Wide pH tolerant microdroplet sers real-time detection system and method

Abstract

The application discloses a wide pH tolerance microdroplet SERS real-time detection system and method, and belongs to the field of surface enhanced Raman scattering detection. The system comprises a sample feeding unit, a microfluidic chip, a Raman excitation and collection unit and a signal acquisition and analysis unit. The sample feeding unit introduces a sample to be detected, a SERS enhancement composite material component and an oil phase continuous phase into the chip; core-shell particles are mixed with polyvinylpyrrolidone to form a wide pH tolerance SERS enhancement composite material composed of a silver nano core, a silica shell layer and a polyvinylpyrrolidone protective layer. The microfluidic chip is sequentially provided with a mixing unit, a droplet generation area and a droplet in-situ capture and SERS detection area along a flow direction; the sample and the composite material form a dispersed phase in the mixing unit, the microdroplets are generated by the intersection of the dispersed phase and the continuous phase in the droplet generation area, and the detection area is provided with a variable cross-section channel to make the droplets stably stay. The problems of easy corrosion and aggregation of a silver-based substrate under pH fluctuation, unstable signal, active regulation of pH to change the original environment of the sample and insufficient integration are solved.

Description

Technical Field

[0001] This invention relates to the field of surface-enhanced Raman scattering (SERS) detection materials and technology, specifically to a wide pH-tolerant microdroplet SERS real-time detection system and method. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) has advantages such as high sensitivity, fingerprint recognition, fast response and in-situ detection. When combined with droplet microfluidics, the sample to be tested can be divided into a large number of independent micro-reaction units, realizing small-volume, high-throughput online analysis. Therefore, it is suitable for real-time detection of metabolites in microbial fermentation processes.

[0003] However, the pH of complex biological systems such as fermentation broth fluctuates significantly during the cultivation process. Although commonly used silver nanoparticles have strong SERS enhancement capabilities, they are prone to surface state changes, corrosion, and particle aggregation when the acid and alkaline environment changes. This leads to changes in the dispersion state and effective hot spots of the enhanced substrate, which in turn causes signal drift, decreased reproducibility, or even detection failure of the same detection system under different pH conditions.

[0004] Existing technologies typically improve stability by externally adjusting the pH of the droplets or samples, or by replacing silver nanoparticles with other noble metal materials. The former requires additional electrochemical regulation, buffer systems, or proton transfer processes, altering the original chemical environment of the sample and making it difficult to synchronize with high-throughput droplet detection; the latter, while improving chemical stability, often reduces SERS enhancement capabilities. Meanwhile, in existing microdroplet SERS platforms, sample mixing, droplet generation, droplet positioning, and Raman detection are often completed in multiple independent stages, making the system highly sensitive to optical path stability, droplet residence time, and signal collection efficiency.

[0005] Therefore, there is an urgent need for a real-time microdroplet SERS detection system that can integrate a wide pH stable SERS signal source, rapid mixing, uniform droplet generation, automatic capture, reflection enhancement optical path, and spectral acquisition and analysis without actively adjusting the sample pH, so as to improve the stability, throughput, and practicality of detection in complex biological environments. Summary of the Invention

[0006] To address the problems of existing microdroplet SERS detection methods, such as easy corrosion and aggregation of silver-based substrates and unstable detection signals under pH fluctuations, as well as the fact that existing active pH control methods can alter the original sample environment and have insufficient system integration, this invention provides a wide pH-tolerant SERS-enhanced composite material, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, this invention discloses a wide pH-tolerant microdroplet SERS real-time detection system, comprising a sample introduction unit, a microfluidic chip, a Raman excitation and collection unit, and a signal acquisition and analysis unit; the sample introduction unit is used to introduce the sample to be tested, the SERS-enhanced composite material component, and an oil phase as a continuous phase into the microfluidic chip; wherein... Core-shell particles and polyvinylpyrrolidone (PVP) are mixed in contact to form a wide pH-tolerant SERS-enhanced composite material. This composite material comprises, from the inside out, a silver nanocore, a silica shell, and a PPVP protective layer. The microfluidic chip is sequentially configured with a mixing unit, a droplet generation region, and a droplet in-situ capture and SERS detection region along the flow direction. The sample to be tested and the SERS-enhanced composite material components are mixed in the mixing unit to form a dispersed phase. The dispersed phase and the continuous phase converge in the droplet generation region to form microdroplets. The droplet in-situ capture and SERS detection region is equipped with a variable cross-section channel structure for stable microdroplet residence. The flow channel layer of the microfluidic chip is bonded to a reflector substrate. The Raman excitation and collection unit focuses excitation light onto the microdroplets in the droplet in-situ capture and SERS detection region and collects the Raman scattered light generated by the microdroplets. The signal acquisition and analysis unit acquires the SERS spectrum corresponding to the Raman scattered light and performs qualitative or quantitative analysis of the analyte based on characteristic Raman peaks.

[0009] In the above technical solution, a wide pH stable SERS signal source, rapid mixing, uniform droplet generation, automatic capture, reflection-enhancing optical path, and spectral acquisition and analysis are integrated into a continuous, unified detection chain. Silver nanocore-silica The SERS-enhanced composite material formed by contact mixing of core-shell particles and polyvinylpyrrolidone maintains stable SERS enhancement activity under fluctuating pH conditions without the need for external electrochemical regulation, buffer systems, or proton transfer processes. This enables high-sensitivity and high-reproducibility detection of analytes in complex biological systems without altering the original chemical environment of the sample. The microfluidic chip integrates mixing, droplet generation, and in-situ detection onto a single chip, avoiding optical path shift, droplet loss, and signal fluctuations introduced by independent operations in multiple stages, significantly improving system integration and detection reliability.

[0010] In one embodiment, the contact mixing is performed in one of the following ways:

[0011] i. will Core-shell particles are dispersed in an aqueous phase to form a core-shell particle dispersion. The core-shell particle dispersion is mixed with an aqueous solution of polyvinylpyrrolidone and incubated at room temperature for a preset time to form a SERS-reinforced composite material dispersion. The sample injection unit is equipped with three injection ports, which are used to deliver the sample to be tested, the SERS-reinforced composite material dispersion, and the oil phase, respectively.

[0012] ii. The sample introduction unit is equipped with four sample inlets, which are used to deliver the sample to be tested, polyvinylpyrrolidone aqueous solution, and so on, respectively. Core-shell particle dispersion and oil phase; the polyvinylpyrrolidone aqueous solution and the... The core-shell particle dispersion is contact-mixed online in the mixing unit to form a wide pH-tolerant SERS-reinforced composite material in situ.

[0013] In the above technical solution, method i involves premixing and static incubation to allow polyvinylpyrrolidone to... The core-shell particles fully adsorb and form a stable steric hindrance protective layer on their surface, resulting in a uniform composition and good batch-to-batch consistency in the SERS-reinforced composite dispersion. This makes it suitable for quantitative detection scenarios with high requirements for composite material maturity. Furthermore, only three inlet ports are needed, resulting in a relatively simple chip flow channel structure. Method ii introduces the polyvinylpyrrolidone aqueous solution and the core-shell particle dispersion as independent aqueous phases into the online mixing unit, achieving instantaneous contact mixing within the chip. This eliminates the need for premixing and incubation steps, shortening the time from sample introduction to result detection. This method is more suitable for high-throughput online detection scenarios requiring rapid response. Moreover, the contact time and mixing degree of the two components in the mixing channel can be precisely controlled by the flow rate, facilitating controllable composite material formation.

