deposited layer is 0.9-1.1 nm, and the thickness of the gold deposited layer is 9.5-10.5 nm; The method for online detection and regeneration of the substrate comprises the following steps: Detection step: a test solution containing target molecules is input through the inlet of the microfluidic channel, the target molecules are adsorbed on the surface of the metal rough film reinforcement layer during the flow process, and the target molecules are detected in real time using a Raman spectrometer; Regeneration step: After the detection step is completed, sodium borohydride solution is input as a cleaning solution through the inlet of the microfluidic channel to remove the target molecules adsorbed on the surface of the enhancement layer, thereby achieving online regeneration of the substrate.

2. The highly sensitive and recyclable microfluidic Raman detection substrate according to claim 1, characterized in that: The substrate is a transparent substrate in the optical visible light band, and the transparent substrate in the optical visible light band is a glass sheet.

3. The highly sensitive and recyclable microfluidic Raman detection substrate according to claim 1, characterized in that: The metal rough film reinforcement layer is prepared by a thermal evaporation process.

4. The highly sensitive and recyclable microfluidic Raman detection substrate according to claim 1, characterized in that: The microfluidic channel is configured as a curved channel.

5. The highly sensitive and recyclable microfluidic Raman detection substrate according to claim 1, characterized in that: It also includes a transparent cover plate for sealing the microfluidic channel to form a closed flow detection cavity.

6. The highly sensitive and recyclable microfluidic Raman detection substrate according to claim 1, characterized in that: After the regeneration step is completed, the detection step is repeated to perform the next round of Raman spectroscopy detection using the regenerated substrate.

7. The highly sensitive and recyclable microfluidic Raman detection substrate according to claim 1, characterized in that: In the regeneration step, the sodium borohydride solution destroys the chemical adsorption between the target molecules and the surface of the enhancement layer through a substitution reaction, thereby removing the target molecules.

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