Highly sensitive recyclable microfluidic raman probing substrate

Abstract

The application relates to the field of nanophotonic fluid control technology, and discloses a high-sensitivity recyclable microfluidic Raman detection substrate, which comprises a substrate, a metal rough film enhancement layer arranged on the surface of the substrate, the enhancement layer comprising a copper deposition layer and a gold deposition layer, and being used for providing a surface-enhanced Raman scattering site for target molecules; and a microfluidic channel arranged on the surface of the metal rough film enhancement layer, the microfluidic channel having at least one inlet and one outlet, and being used for guiding a liquid sample to flow through the surface of the enhancement layer. The metal rough film enhancement layer formed by depositing the copper layer and the gold layer on the substrate has a nanoscale rough structure on the surface, high-density local surface plasmon resonance hot spots are generated, the Raman scattering signal of the target molecules adsorbed on the surface is greatly enhanced, and therefore, the detection sensitivity can be obviously improved, and the effective detection demand for ultratrace 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:

[0004] (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.

[0005] (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.

[0006] (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.

[0007] (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.

[0008] (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

[0009] 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.

[0010] To achieve the above object, the present application is realized by the following technical scheme:

[0011] 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.

[0012] 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 structures can produce a localized surface plasmon resonance (LSPR) effect, providing high-intensity Raman signal enhancement sites for target molecules adsorbed thereon.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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).

[0017] In a specific embodiment, the metal rough film enhancement layer is formed by a thermal evaporation process.

[0018] In a specific embodiment, the shape of the microfluidic channel is set as a curved channel.

[0019] In a specific embodiment, the substrate further comprises a transparent cover plate. The transparent cover plate covers the microfluidic channel and seals the microfluidic channel, thereby forming a closed flow detection cavity together with the surface of the metal rough film enhancement layer.

[0020] The second aspect of the present application provides an online detection and regeneration method using the microfluidic Raman detection substrate described above. The method comprises a detection step and a regeneration step.

[0021] The detection step specifically comprises: inputting a solution to be tested 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.

[0022] 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.

[0023] The technical mechanism of this regeneration step is that the sodium borohydride solution can undergo a substitution reaction with the surface of the gold deposition layer, which will 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 detached 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.

[0024] In an 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 realizing the multiple cycles of the substrate.

[0025] The present application provides a high-sensitivity recyclable microfluidic Raman detection substrate. It has the following advantages:

[0026] 1. The metal rough film enhancement layer formed by depositing a copper layer and a gold layer on the substrate utilizes 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.

[0027] 2. This invention uses sodium borohydride solution as a cleaning medium to initiate a substitution reaction on the gold deposition layer surface, thereby breaking the chemical bonds formed between the adsorbed target molecules and the surface. This process effectively removes residual molecules from the surface, thus suppressing the memory effect commonly found on conventional SERS substrates. Therefore, this substrate can support multiple cycles of use and maintain signal consistency and stability during continuous detection, thereby reducing analysis costs and improving the long-term operational capability of the system.

[0028] 3. This invention combines a rough metal film reinforcement layer with a microfluidic channel to create a dynamic flow detection environment. It also promotes full contact between target molecules in the flowing sample and the reinforcement sites on the surface of the reinforcement layer. Compared with static detection methods, it improves the efficiency and sensitivity of real-time detection and enables online Raman spectroscopy acquisition of flowing samples, meeting the technical requirements of high-throughput and automated detection and expanding the application scope of surface-enhanced Raman scattering technology in the field of continuous monitoring.

[0029] 4. The overall system structure of this invention is simple, the manufacturing process is easy to implement, and the design of the microfluidic channel is flexible. In addition, the prepared metal rough film reinforcement layer has high light transmittance, which allows the substrate to be compatible with detection methods using high numerical aperture. Therefore, it can further improve the acquisition efficiency and imaging quality of Raman signals, and expand the application potential of this system in high-resolution detection and integrated microfluidic chip detection. Attached Figure Description

[0030] Figure 1 The diagram shows the conventional Raman scattering process and the surface-enhanced Raman scattering process of the present invention. In the diagram, (a) is the conventional Raman scattering process and (b) is the surface-enhanced Raman scattering process.