[0014] In one embodiment, the mixing unit includes an aqueous inlet, a premixing zone, and a mixing channel communicating with the premixing zone;

[0015] When mode i is used, the mixing unit includes two aqueous phase inlets, and the sample to be tested and the SERS-reinforced composite material dispersion enter through the two aqueous phase inlets and are mixed in the mixing channel.

[0016] When mode ii is used, the mixing unit includes three aqueous inlets, wherein the sample to be tested, the polyvinylpyrrolidone aqueous solution, and... The core-shell particle dispersion enters through the three aqueous inlets and is rapidly mixed in the mixing channel;

[0017] The droplet generation region is a T-shaped droplet generation structure;

[0018] The droplet in-situ capture and SERS detection area includes a circular detection cavity and a variable cross-section channel connected thereto, so that microdroplets entering the detection area are automatically captured and stably remain during Raman exposure;

[0019] The reflector substrate is used to reflect the downward-propagating Raman scattered light back to the upward collection optical path, thereby improving the effective collection efficiency of the Raman signal.

[0020] In the above technical solution, the mixing unit, through the cooperation of multiple aqueous phase inlets and mixing channels, enables the sample to be mixed rapidly and uniformly with the SERS-reinforced composite material components before droplet formation, avoiding signal fluctuations caused by insufficient mixing. The T-shaped droplet formation structure utilizes the shearing effect of the continuous relative dispersed phase to generate microdroplets of uniform size and controllable spacing, ensuring the representativeness of the analyte concentration in each droplet and the consistency of the SERS signal in subsequent detection. The in-situ droplet capture and SERS detection area, through the cooperation of a circular detection cavity and a variable cross-section channel, utilizes the abrupt change in fluid resistance to automatically decelerate and stabilize the microdroplets in the detection cavity, providing a sufficient and stable time window for Raman exposure. Passive droplet capture can be achieved without the need for external valves or active control mechanisms, simplifying the system structure and improving detection throughput. The reflector substrate reflects the downward-propagating Raman scattered light back to the upper collection optical path, allowing the scattered light that was originally absorbed by the substrate or lost through transmission to be collected again by the objective lens, effectively improving the collection efficiency and signal-to-noise ratio of the Raman signal and enhancing the system's ability to detect low-concentration analytes.

[0021] In one embodiment, the average particle size of the silver nanonuclei is 40-80 nm, the thickness of the silica shell is 2-43 nm, and the average molecular weight of the polyvinylpyrrolidone is 8000-58000. The polyvinylpyrrolidone protective layer is adsorbed on the outer surface of the silica shell to form a steric barrier. The steric barrier and the silica shell work together to inhibit the corrosion and aggregation of the silver nanonuclei under pH fluctuation conditions, so that the wide pH tolerant SERS-enhanced composite material maintains SERS-enhancing activity in the pH range of 3-11.

[0022] In the above technical solution, the average particle size of the silver nanonuclei is controlled within the range of 40-80 nm, which can maintain good colloidal dispersion while ensuring strong local surface plasmon resonance effect and SERS enhancement capability. The thickness of the silica shell is controlled within the range of 2-43 nm, which provides an effective physical barrier for the silver nanonuclei to prevent direct erosion of the silver nuclei by external acid and alkaline environments, and does not significantly attenuate the electromagnetic field enhancement effect due to excessive shell thickness. The polyvinylpyrrolidone protective layer is adsorbed on the outer surface of the silica shell to form a steric barrier, further inhibiting close contact and aggregation between particles. This synergistic protection with the physical isolation effect of the silica shell allows the composite material to maintain structural integrity and SERS enhancement activity within a wide pH range of 3-11, eliminating the need for pH adjustment of the sample before detection and preserving the original chemical state of the analyte.

[0023] In one embodiment, the Raman excitation and collection unit includes a laser, a first collimator, a first filter, a dichroic mirror, an objective lens, a second collimator, a second filter, and a Raman spectrometer. The excitation light output from the laser is collimated by the first collimator and stray light is filtered out by the first filter before being incident on the dichroic mirror. The light is reflected by the dichroic mirror to the objective lens, which focuses it onto the microdroplet in the in-situ droplet capture and SERS detection area. The Raman scattered light generated by the microdroplet is collected by the objective lens, transmitted through the dichroic mirror, collimated by the second collimator, and Rayleigh scattered light is filtered out by the second filter before being introduced into the Raman spectrometer for spectral acquisition.

[0024] The microfluidic chip is fixed on a three-dimensional displacement stage to adjust the relative position of the objective lens focus and the droplet in-situ capture and SERS detection area; the signal acquisition and analysis unit is electrically connected to the Raman spectrometer to perform characteristic peak identification, qualitative discrimination and quantitative analysis on the acquired SERS spectrum, and to display the corresponding spectrum in real time on the display terminal.

[0025] In the above technical solution, a first collimator and a first filter are set in the excitation optical path to ensure that the excitation light incident on the dichroic mirror has good collimation and monochromaticity, reducing the interference of stray light on the SERS signal. A second collimator and a second filter are set in the scattering optical path to effectively filter out Rayleigh scattering light and improve the collimation of Raman scattering light, thereby improving the signal-to-noise ratio and resolution of the spectrum. The dichroic mirror realizes the spatial separation of the excitation light and the scattered light, allowing excitation and collection to share the same objective lens without interference, simplifying the optical path structure. The microfluidic chip is fixed on a three-dimensional displacement stage, which can finely adjust the relative position of the objective lens focus and the droplet capture area to ensure that the excitation light is precisely focused on the center of the microdroplet, obtaining the best excitation efficiency and signal intensity. The signal acquisition and analysis unit displays the corresponding spectrum in real time. Once the microdroplet leaves the in-situ droplet capture area, the corresponding spectrum disappears automatically, intuitively reflecting the in-situ nature and reliability of signal acquisition, and facilitating real-time monitoring of the detection process by operators.

[0026] Secondly, the present invention provides a method for real-time detection of wide pH-tolerant microdroplet SERS, based on the aforementioned real-time detection system for wide pH-tolerant microdroplet SERS, comprising:

[0027] The sample to be tested and the SERS-reinforced composite material component were introduced into the microfluidic chip, so that... Core-shell particles and polyvinylpyrrolidone are mixed in contact to form a wide pH-tolerant SERS-reinforced composite material, which allows the wide pH-tolerant SERS-reinforced composite material to be in full contact with the analyte;

[0028] The mixed dispersed phase and the continuous phase converge in the droplet formation region to form microdroplets of uniform size;

[0029] The microdroplets are brought into the in-situ droplet capture and SERS detection zone, and the microdroplets are automatically captured and stably held by the variable cross-section channel structure.

[0030] Excitation light is irradiated onto the captured microdroplets, and the Raman scattering signals generated by the microdroplets are collected. Qualitative identification is performed based on the characteristic Raman peaks of the analyte, or quantitative analysis is performed based on a standard curve. After the detection is completed, the microdroplets are discharged from the chip outlet.

[0031] In the above technical solution, this method integrates the formation of SERS-reinforced composite materials, the mixing of analytes, the generation and capture of microdroplets, and Raman detection into a continuous online process. Each step is completed sequentially within the microfluidic chip, eliminating the need for sample transfer or environmental switching between steps, thus avoiding external contamination and signal loss. The wide pH-tolerant SERS-reinforced composite material eliminates the need for additional sample pH adjustment during detection, maintaining the original chemical environment of the analyte. This makes it particularly suitable for online detection of complex biological systems such as microbial fermentation broths where pH fluctuates during the cultivation process. After detection, the microdroplets are automatically discharged from the chip outlet, freeing up detection space for subsequent droplets and enabling continuous, high-throughput real-time detection.

