A sers microfluidic detection platform and a preparation method and application thereof

Abstract

This invention discloses a surface-enhanced Raman scattering (SERS) microfluidic detection platform, its fabrication method, and its applications. The platform comprises a silicon substrate, a SERS functional component composed of a patterned ultralong metal oxide nanowire array, and a bonded polydimethylsiloxane prepolymer (PDMS) microfluidic layer. The nanowire array is exposed within the channel and sealed via plasma activation. The nanowire array SERS substrate is grown using a mask-assisted growth process, a negative mold is prepared using standard photolithography, and the microfluidic layer is obtained by PDMS molding. In application, the size-selective filtering and SERS enhancement effect of the nanowire array, combined with on-chip pretreatment to remove plasma protein interference, and individualized matrix matching calibration, enable rapid, highly sensitive, label-free quantitative detection of trace small molecule drugs, suitable for point-of-care clinical testing.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and more specifically, to a SERS microfluidic detection platform, its preparation method, and its application. Background Technology

[0002] Imatinib (IMT), a first-generation tyrosine kinase inhibitor, is widely used to treat chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Despite its significant clinical efficacy, pharmacokinetic variability varies considerably among patients, stemming from factors such as transporter-mediated efflux, genetic polymorphisms of hepatic metabolic enzymes (e.g., CYP3A4 / 5), and drug-drug interactions. Clinical studies have confirmed a clear exposure-response (ER) relationship between drug exposure levels and efficacy and toxicity. Therefore, therapeutic drug monitoring (TDM) to maintain steady-state trough concentrations within a therapeutic window of approximately 1000 ng / mL is crucial for dose optimization.

[0003] Currently, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is considered the clinical gold standard for therapeutic diagnostics (TDM) due to its high sensitivity and specificity. However, this method faces serious challenges in practical applications: firstly, the high cost of the instruments, complex operation, and heavy reliance on skilled technicians limit its application in primary healthcare institutions and resource-constrained environments; secondly, the sample pretreatment process is cumbersome and time-consuming, often requiring complex protein precipitation or liquid-liquid extraction steps, which not only delays clinical decision-making but also fails to meet the timeliness requirements of point-of-care testing (POCT).

[0004] Surface-enhanced Raman scattering (SERS) technology, with its advantages of high sensitivity, molecular fingerprinting ability, and rapid detection, is considered a strong candidate to replace LC-MS / MS for drug monitoring. However, translating SERS technology from the laboratory to routine clinical applications still faces two major bottlenecks. First, the reproducibility and stability of SERS substrates are insufficient. Traditional colloidal substrates, while generating strong signals, suffer from problems such as aggregation and spatiotemporal heterogeneity; while solid substrates prepared by photolithography or electrodeposition, although ordered, are often expensive and have limited accessibility of analytes to hot spots. Second, interference from complex biological matrices is severe. High abundances of proteins, lipids, and endogenous metabolites in plasma competitively adsorb onto the substrate surface, and IMT itself has a high binding rate with plasma proteins, resulting in low free drug concentrations, large signal fluctuations, and difficulty in achieving accurate quantification.

[0005] To address these issues, microfluidics has been introduced into SERS detection systems, aiming to integrate sample pretreatment and detection through miniaturization. However, while the application of microfluidic-SERS systems in biological sample analysis has been proven, their potential in label-free quantitative analysis remains underutilized. Furthermore, most current platforms focus only on single detection functions, lacking efficient removal of plasma proteins and size-selective filtration capabilities. This results in significant matrix effects in actual clinical sample testing, and quantitative accuracy fails to meet clinical requirements.

[0006] Therefore, there is an urgent need to develop a low-cost, highly reproducible SERS microfluidic detection platform that integrates efficient on-chip preprocessing functions to resolve the above-mentioned technical contradictions and achieve rapid, accurate, and clinical-grade quantitative detection of IMT in human plasma. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a SERS microfluidic detection platform, its preparation method and application.

[0008] The first objective of this invention is to provide a SERS microfluidic detection platform, comprising: Silicon substrate; SERS functional components are disposed on the surface of the silicon substrate. The SERS functional components include a patterned array of ultralong metal oxide nanowires and noble metal nanoparticles modified on the surface of the ultralong metal oxide nanowire array. A PDMS microfluidic layer, wherein a microfluidic channel is formed within the PDMS microfluidic layer, and the SERS functional component is exposed within the microfluidic channel; The PDMS microfluidic layer and the silicon substrate are irreversibly sealed together through plasma surface activation treatment.

[0009] In one possible implementation, the material of the ultralong metal oxide nanowire array is zinc oxide, and the material of the noble metal nanoparticles is silver.

[0010] In one possible implementation, the height of the microfluidic channel is 30–50 μm, and multiple inlets are provided upstream of the microfluidic channel for introducing samples and auxiliary reagents respectively.

[0011] The second objective of this invention is to provide a method for preparing a SERS microfluidic detection platform, comprising the following steps: SERS active substrate preparation steps: A patterned ultralong nanowire array is prepared on a silicon substrate by mask-assisted growth, and the nanowire array is modified with noble metal nanoparticles. Microfluidic mold fabrication steps: Prepare the female mold using SU-8 photoresist and standard photolithography process; PDMS microfluidic layer molding and bonding steps: PDMS is cast onto the negative mold and cured to form a PDMS microfluidic layer. Then, the PDMS microfluidic layer is aligned with the SERS active substrate, and the two are sealed together by plasma bonding process to obtain the SERS microfluidic detection platform.

[0012] In one possible implementation, the mask-assisted growth method in the SERS active substrate preparation step includes: The seed solution is cyclically dipped into the silicon substrate surface and subjected to high-temperature heat treatment, which is repeated multiple times to form a uniform seed layer. A patterned growth region is defined on the seed layer using a custom metal mask; The ultra-long nanowire array is grown in the mask area using a hydrothermal method, and the nanowires are oriented perpendicular to the silicon substrate surface.