[0031] Figure 2 This is a schematic diagram of the experimental detection of the present invention. In the diagram: In represents the inlet, Out represents the inlet, and Molecular represents the solution to be tested.

[0032] Figure 3 This is a schematic diagram of the Raman signal intensity changes during multiple rounds of detection-regeneration cycle testing according to the present invention. In the figure: the horizontal axis Cycle number represents the number of cycles, the vertical axis Intensity (au) represents the Raman signal intensity, BPT represents the Raman signal intensity measured when the biphenyl-4-thiol test solution 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 subjected to Raman spectroscopy detection again. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] To better understand the present invention, the above content will be described in detail below with reference to specific embodiments.

[0035] Please see the appendix Figure 1 The present invention provides a highly sensitive and reusable microfluidic Raman detection substrate.

[0036] Example 1

[0037] This embodiment provides a method for fabricating a highly sensitive, recyclable microfluidic Raman detection substrate, including:

[0038] Substrate pretreatment

[0039] Take a glass slide (glass substrate, size 2cm × 2cm) and place it sequentially into beakers containing acetone, anhydrous ethanol, and deionized water, and perform ultrasonic cleaning for 15 minutes in each. After cleaning, place the slide in a nitrogen stream to dry it, obtaining a clean and dry substrate, and store it in a clean environment for later use.

[0040] Preparation of metal roughened film reinforcement layer

[0041] The clean substrate treated in step 1 is fixed onto the sample stage of the thermal evaporation coating equipment. The vacuum system is activated, and the background vacuum level of the vacuum chamber is evacuated to 5.0 × 10⁻⁶. -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 deposition was complete, without disrupting the vacuum environment, the evaporation source parameters were adjusted to deposit a 10.0 nm thick gold layer on the copper layer surface at a deposition rate of 0.2 Å / s. 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 roughening film reinforcement layer on its surface was obtained.

[0042] Microfluidic system assembly

[0043] A microfluidic channel structure is placed on the surface of the metal roughened film reinforcement layer prepared in step 2. Then, a transparent acrylic plate of matching size is used as a cover plate and placed over the microfluidic channel structure. Uniform pressure is applied to the entire structure using external clamps, ensuring a tight fit between the cover plate, the microfluidic channel structure, and the substrate, forming a closed flow detection chamber with a fluid inlet and outlet. This completes the fabrication of the highly sensitive, reusable microfluidic Raman detection substrate described in this embodiment. Simultaneously, detection is performed using an oil immersion lens; a schematic diagram of the experiment is shown below. Figure 2 As shown;

[0044] Comparative Example 1:

[0045] Compared to Example 1, the difference lies in that: in the preparation of the metal roughening film reinforcement layer in step 2, no copper layer is deposited; instead, a gold layer with a thickness of 10.0 nm is directly deposited on the clean and dry substrate surface. The remaining preparation steps and conditions are the same as in Example 1.

[0046] Comparative Example 2:

[0047] The probe substrate used in this comparative example is exactly the same as the substrate prepared in Example 1. The difference between this and the regeneration method described in this invention is that deionized water is used as the cleaning solution in the subsequent online regeneration step, instead of the sodium borohydride solution.

[0048] Test Example 1

[0049] Substrate detection sensitivity performance test

[0050] To verify the performance of the technical solution of the present invention in terms of detection sensitivity, the detection substrate prepared in Example 1 and the detection substrate prepared in Comparative Example 1 were compared and tested.

[0051] The experimental procedure is as follows: First, biphenyl-4-thiol was prepared into a low-concentration test solution using anhydrous ethanol. Then, the test solution was injected into the microfluidic channels of the substrates of Example 1 and Comparative Example 1 at a constant flow rate using a microinjection pump.