[0032] In one embodiment, the contact mixing is performed in one of the following ways:

[0033] i. will Core-shell particles are dispersed in an aqueous phase to form a core-shell particle dispersion. The core-shell particle dispersion is mixed with a polyvinylpyrrolidone aqueous solution and incubated at room temperature for a preset time to form a SERS-reinforced composite material dispersion. Then, the dispersion is input into a microfluidic chip along with the sample to be tested.

[0034] ii. Combine the sample to be tested, polyvinylpyrrolidone aqueous solution, and... The core-shell particle dispersions are input as independent aqueous phases into the microfluidic chip, where they are mixed online in the mixing unit to form a wide pH-tolerant SERS-reinforced composite material in situ.

[0035] In the above technical solutions, method i's premixing and incubation allows for the full adsorption of polyvinylpyrrolidone on the surface of core-shell particles, resulting in a composite material dispersion with good stability and batch-to-batch consistency, making it suitable for detection tasks requiring high quantitative accuracy. Method ii's online mixing eliminates the premixing step, completing the entire process from sample introduction to composite material formation and droplet generation continuously within the chip, shortening the detection cycle and making it more suitable for online monitoring scenarios requiring rapid response and continuous sample introduction. Both methods can be flexibly selected according to actual detection needs, enhancing the applicability of the method.

[0036] In one embodiment, the The preparation of core-shell particles includes: preparation of silver nanonuclei: adding a reducing agent to an aqueous solution of soluble silver salts, heating under reflux to obtain silver nanonuclei colloids; coating with a silica shell: centrifuging and concentrating the obtained silver nanonuclei colloids, dispersing them in an alcohol solvent, adding an alkaline catalyst, then adding a silicon source precursor, stirring the reaction at room temperature, and centrifuging to obtain... Core-shell particles.

[0037] In the above technical solution, silver nanonuclei are prepared using a chemical reduction method, a mature and simple process. By controlling the amount of reducing agent and the reflux time, silver nanonuclei with uniform particle size and regular morphology can be obtained. The silica shell is coated in an alcohol solvent using a sol-gel method under mild reaction conditions (room temperature), avoiding the damage to the morphology and plasmon resonance characteristics of the silver nanonuclei caused by high temperatures. The thickness of the silica shell can be precisely controlled by adjusting the amount of silicon source precursor added, thus achieving a balance between physical isolation effect and SERS enhancement efficiency. The entire preparation process does not require complex equipment and is suitable for batch preparation.

[0038] In one embodiment, the soluble silver salt is silver nitrate, and the reducing agent is trisodium citrate; the concentration of the soluble silver salt aqueous solution is 0.5~5mM, and 2~8mL of trisodium citrate solution with a mass fraction of 0.5~2wt% is added to every 200mL of the solution, and the heating and reflux time is 30~120min; the alkaline catalyst is ammonia water, and the silicon source precursor is tetraethyl orthosilicate; after centrifuging and concentrating the silver nanoparticle colloid, the concentrate is mixed and dispersed with anhydrous ethanol at a volume ratio of 1:2~1:6, and 25%~35% ammonia water with a mass fraction of 25%~35% is added. A 5~50mM tetraethyl orthosilicate solution prepared with ethanol is used as the silicon source, and the reaction is stirred at room temperature for 12~48h, and the thickness of the silica shell is controlled by adjusting the amount of tetraethyl orthosilicate added.

[0039] In the above technical solution, silver nitrate is used as the silver source and trisodium citrate as the reducing agent, resulting in silver nanoparticles with controllable particle size and clean surface, which is beneficial for the uniform coating of the subsequent silica shell. The silica shell coating is carried out in a room-temperature alcohol phase system using ammonia as an alkaline catalyst and tetraethyl orthosilicate as the silicon source, with mild and controllable reaction conditions. By adjusting the concentration and amount of tetraethyl orthosilicate, the shell thickness can be precisely controlled within the range of 2-43 nm, meeting the differentiated requirements of isolation effect and enhancement efficiency in different detection scenarios. The above parameter range has been experimentally verified to ensure the morphological uniformity of the core-shell particles and the batch stability of SERS enhancement activity.

[0040] In one embodiment, the method further includes: controlling the size, generation frequency, and residence time of microdroplets in the detection zone by adjusting the flow rates of the dispersed phase and the continuous phase, so that the residence time of microdroplets matches the Raman exposure time; the Raman exposure time is 15~500ms, and the corresponding droplet detection throughput is 120~4000 drops / minute.

[0041] In the above technical solution, by adjusting the flow rate ratio of the dispersed phase to the continuous phase, the size and generation frequency of microdroplets can be flexibly controlled, thereby regulating the residence time of droplets in the detection zone to precisely match the Raman exposure time. This ensures that each droplet receives sufficient spectral acquisition during its capture period, while preventing excessive droplet retention from affecting the entry of subsequent droplets and the detection throughput. The Raman exposure time is 15~500ms, corresponding to a droplet detection throughput of 120~4000 droplets / minute, balancing the spectral signal-to-noise ratio with high-throughput detection requirements, and meeting the real-time monitoring requirements for rapid changes in metabolite concentration during microbial fermentation.

[0042] The present invention has the following beneficial effects:

[0043] 1. This invention takes a wide pH-tolerant microdroplet SERS real-time detection system as the overall protection object, and integrates a wide pH stable SERS signal source, rapid mixing, uniform droplet generation, automatic capture, reflection enhancement optical path and spectral acquisition and analysis into a continuous integrated detection link. Each functional unit is connected sequentially on the microfluidic chip, avoiding optical path offset, droplet loss and signal fluctuation introduced by independent operation of multiple links, and significantly improving the stability and operability of detection.

[0044] 2. This invention utilizes the synergistic protection of the physical isolation effect of the silica shell and the steric hindrance effect of the polyvinylpyrrolidone protective layer to ensure that the silver nanonuclei maintain structural integrity and SERS-enhanced activity over a wide pH range of 3 to 11. This eliminates the need for external electrochemical regulation, buffer systems, or proton transfer processes, preserving the original chemical environment of the analyte. It is particularly suitable for online detection of complex biological systems such as microbial fermentation broths where the pH fluctuates during the culture process.

[0045] 3. The mixing unit of the present invention enables rapid and uniform mixing of the sample to be tested and the SERS-reinforced composite material components. The T-shaped droplet generation structure utilizes the shearing effect of the continuous phase to generate microdroplets with uniform size and controllable spacing. The variable cross-section capture structure enables the microdroplets to automatically decelerate and remain stably in the detection chamber, providing a stable and sufficient time window for in-situ Raman detection. The three elements work together to ensure the signal consistency and reproducibility of droplet-by-droplet detection.

[0046] 4. The microfluidic chip channel layer of the present invention is bonded to the reflector substrate, which reflects the downward propagating Raman scattered light back to the upper collection optical path, so that the scattered light that was originally absorbed by the substrate or lost by transmission can be collected again by the objective lens, which effectively improves the Raman signal collection efficiency and signal-to-noise ratio, and enhances the system's ability to detect low concentrations of analytes.

[0047] 5. By adjusting the flow rates of the dispersed and continuous phases, this invention can flexibly match the residence time of microdroplets and the Raman exposure time, achieving a droplet detection throughput of 120~4000 droplets / minute. This meets the needs of continuous and real-time monitoring when metabolite concentrations change rapidly during microbial fermentation. It can also be used for qualitative and quantitative analysis of metabolites (such as L-DOPA, L-tyrosine, arbutin, etc.) in standard solutions and real microbial fermentation broths. Attached Figure Description

[0048] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0049] Figure 1 This is a schematic diagram of the structure of a wide pH-tolerant microdroplet SERS real-time detection system provided in an embodiment of the present invention.

[0050] Figure 2 This is a schematic diagram of the structure of a microfluidic chip provided in an embodiment of the present invention.

[0051] Figure 3 This is a schematic diagram of the structure of the wide pH-tolerant SERS-reinforced composite material provided in an embodiment of the present invention.

[0052] Figure 4 In the embodiments of the present invention, Ag NPs, and Comparison photos of dispersion states in solutions with pH values ​​of 3, 7, and 11.

[0053] Figure 5 This is a standard curve for SERS semi-quantitative analysis of L-DOPA in the actual fermentation broth in this embodiment of the invention.

[0054] Figure 6 This is a statistical bar chart showing the SERS Raman intensity of microdroplets at different L-DOPA concentrations during 60 min of continuous monitoring of the actual fermentation broth in this embodiment of the invention.

[0055] Figure 7 Standard curves for SERS quantitative detection of L-DOPA under different pH conditions.

[0056] Figure 8 A statistical graph showing the inter-droplet reproducibility of SERS Raman intensities of 20 microdroplets under different pH conditions.

[0057] Explanation of reference numerals in the attached figures:

[0058] 100 - Injection unit, 110 - First injection port, 120 - Second injection port, 130 - Third injection port, 140 - Fourth injection port.

[0059] 200 - Microfluidic chip; 210 - Mixing unit; 211 - Aqueous phase inlet; 212 - Premixing zone; 213 - Mixing channel; 220 - Droplet generation zone; 230 - In-situ droplet capture and SERS detection zone.

[0060] 300 - Raman excitation and collection unit; 310 - Laser; 320 - First collimator; 330 - First filter; 340 - Dichroic mirror; 350 - Objective lens; 360 - Second collimator; 370 - Second filter; 380 - Raman spectrometer.

[0061] 400 - Signal Acquisition and Analysis Unit

[0062] 500-droplet outlet,

[0063] 600-Silver nanocore, 700-Silica shell, 800-Polyvinylpyrrolidone protective layer. Detailed Implementation

[0064] The embodiments of the present invention will be described below with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention and not for limiting the scope of protection of the present invention.

[0065] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. The drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component may be appropriately changed, and the layout of the components may also be more complex.

[0066] Surface-enhanced Raman scattering (SERS) offers advantages such as high sensitivity, fingerprint recognition, rapid response, and in-situ detection. When combined with droplet microfluidics, it can segment the sample into numerous independent microreaction units, enabling small-volume, high-throughput online analysis, thus making it suitable for real-time detection of metabolites during microbial fermentation. However, the pH of complex biological systems such as fermentation broths fluctuates significantly during cultivation. While commonly used silver nanoparticles possess strong SERS enhancement capabilities, they are prone to surface state changes, corrosion, and particle aggregation under varying acid-base environments. This alters the dispersion state and effective hot spots of the enhanced substrate, leading to signal drift, decreased reproducibility, or even detection failure under different pH conditions. Existing technologies typically improve stability by externally adjusting the pH of the droplet or sample, or by replacing silver nanoparticles with other noble metal materials. The former requires additional electrochemical regulation, buffer systems, or proton transfer processes, altering the original chemical environment of the sample and making synchronization with high-throughput droplet detection difficult; the latter, while improving chemical stability, often reduces SERS enhancement capabilities. Meanwhile, in existing microdroplet SERS platforms, sample mixing, droplet generation, droplet positioning, and Raman detection are often completed by multiple independent steps, making the system highly sensitive to optical path stability, droplet residence time, and signal collection efficiency. Based on this, embodiments of the present invention provide a wide pH-tolerant SERS-enhanced composite material, its preparation method, and its application. This system integrates a wide pH-stable SERS signal source, rapid mixing, uniform droplet generation, automatic capture, reflection-enhanced optical path, and spectral acquisition and analysis into a single real-time microdroplet SERS detection system without actively adjusting the sample pH. The following detailed description, in conjunction with the accompanying drawings and embodiments, further illustrates this concept.

[0067] Example 1, refer to Figures 1 to 3 This embodiment provides a wide pH-tolerant microdroplet SERS real-time detection system, including a sample introduction unit 100, a microfluidic chip 200, a Raman excitation and collection unit 300, and a signal acquisition and analysis unit 400.

[0068] The sample introduction unit 100 is used to introduce the sample to be tested, the SERS-reinforced composite material components, and the oil phase as a continuous phase into the microfluidic chip 200. The sample introduction unit 100 includes a first inlet 110, a second inlet 120, a third inlet 130, and a fourth inlet 140. Depending on the mixing method, three or all four inlets may be selectively activated. In this embodiment, the sample to be tested is a microbial fermentation broth or standard solution containing the metabolite to be tested, such as L-DOPA, L-tyrosine, or arbutin; the oil phase is a fluorinated oil or silicone oil containing a surfactant, used as the continuous phase.

[0069] In this process, Ag@SiO2 core-shell particles are mixed with polyvinylpyrrolidone to form a wide pH-tolerant SERS-reinforced composite material. (Refer to...) Figure 3 The wide pH-tolerant SERS-reinforced composite material comprises, from the inside out, silver nanonuclei 600, a silica shell 700, and a polyvinylpyrrolidone protective layer 800. The average particle size of the silver nanonuclei 600 is 40–80 nm, the thickness of the silica shell 700 is 2–43 nm, and the average molecular weight of the polyvinylpyrrolidone is 8,000–58,000. The polyvinylpyrrolidone protective layer 800 adsorbs onto the outer surface of the silica shell 700, forming a steric barrier. This steric barrier, together with the silica shell 700, synergistically inhibits the corrosion and aggregation of the silver nanonuclei 600 under fluctuating pH conditions, enabling the wide pH-tolerant SERS-reinforced composite material to maintain its SERS-enhancing activity within a pH range of 3–11.

[0070] The microfluidic chip 200 is sequentially configured with a mixing unit 210, a droplet generation region 220, and a droplet in-situ capture and SERS detection region 230 along the flow direction. The sample to be tested and the SERS-reinforced composite material components are mixed in the mixing unit 210 to form a dispersed phase, and the dispersed phase and the continuous phase meet in the droplet generation region 220 to form microdroplets.

[0071] Reference Figure 2 The mixing unit 210 includes an aqueous inlet 211, a premixing zone 212, and a mixing channel 213 connected to the premixing zone 212. The droplet generation zone 220 is a T-shaped droplet generation structure, which utilizes the shearing action of the continuous relative dispersed phase to generate microdroplets with uniform size and controllable spacing. The droplet in-situ capture and SERS detection zone 230 includes a circular detection cavity and a variable cross-section channel connected to it. That is, the droplet in-situ capture and SERS detection zone 230 is provided with a variable cross-section channel structure for the stable residence of microdroplets. The sudden change in fluid resistance allows the microdroplets entering the detection zone to be automatically captured and stably residence during Raman exposure, achieving passive capture without the need for external valves or active control mechanisms. The flow channel layer of the microfluidic chip 200 is bonded to the reflector substrate, which is used to reflect the downward propagating Raman scattered light back to the upper collection optical path to improve the effective collection efficiency of the Raman signal. The microfluidic chip 200 is also provided with a droplet outlet 500 that is connected to the droplet in-situ capture and SERS detection area 230. After the detection is completed, the microdroplets are discharged from the droplet outlet 500.