[0052] During solution flow, the same Raman spectrometer was used with identical instrument parameters (laser wavelength, power, integration time, and objective magnification) to acquire spectra of the same region on the metal roughened film reinforcement layer of both substrates. Biphenyl-4-thiol was recorded at 1288 cm⁻¹. -1 The signal intensity of the Raman characteristic peak was measured three times for each sample, and the results are recorded in Table 1.

[0053] Table 1: Comparison of Raman signal intensity of characteristic peaks of p-biphenyl-4-thiol in the substrates of Example 1 and Comparative Example 1

[0054]

[0055] Experiment Summary:

[0056] As shown in Table 1, under identical detection conditions, the Raman signal intensity obtained from the detection substrate prepared in Example 1 was significantly higher than that obtained from the detection substrate prepared in Comparative Example 1. This result indicates that introducing a copper deposition layer beneath the gold deposition layer plays a decisive role in enhancing the signal enhancement effect of the substrate. The underlying mechanism is that the pre-deposited copper layer acts as a morphology-inducing layer, altering the growth pattern of the subsequent gold layer. This results in the formation of a nanoscale rough structure with higher density and superior enhancement effect on the surface of the final metal rough film enhancement layer. These structures can generate stronger localized surface plasmon resonances, thereby significantly amplifying the Raman scattering signal of biphenyl-4-thiol molecules adsorbed on them, achieving higher detection sensitivity.

[0057] Test Example 2

[0058] Baseboard Cyclic Performance and Stability Testing

[0059] To verify the performance of the technical solution described in this invention in terms of recyclability, the detection substrate prepared in Example 1 was subjected to multiple rounds of detection-regeneration cycle tests.

[0060] The experimental procedure is as follows: First, the biphenyl-4-thiol test solution was injected into the microfluidic channel, and Raman spectra were acquired under the same spectrometer parameters as in Test Example 1. The concentration of biphenyl-4-thiol at 1288 cm⁻¹ was recorded. -1 The signal intensity of the characteristic peak is shown; this is the detection result of the first cycle. Subsequently, sodium borohydride solution is injected into the channel as a cleaning solution, and the substrate is continuously rinsed at a constant flow rate for a certain period of time to complete the online regeneration of the substrate.

[0061] After the regeneration step is completed, deionized water is injected to rinse away any residual cleaning solution. After completing the above steps, the biphenyl-4-thiol test solution is injected again for a second detection. This "detection-regeneration-rinse" process is repeated 10 times, and the signal intensity detected in each cycle is recorded. The results are shown in Table 2.

[0062] Table 2: Base Cyclic Performance Test Data

[0063]

[0064] Experiment Summary:

[0065] As can be seen from the test data in Table 2 (e.g.) Figure 3As shown in the figure, after 10 detection-regeneration cycles, the Raman signal intensity remained above 95% of the initial intensity without significant attenuation, demonstrating the system's high signal consistency and detection stability. This result confirms the high efficiency of the regeneration method. The technical mechanism lies in the fact that sodium borohydride solution can effectively break the chemical bonds formed by 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 reinforcement layer. This regeneration process can restore the detection activity of the reinforcement layer surface without damaging the nanostructure of the rough metal film, thus ensuring that the substrate can still provide a stable and reliable Raman signal enhancement effect during multiple rounds of continuous use, solving the technical problem of the difficulty in recycling traditional substrates.

[0066] Test Example 3

[0067] Memory effect inhibition test

[0068] To verify the effectiveness of the regeneration method described in this invention in suppressing signal residue, this test was conducted. Two identical detector substrates prepared according to Example 1 were taken and labeled as Group A and Group B, respectively.