[0072] The Raman excitation and collection unit 300 is used to focus the excitation light onto the microdroplet within the in-situ droplet capture and SERS detection area 230, and to collect the Raman scattered light generated by the microdroplet. The signal acquisition and analysis unit 400 is used to acquire the SERS spectrum corresponding to the Raman scattered light, and to perform qualitative or quantitative analysis of the analyte based on the characteristic Raman peaks.

[0073] As an optional embodiment of the present invention, contact mixing is performed in one of the following ways:

[0074] Method i: will Core-shell particles are dispersed in an aqueous phase to form a core-shell particle dispersion. This dispersion is then mixed with an aqueous solution of polyvinylpyrrolidone (PVP) and incubated at room temperature for a preset time to form a SERS-reinforced composite material dispersion. At this time, the sample introduction unit 100 uses three inlets: the first inlet 110, the second inlet 120, and the third inlet 130, which are used to deliver the test sample, the SERS-reinforced composite material dispersion, and the oil phase, respectively. Correspondingly, the mixing unit 210 includes two aqueous inlets 211. The test sample and the SERS-reinforced composite material dispersion enter through the two aqueous inlets 211 and are mixed in the mixing channel 213. Method i involves pre-mixing and static incubation to allow the PEP to... The core-shell particles fully adsorb and form a stable steric hindrance protective layer on their surface, resulting in a dispersion with uniform composition and good batch-to-batch consistency. This makes it suitable for quantitative detection scenarios with high requirements for the maturity of composite materials, and the chip flow channel structure is relatively simple.

[0075] Method ii: The sample injection unit 100 uses four injection ports, namely the first injection port 110, the second injection port 120, the third injection port 130, and the fourth injection port 140, which are used to deliver the sample to be tested, polyvinylpyrrolidone aqueous solution, and so on, respectively. Core-shell particle dispersion and oil phase; polyvinylpyrrolidone aqueous solution and The core-shell particle dispersion is online contact-mixed in mixing unit 210 to form a wide pH-tolerant SERS-reinforced composite material in situ for detection. Accordingly, mixing unit 210 includes three aqueous inlets 211 for the sample to be tested, polyvinylpyrrolidone aqueous solution, and... The core-shell particle dispersions enter through three aqueous inlets 211 and are rapidly mixed within the mixing channel 213. Method ii eliminates the premixing and incubation step, shortening the time from sample introduction to detection results. It is more suitable for high-throughput online detection scenarios requiring rapid response, and the contact time and mixing degree of the two components can be precisely controlled by the flow rate.

[0076] As an optional embodiment of the present invention, refer to Figure 1The Raman excitation and collection unit 300 includes a laser 310, a first collimator 320, a first filter 330, a dichroic mirror 340, an objective lens 350, a second collimator 360, a second filter 370, and a Raman spectrometer 380. The excitation light output from the laser 310 is collimated by the first collimator 320 and stray light is filtered out by the first filter 330 before being incident on the dichroic mirror 340. The light is reflected by the dichroic mirror 340 to the objective lens 350, where it is focused onto the microdroplets within the in-situ droplet capture and SERS detection area 230. The Raman scattered light generated by the microdroplets is collected by the objective lens 350, transmitted through the dichroic mirror 340, collimated by the second collimator 360, and Rayleigh scattered light is filtered out by the second filter 370 before being introduced into the Raman spectrometer 380 for spectral acquisition.

[0077] The microfluidic chip 200 is fixed on a three-dimensional displacement stage to adjust the relative position of the focal point of the objective lens 350 and the droplet in-situ capture and SERS detection area 230, ensuring that the excitation light is precisely focused on the center of the microdroplet. The signal acquisition and analysis unit 400 is electrically connected to the Raman spectrometer 380 to identify characteristic peaks, perform qualitative and quantitative analysis on the acquired SERS spectra, and display the corresponding spectra in real time on the display terminal; once the microdroplet leaves the droplet in-situ capture and SERS detection area 230, the corresponding spectrum automatically disappears, intuitively demonstrating the in-situ nature and reliability of signal acquisition.

[0078] Example 2: This example provides a method for preparing the above-mentioned wide pH-tolerant SERS-reinforced composite material, including the following steps:

[0079] Step 1, Preparation of Silver Nanonuclei 600: A reducing agent is added to a soluble silver salt aqueous solution, and the mixture is heated under reflux to obtain silver nanonuclei colloids. Preferably, the soluble silver salt is silver nitrate, and the reducing agent is trisodium citrate; the concentration of the soluble silver salt aqueous solution is 0.5~5mM, and 2~8mL of 0.5~2wt% trisodium citrate solution is added to every 200mL of this solution, with a heating and reflux time of 30~120min. By controlling the amount of reducing agent and the reflux time, silver nanonuclei 600 with uniform particle size, regular morphology, and an average particle size of 40~80nm can be obtained.

[0080] Step 2, Silica shell coating 700: The obtained silver nanoparticle colloids were centrifuged and concentrated, then dispersed in an alcohol solvent. An alkaline catalyst was added, followed by a silicon source precursor. The mixture was stirred at room temperature and then centrifuged to obtain... Core-shell particles. Preferably, the alkaline catalyst is ammonia water, and the silicon source precursor is tetraethyl orthosilicate. After centrifuging and concentrating the silver nanoparticle colloid, the concentrate is mixed and dispersed with anhydrous ethanol at a volume ratio of 1:2 to 1:6. Ammonia water with a mass fraction of 25% to 35% is added, and a 5 to 50 mM tetraethyl orthosilicate solution prepared with ethanol is used as the silicon source. The reaction is carried out at room temperature with stirring for 12 to 48 hours, and the thickness of the silica shell 700 is controlled within the range of 2 to 43 nm by adjusting the amount of tetraethyl orthosilicate added.

[0081] Step 3, Formation of the polyvinylpyrrolidone protective layer 800: Following method i or method ii as described in Example 1, ... Core-shell particles are mixed with polyvinylpyrrolidone (PVP) with an average molecular weight of 8,000 to 58,000. PPVP adsorbs onto the outer surface of the silica shell 700 to form a steric barrier, thus obtaining a wide pH-tolerant SERS-reinforced composite material.

[0082] In a specific example, 200 mL of 1 mM silver nitrate solution was taken, and 4 mL of 1 wt% trisodium citrate solution was added. The mixture was heated under reflux for 60 min to obtain silver nanoparticles with an average particle size of approximately 60 nm. After centrifugation and concentration, the concentrate was mixed with anhydrous ethanol at a volume ratio of 1:4 and dispersed. Ammonia solution with a mass fraction of approximately 28% was added, and the mixture was stirred at room temperature for 24 h using 20 mM tetraethyl orthosilicate ethanol solution as the silicon source to obtain a shell thickness of approximately 10-15 nm. Core-shell particles are then mixed with an aqueous solution of polyvinylpyrrolidone with an average molecular weight of approximately 40,000 and incubated at room temperature to obtain a wide pH-tolerant SERS-reinforced composite material.

[0083] Reference Figure 4 To verify the colloidal stability of the wide pH-tolerant SERS-reinforced composite material of this invention under acid-base fluctuations, bare silver nanoparticles (Ag NPs) and silica-coated core-shell particles were respectively... and further introduction of a polyvinylpyrrolidone protective layer The samples were dispersed at equal concentrations in aqueous solutions with pH=3, pH=7, and pH=11, and their dispersion was observed after standing at room temperature for the same amount of time.

[0084] Wherein: Ag NPs refers to bare silver nanoparticles without any surface modification, that is, control samples composed only of silver nanonuclei 600, used to characterize the response of silver nuclei to pH fluctuations in an unprotected state; The term refers to core-shell particles coated with a silica shell 700 but without the introduction of a polyvinylpyrrolidone protective layer 800. These are intermediate products composed of silver nano-cores 600 and silica shells 700, used to evaluate the physical isolation effect of silica shells 700 alone. In The final product, which is a wide pH-tolerant SERS-reinforced composite material of the present invention, further adsorbs polyvinylpyrrolidone with an average molecular weight of 8,000 to 58,000 to form a steric barrier, and consists of silver nano-core 600, silica shell 700 and polyvinylpyrrolidone protective layer 800 from the inside out.

[0085] Depend on Figure 4 It is evident that bare silver nanoparticles (Ag NPs) can maintain uniform dispersion under neutral conditions (pH=7), but under acidic (pH=3) and alkaline (pH=11) conditions, obvious black agglomerates and precipitates appear at the bottom of the tube. This indicates that the surface charge of the exposed silver nanonuclei 600 is neutralized or corroded in acidic or alkaline environments, and van der Waals forces dominate, leading to rapid agglomeration and sedimentation, resulting in poor dispersion stability. The silver nanoparticles coated with a silica shell 700... Under neutral conditions, the dispersion was uniform. While under acidic and alkaline conditions, it showed significant improvement compared to bare silver particles, with no significant precipitation. However, slight aggregation was still visible at the bottom, indicating that the silica shell 700, through physical isolation, to some extent prevented the direct erosion of the silver nanocore 600 by the external acidic or alkaline environment. However, the single shell's inhibition of close-range contact between particles was still insufficient under extreme pH conditions. Further introduction of a polyvinylpyrrolidone protective layer 800... The solution remained uniformly and stably dispersed throughout the pH range of 3, 7, and 11, with clear and homogeneous solutions in each centrifuge tube and no visible precipitate at the bottom of the tube.

[0086] The above results demonstrate that the physical isolation effect of the silica shell 700 and the steric hindrance effect of the polyvinylpyrrolidone protective layer 800 form a synergistic protection: the silica shell 700 blocks the direct corrosion of the silver nanonucleus 600 by acid and alkaline media, while the polyvinylpyrrolidone protective layer 800 further inhibits close contact and aggregation between particles by forming a steric barrier adsorbed on the outer surface of the shell. The synergistic effect of the two enables the wide pH-tolerant SERS-enhanced composite material of this invention to maintain structural integrity and stable colloidal dispersion in a wide pH range of 3 to 11, without the need for pH adjustment of the sample before detection. This provides a stable reinforcing substrate for in-situ, online SERS detection of complex biological systems (such as microbial fermentation broth with pH fluctuations during the culture process).

[0087] Example 3: This example provides a real-time detection method for wide pH-tolerant microdroplet SERS. Based on the system of Example 1, the detection method of this example includes:

[0088] S1, the sample to be tested and the SERS-reinforced composite material component are introduced into the microfluidic chip 200, so that... Core-shell particles and polyvinylpyrrolidone are mixed in contact to form a wide pH-tolerant SERS-reinforced composite material, and the wide pH-tolerant SERS-reinforced composite material is brought into full contact with the analyte.

[0089] S2, the mixed dispersed phase and the continuous phase meet in the droplet generation region 220 to form microdroplets of uniform size.

[0090] S3 allows microdroplets to enter the in-situ droplet capture and SERS detection zone 230, where the microdroplets are automatically captured and stably held through the variable cross-section channel structure.

[0091] S4 irradiates the captured microdroplets with excitation light, collects the Raman scattering signals generated by the microdroplets, and performs qualitative identification based on the characteristic Raman peaks of the analyte or quantitative analysis based on the standard curve; after detection, the microdroplets are discharged from the droplet outlet 500 to make room for subsequent droplets, realizing continuous, high-throughput real-time detection.

[0092] Furthermore, by adjusting the flow rates of the dispersed and continuous phases, the size, generation frequency, and residence time of the microdroplets in the detection zone are controlled, so that the residence time of the microdroplets is matched with the Raman exposure time. The Raman exposure time is 15~500ms, corresponding to a droplet detection throughput of 120~4000 droplets / minute, which takes into account both the spectral signal-to-noise ratio and the high-throughput detection requirements, and can meet the real-time monitoring requirements when the concentration of metabolites changes rapidly during microbial fermentation.

[0093] Reference Figure 5 and Figure 6 To verify the actual detection capability and temporal stability of the system of the present invention in complex biological systems, the wide pH-tolerant microdroplet SERS real-time detection system of Example 1 was used to detect L-DOPA in real microbial fermentation broth that had not undergone pretreatment such as pH adjustment and dilution. A mixture was prepared into the fermentation broth background... , , , , Five L-DOPA samples with different concentration gradients were injected separately. During a 60-minute continuous monitoring process, the intensity of the characteristic Raman peaks of multiple microdroplets at each concentration level was collected. Based on this, a semi-quantitative analysis standard curve was established and a statistical bar chart was plotted.

[0094] Reference Figure 5 The negative logarithm of L-DOPA concentration The x-axis represents the logarithm of the corresponding characteristic Raman peak intensity. A linear fit was performed on the ordinate, and the resulting standard curve equation is y = 3.305 - 0.160x. As L-DOPA concentration increases, the Raman intensity increases monotonically logarithmically, and all five concentration points are clearly distinguishable; even at the lowest concentration... Even at low concentrations, it can still stably detect characteristic peak signals that are significantly higher than the background. The slope of -0.160 indicates that for every order of magnitude change in concentration, the Raman intensity changes logarithmically by approximately 0.160 units. The signal response spans approximately 0.73 log units across the entire concentration range, covering a concentration interval of 5 orders of magnitude, and exhibits good distinguishability.

[0095] Figure 6 This is a statistical bar chart of the SERS Raman intensities of microdroplets at various concentration levels during the 60-minute continuous monitoring process described above. The error bars represent the standard deviation. Figure 6 As shown, , , , , The corresponding average Raman intensities were approximately 427, 353, 230, 158, and 93 a.u., respectively, increasing monotonically with increasing concentration. Column heights differed significantly between adjacent concentration levels without overlap, with the lowest concentration... The signal at each concentration point remained resolvable at approximately 93 a.u.; the relative column height of the error bars at each concentration point was relatively small, indicating that during the continuous monitoring process of up to 60 minutes, the SERS signal at the same concentration level fluctuated little and had good temporal stability, and there was no signal attenuation or abnormal jump caused by substrate inactivation, unstable droplet capture, or optical path drift.

[0096] Figure 5 The log-log standard curve and Figure 6 The linear intensity statistical histogram characterizes the system's detection performance from two dimensions: Figure 6 The absolute signal magnitude of each concentration level and its stability under long-term continuous monitoring were visually displayed using the original Raman intensity, proving that the system has good signal reproducibility and strong anti-drift capability on a 60-minute scale. Figure 5 A quantitative relationship is then provided through linear fitting after logarithmic transformation, proving a reliable monotonic correspondence between signal and concentration, which can be used for inverse calculation of unknown sample concentrations. Both conclusions are consistent; as L-DOPA concentration increases... Rise to The SERS signal increases monotonically and steadily, and the concentration levels are distinguishable from each other.

[0097] It should be noted that, with Figure 7 Standard solutions at pH 3, 7, and 11 Compared to quantitative standard curves where all values ​​are greater than 0.99, Figure 5The goodness of fit of the standard curve of the real fermentation broth was slightly lower. This is because the real fermentation broth contains complex components such as cells, proteins, and coexisting metabolites, which produce a certain matrix effect on the adsorption of the analyte on the enhanced substrate surface and on the SERS signal; however, Combination Figure 6 The small standard deviation shown still meets the accuracy and stability requirements of semi-quantitative analysis, indicating that the wide pH-tolerant SERS-reinforced composite material of the present invention can withstand interference from complex biological matrices and maintain stable reinforcing activity during long-term continuous monitoring without pretreatment such as pH adjustment of the sample.