[0069] The experimental steps are as follows: First, a biphenyl-4-thiol test solution of the same concentration was injected into the microfluidic channels of the substrates in groups A and B, respectively, and spectral acquisition was completed. Then, the two substrates were cleaned and regenerated: group A corresponded to Example 1 (using sodium borohydride solution as the cleaning solution), and group B corresponded to Example 2 (using deionized water as the cleaning solution). After the cleaning and regeneration steps were completed, pure solvent (anhydrous ethanol) without biphenyl-4-thiol was injected into the microfluidic channels of both substrates. Under the same instrument parameters as the initial detection, Raman spectra were again acquired on the surface of the reinforcement layer of both substrates, and the readings were recorded at 1288 cm⁻¹. -1 The residual signal intensity of the characteristic peak of biphenyl-4-thiol is recorded in Table 3.

[0070] Table 3: Test data on the performance of memory effect inhibition

[0071]

[0072] Experiment Summary:

[0073] As shown in Table 3, the test data of Group A substrates, after being cleaned with sodium borohydride solution, revealed that the signal intensity of the biphenyl-4-thiol characteristic peak in the subsequent spectra had decreased to the background noise level, with no effective signal residue observed. In contrast, the Group B substrates, cleaned only with deionized water, still showed a significant biphenyl-4-thiol characteristic peak signal in their subsequent spectra. This result clearly demonstrates that the regeneration method employed in this invention can effectively remove surface-adsorbed molecules, thereby completely suppressing the memory effect.

[0074] The technical mechanism of this phenomenon lies in the fact that biphenyl-4-thiol molecules form stable chemical adsorption bonds with the gold deposition layer surface through sulfur atoms. Conventional solvent rinsing (such as group B) cannot provide sufficient energy to break these chemical bonds, resulting in molecular residue. The sodium borohydride solution used in this invention can initiate a chemical substitution reaction on the gold deposition layer surface. This reaction can actively break the chemical bonds between the target molecules and the gold surface, thereby completely removing the target molecules from the reinforcement layer surface, restoring the substrate surface to a clean state, and providing interference-free surface conditions for subsequent accurate detection.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An on-line detection and regeneration method of a high-sensitivity recyclable microfluidic Raman probing substrate, characterized in that, The application relates to a substrate, a metal rough film enhancement layer, a microfluidic channel, and a method for on-line detection and regeneration of the substrate. The substrate is provided with a metal rough film enhancement layer on the surface, which comprises a copper deposition layer and a gold deposition layer for providing surface enhanced Raman scattering sites for target molecules. The microfluidic channel is provided on the surface of the metal rough film enhancement layer, and has at least one inlet and one outlet for guiding liquid sample to flow through the surface of the enhancement layer. The copper deposition layer is located between the substrate and the gold deposition layer, and the metal rough film enhancement layer is transparent. The thickness of the copper deposition layer is 0.9-1.1 nm, and the thickness of the gold deposition layer is 9.5-10.5 nm. The method for on-line detection and regeneration of the substrate comprises the following steps: The detection step is that a solution to be detected containing target molecules is input through the inlet of the microfluidic channel, so that the target molecules are adsorbed on the surface of the metal rough film enhancement layer during the flow process, and the target molecules are detected in real time by using a Raman spectrometer. The regeneration step is that after the detection step is completed, a sodium borohydride solution is input as a cleaning liquid through the inlet of the microfluidic channel, so that the target molecules adsorbed on the surface of the enhancement layer are removed, and the on-line regeneration of the substrate is realized. The substrate is a transparent substrate in the optical visible light wave band.

2. The method of claim 1, wherein the method is characterized by, The transparent substrate in the optical visible light wave band is a glass sheet.

3. The method of claim 2, wherein the method further comprises: The metal rough film enhancement layer is prepared by a thermal evaporation process.

4. The method of claim 1, wherein the method is characterized by, The microfluidic channel is provided in a curved type.

5. The method of claim 1, wherein the method is characterized by, A transparent cover plate is further provided for sealing the microfluidic channel to form a closed flow detection cavity.

6. The method of claim 1, wherein the method is characterized by, After the regeneration step is completed, the detection step is repeated, so that the regenerated substrate is used for the next round of Raman spectrum detection.

7. The method of claim 1, wherein the method is characterized by, 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, so that the target molecules are removed.

8. The method of claim 1, wherein the method is characterized by, ​

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