[0098] The above results demonstrate that the system of this invention can be directly used for semi-quantitative detection and continuous monitoring of L-DOPA in real microbial fermentation broths. The established standard curve can be used to back-calculate the L-DOPA concentration in unknown fermentation broth samples, thereby supporting real-time, online monitoring of the microbial fermentation process. The same method can also be used for qualitative and quantitative analysis of metabolites such as L-tyrosine and arbutin.

[0099] Reference Figure 7 To verify the quantitative detection capability of the wide pH-tolerant SERS-reinforced composite material of this invention for levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) under different acid and alkaline conditions, L-DOPA standard solutions were adjusted to pH=3, pH=7 and pH=11 respectively to prepare... , , , , Five concentration gradient samples were analyzed using the microdroplet SERS real-time detection system described in Example 1, with the negative logarithm of L-DOPA concentration as the criterion. The x-axis represents the logarithm of the corresponding characteristic Raman peak intensity. Linear fitting is performed on the ordinate, and the error bars at each concentration point represent the standard deviation of the SERS signals of multiple microdroplets.

[0100] like Figure 7 As shown, the standard curves obtained under the three pH conditions are as follows: y = 3.877 - 0.274x at pH = 3, At pH=7, y=4.079-0.288x At pH=11, y=4.064-0.303x Goodness of fit of the three curves All values ​​are greater than 0.99, indicating that there is an excellent linear relationship between the SERS signal logarithm and the negative logarithm of L-DOPA concentration under acidic, neutral and alkaline conditions. This relationship can be used for accurate back-calculation of unknown sample concentrations and meets the accuracy requirements of quantitative analysis.

[0101] Further comparison of the three curves reveals that their slopes range from -0.274 to -0.303, and their intercepts range from 3.877 to 4.079, with highly similar values. This indicates that within a wide pH range of 3 to 11, the SERS signal response amplitude caused by a unit concentration change is essentially consistent, and the detection sensitivity does not fluctuate significantly due to changes in acidity or alkalinity. Simultaneously, the lowest concentration at each pH... The system consistently detected characteristic peak signals significantly higher than the background at various concentration points, with the linear range covering five orders of magnitude. The relatively small error bars at each concentration point indicate good signal reproducibility in multiple microdroplet detections under the same pH conditions.

[0102] The above results and Figure 4 The wide pH dispersion stability shown corroborates each other: it is precisely due to the synergistic protection of the physical isolation effect of the silica shell 700 and the steric hindrance effect of the polyvinylpyrrolidone protective layer 800 that the silver nanonuclei 600 neither aggregate and settle nor are corroded by acidic or alkaline media within a pH range of 3 to 11. This allows them to maintain stable surface-enhancing activity and consistent concentration response under a wide pH range. Therefore, when detecting complex systems such as microbial fermentation broths where pH fluctuates during the culture process, the system of this invention can directly obtain reliable quantitative results without prior pH adjustment of the sample, overcoming the limitations of traditional SERS substrates which are only suitable for narrow pH ranges and require pre-adjustment of sample pH.

[0103] Reference Figure 8 To evaluate the signal reproducibility and inter-droplet consistency of the microdroplet SERS real-time detection system of this invention under wide pH conditions, concentrations were set at pH=3, pH=7, and pH=11, respectively. L-DOPA standard solution (i.e. Figure 7 The lowest concentration point of the standard curve was used to continuously generate and detect 20 microdroplets using the detection system of Example 1. The intensity of the characteristic Raman peak of each microdroplet was recorded, and the relative standard deviation (RSD) was used to characterize the signal fluctuation between droplets.

[0104] like Figure 8 As shown, under pH=3, pH=7, and pH=11 conditions, the Raman intensity of the 20 microdroplets fluctuated slightly around the average value of approximately 56, 55, and 56 a.u. (dashed line in the figure), with corresponding RSDs of approximately 4.9%, 4.5%, and 4.4%, respectively, all below 5%. The column height distribution of the 20 droplets was uniform at each pH, ​​and no signal attenuation, drift, or abnormal jumps were observed with increasing droplet number. This indicates that the system maintains stable droplet formation during continuous sample introduction, the enhancing substrate is uniformly dispersed within the droplets, and the optical path acquisition is consistent. A single microdroplet can serve as an independent and reliable detection unit.

[0105] Further comparison of the three pH conditions revealed highly similar RSD values ​​(4.4%–4.9%), indicating that the reproducibility of microdroplet detection does not deteriorate with changes in pH levels in acidic, neutral, and alkaline environments. This result is consistent with… Figure 4 Wide pH dispersion stability Figure 7 The wide pH quantitative linearity mutually confirms that it is precisely because of the physical isolation effect of the silica shell 700 and the steric hindrance effect of the polyvinylpyrrolidone protective layer 800 that the silver nanonucleus 600 maintains a stable dispersion state and surface enhancement activity in the pH range of 3 to 11, thereby ensuring that the enhanced "hot spots" in each microdroplet are distributed in a consistent manner, which ultimately manifests as a high degree of reproducibility of the signal between droplets.

[0106] It should be noted that, Figure 8 At the lowest concentration Even at concentration levels where the signal is weakest and most susceptible to noise, the system still achieves an RSD of less than 5%, meaning it maintains good precision. Figure 7 The fact that the concentration remains within the linear range further demonstrates that the system of this invention possesses both high sensitivity and high reproducibility under a wide pH range, meeting the precision requirements for trace metabolite detection. Simultaneously, the dropwise independent detection method matches the high detection throughput of 120~4000 samples / minute of this invention, enabling high-throughput statistical analysis while ensuring the reliability of single-drop signals, effectively reducing random errors.

[0107] The above results demonstrate that the microdroplet SERS detection system of the present invention exhibits excellent inter-droplet reproducibility (RSD<5%) over a wide pH range of 3 to 11. A single microdroplet can serve as an independent quantitative detection unit, providing reliable precision assurance for real-time, online, and high-throughput monitoring of metabolites in complex systems such as microbial fermentation broths.

[0108] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A wide pH-tolerant microdroplet SERS real-time detection system, characterized in that, It includes a sample introduction unit, a microfluidic chip, a Raman excitation and collection unit, and a signal acquisition and analysis unit; The sample introduction unit is used to introduce the sample to be tested, the SERS-reinforced composite material components, and the oil phase as a continuous phase into the microfluidic chip; wherein... Core-shell particles and polyvinylpyrrolidone are mixed in contact to form a wide pH-tolerant SERS-reinforced composite material, which consists of a silver nano-core, a silica shell, and a polyvinylpyrrolidone protective layer from the inside out. The microfluidic chip is sequentially configured with a mixing unit, a droplet generation region, and a droplet in-situ capture and SERS detection region along the flow direction. The sample to be tested and the SERS-reinforced composite material component are mixed in the mixing unit to form a dispersed phase. The dispersed phase and the continuous phase converge in the droplet generation region to form microdroplets. The droplet in-situ capture and SERS detection region is provided with a variable cross-section channel structure for stabilizing the microdroplets. The flow channel layer of the microfluidic chip is bonded to the reflector substrate. The Raman excitation and collection unit is used to focus the excitation light onto the microdroplet in the in-situ capture and SERS detection area of ​​the droplet, and collect the Raman scattered light generated by the microdroplet; the signal acquisition and analysis unit is used to acquire the SERS spectrum corresponding to the Raman scattered light, and perform qualitative or quantitative analysis on the analyte based on the characteristic Raman peaks.

2. The wide pH tolerance microdroplet SERS real-time detection system according to claim 1, characterized in that: The contact mixing is performed in one of the following ways: i. will Core-shell particles are dispersed in an aqueous phase to form a core-shell particle dispersion. The core-shell particle dispersion is mixed with an aqueous solution of polyvinylpyrrolidone and incubated at room temperature for a preset time to form a SERS-reinforced composite material dispersion. The sample injection unit is equipped with three injection ports, which are used to deliver the sample to be tested, the SERS-reinforced composite material dispersion, and the oil phase, respectively. ii. The sample introduction unit is equipped with four sample inlets, which are used to deliver the sample to be tested, polyvinylpyrrolidone aqueous solution, and so on, respectively. Core-shell particle dispersion and oil phase; the polyvinylpyrrolidone aqueous solution and the... The core-shell particle dispersion is contact-mixed online in the mixing unit to form a wide pH-tolerant SERS-reinforced composite material in situ.

3. The wide pH tolerance microdroplet SERS real-time detection system according to claim 2, characterized in that: The mixing unit includes an aqueous inlet, a premixing zone, and a mixing channel communicating with the premixing zone; When mode i is used, the mixing unit includes two aqueous phase inlets, and the sample to be tested and the SERS-reinforced composite material dispersion enter through the two aqueous phase inlets and are mixed in the mixing channel. When mode ii is used, the mixing unit includes three aqueous inlets, wherein the sample to be tested, the polyvinylpyrrolidone aqueous solution, and... The core-shell particle dispersions enter through the three aqueous inlets and are rapidly mixed within the mixing channel. The droplet generation region is a T-shaped droplet generation structure; The droplet in-situ capture and SERS detection area includes a circular detection cavity and a variable cross-section channel connected thereto, so that microdroplets entering the detection area are automatically captured and stably remain during Raman exposure; The reflector substrate is used to reflect the downward-propagating Raman scattered light back to the upward collection optical path, thereby improving the effective collection efficiency of the Raman signal.

4. The wide pH tolerance microdroplet SERS real-time detection system according to claim 1, characterized in that: The average particle size of the silver nanonucleus is 40~80nm, the thickness of the silica shell is 2~43nm, and the average molecular weight of the polyvinylpyrrolidone is 8000~58000. The polyvinylpyrrolidone protective layer is adsorbed onto the outer surface of the silica shell to form a steric barrier. The steric barrier and the silica shell work together to inhibit the corrosion and aggregation of silver nanonuclei under pH fluctuation conditions, so that the wide pH tolerant SERS-enhanced composite material maintains SERS-enhancing activity in the pH range of 3 to 11.

5. The wide pH tolerance microdroplet SERS real-time detection system according to claim 1, characterized in that: The Raman excitation and collection unit includes a laser, a first collimator, a first filter, a dichroic mirror, an objective lens, a second collimator, a second filter, and a Raman spectrometer. The excitation light output from the laser is collimated by the first collimator and stray light is filtered out by the first filter before being incident on the dichroic mirror. The light is reflected by the dichroic mirror to the objective lens, which focuses it onto the microdroplets in the in-situ droplet capture and SERS detection area. The Raman scattered light generated by the microdroplets is collected by the objective lens, transmitted through the dichroic mirror, collimated by the second collimator, and Rayleigh scattered light is filtered out by the second filter before being introduced into the Raman spectrometer for spectral acquisition. The microfluidic chip is fixed on a three-dimensional displacement stage to adjust the relative position of the objective lens focal point and the droplet in-situ capture and SERS detection area; The signal acquisition and analysis unit is electrically connected to the Raman spectrometer and is used to identify characteristic peaks, perform qualitative and quantitative analysis on the acquired SERS spectra, and display the corresponding spectra in real time on the display terminal.

6. A method for real-time detection of wide pH-tolerant microdroplet SERS, characterized in that, The wide pH-tolerant microdroplet SERS real-time detection system according to any one of claims 1 to 5 includes: The sample to be tested and the SERS-reinforced composite material component were introduced into the microfluidic chip, so that... Core-shell particles and polyvinylpyrrolidone are mixed in contact to form a wide pH-tolerant SERS-reinforced composite material, which allows the wide pH-tolerant SERS-reinforced composite material to be in full contact with the analyte; The mixed dispersed phase and the continuous phase converge in the droplet formation region to form microdroplets of uniform size; The microdroplets are brought into the in-situ droplet capture and SERS detection zone, and the microdroplets are automatically captured and stably held by the variable cross-section channel structure. Excitation light is irradiated onto the captured microdroplets, and the Raman scattering signals generated by the microdroplets are collected. Qualitative identification is performed based on the characteristic Raman peaks of the analyte, or quantitative analysis is performed based on a standard curve. After the detection is completed, the microdroplets are discharged from the chip outlet.

7. The method for real-time detection of wide pH-tolerant microdroplet SERS according to claim 6, characterized in that: The contact mixing is performed in one of the following ways: i. will Core-shell particles are dispersed in an aqueous phase to form a core-shell particle dispersion. The core-shell particle dispersion is mixed with a polyvinylpyrrolidone aqueous solution and incubated at room temperature for a preset time to form a SERS-reinforced composite material dispersion. Then, the dispersion is input into a microfluidic chip along with the sample to be tested. ii. Combine the sample to be tested, polyvinylpyrrolidone aqueous solution, and... The core-shell particle dispersions are input as independent aqueous phases into the microfluidic chip, where they are mixed online in the mixing unit to form a wide pH-tolerant SERS-reinforced composite material in situ.

8. The method for real-time detection of wide pH-tolerant microdroplet SERS according to claim 6, characterized in that: The The preparation of core-shell particles includes: preparation of silver nanonuclei: adding a reducing agent to an aqueous solution of soluble silver salts, heating and refluxing to obtain silver nanonuclei colloids; Silica shell coating: The obtained silver nanoparticle colloids were concentrated by centrifugation and dispersed in an alcohol solvent. An alkaline catalyst was added, followed by a silicon source precursor. The mixture was stirred at room temperature and then centrifuged to obtain the desired product. Core-shell particles.

9. The method for real-time detection of wide pH-tolerant microdroplet SERS according to claim 8, characterized in that: The soluble silver salt is silver nitrate, and the reducing agent is trisodium citrate; the concentration of the aqueous solution of the soluble silver salt is 0.5~5mM, and 2~8mL of trisodium citrate solution with a mass fraction of 0.5~2wt% is added to every 200mL of the solution, and the reflux time is 30~120min; The alkaline catalyst is ammonia water, and the silicon source precursor is tetraethyl orthosilicate. After centrifuging and concentrating the silver nanoparticle colloid, the concentrate is mixed and dispersed with anhydrous ethanol at a volume ratio of 1:2 to 1:

6. Ammonia water with a mass fraction of 25% to 35% is added. A 5 to 50 mM tetraethyl orthosilicate solution prepared with ethanol is used as the silicon source. The reaction is stirred at room temperature for 12 to 48 hours, and the thickness of the silica shell is controlled by adjusting the amount of tetraethyl orthosilicate added.

10. The method for real-time detection of wide pH-tolerant microdroplet SERS according to claim 6, characterized in that, Also includes: The size, generation frequency, and residence time of microdroplets in the detection zone are controlled by adjusting the flow rates of the dispersed phase and the continuous phase, so that the residence time of microdroplets is matched with the Raman exposure time. The Raman exposure time is 15~500ms, corresponding to a droplet detection throughput of 120~4000 droplets / minute.

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