[0013] In one possible implementation, the standard photolithography process in the microfluidic mold fabrication step includes: PDMS is mixed with a curing agent and poured onto the negative mold. After curing, it is peeled off to form a microgroove structure on the surface of the cured PDMS that corresponds to the pattern of the negative mold, thus obtaining the PDMS microfluidic layer. In the PDMS microfluidic layer forming and bonding steps, the irreversible sealing bonding is achieved through oxygen plasma surface treatment, so that the PDMS microfluidic layer is tightly attached to the silicon substrate.

[0014] The third objective of this invention is to provide a method for detecting trace substances using a SERS microfluidic detection platform, comprising the following steps: The sample solution containing the analyte is introduced into the microfluidic channel of the SERS microfluidic detection platform; The patterned ultralong metal oxide nanowire array was used to perform size-selective filtration and surface-enhanced Raman scattering detection of target analytes in the sample solution. Raman spectral signals were acquired, and the concentration of the analyte was quantitatively analyzed based on the intensity of characteristic peaks.

[0015] In one possible implementation, the size-selective filtration is achieved through the gaps in the ultralong metal oxide nanowire array or the diameter of the ultralong metal oxide nanowire array, for retaining large protein molecules while allowing small molecule analytes to pass through.

[0016] In one possible implementation, the trace substance is IMT in plasma, the detection limit of the detection method is less than 10 ng / mL, and the sample volume required for a single detection is less than 50 μL.

[0017] In one possible implementation, the quantitative analysis employs an individualized matrix-matched calibration method, using a blank matrix from which the sample to be tested is derived to construct a standard curve.

[0018] Compared with the prior art, the present invention has the following advantages: 1. Low cost and high reproducibility manufacturing: By combining mask-assisted hydrothermal growth method, patterned large-area fabrication of ultra-long ZnO@Ag nanowire arrays was achieved, which significantly reduced the manufacturing cost of SERS microfluidic detection platform, while ensuring batch-to-batch consistency (RSD < 10%).

[0019] 2. Highly efficient on-chip preprocessing and anti-interference capabilities: Through the unique three-dimensional mesh structure of the "ultra-long nanowire array," this invention achieves a dual cascade purification mechanism of "chemical precipitation + physical filtration." Utilizing methanol-induced protein precipitation, combined with the size exclusion effect of the nanowire gaps, it effectively removes interference from high-abundance macromolecular proteins in plasma, solving the signal drift and matrix effect problems caused by protein adsorption in traditional SERS detection.

[0020] 3. Excellent SERS detection performance: By precisely modifying ultralong ZnO nanowires with silver nanoparticles (Ag NPs), abundant three-dimensional electromagnetic "hot spots" are constructed at the nanowire junctions and sidewalls. Although some intrinsic enhancement is sacrificed, this results in highly efficient processing capabilities for complex biological matrices. Experiments have shown that this platform has a detection limit for IMT as low as 2.5 ng / mL, covering the clinical therapeutic window (1000 ng / mL).

[0021] 4. Minimal Volume, Rapid Flow, and Clinical Applicability: This invention integrates the entire process of sample pretreatment, filtration and enrichment, and SERS detection into a single SERS microfluidic detection platform. A single test requires only <40 μL of plasma, eliminating the need for cumbersome centrifugation and extraction steps, reducing the total analysis time to less than 5 minutes. Combined with the "personalized matrix matching calibration method," a standard curve is constructed using the patient's own blank plasma, effectively eliminating quantitative errors caused by individual differences in plasma composition. The average relative deviation from the LC-MS / MS gold standard is only 6.864%, demonstrating extremely high potential for clinical translation. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the SERS microfluidic detection platform of the present invention and an enlarged view of its working principle.

[0023] Figure 2This is a schematic diagram illustrating the fabrication process of the SERS microfluidic detection platform of the present invention; Figure 3 The microstructure and elemental composition characterization diagram of the ultralong ZnO@Ag nanowires prepared in Example 1 are shown below. Figure 4 This is a diagram verifying the size-selective filtration performance of the SERS microfluidic detection platform for ultra-long ZnO@Ag nanowires in Example 2 of this invention. Figure 5 This is a verification diagram of the SERS enhancement performance of the SERS microfluidic detection platform in Embodiment 2 of the present invention; Figure 6 This is a diagram showing the clinical application results of plasma IMT detection using the individualized matrix matching calibration method in Example 3 of the present invention. Detailed Implementation

[0024] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0025] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0027] like Figure 1 As shown, the first embodiment of the present invention provides a SERS microfluidic detection platform, comprising: Silicon substrate; SERS functional components are disposed on the surface of a silicon substrate. The SERS functional components include a patterned array of ultralong metal oxide nanowires and noble metal nanoparticles modified on the surface of the ultralong metal oxide nanowire array. The PDMS microfluidic layer contains microfluidic channels, and the SERS functional components are exposed within these channels. In this process, the PDMS microfluidic layer and the silicon substrate are irreversibly sealed together through plasma surface activation treatment.

[0028] The material of the ultralong metal oxide nanowire array is zinc oxide, and the material of the noble metal nanoparticles is silver.

[0029] In practice, the microfluidic channel has a height of 30–50 μm, and multiple inlets are located upstream of the channel for introducing samples and auxiliary reagents. During the detection experiment, the sample and auxiliary reagents are pumped into three circular inlet reservoirs (each with a diameter of 2 mm) located at the upstream end of the SERS microfluidic detection platform. Notably, the dual-inlet design of methanol is specifically designed to homogenize the fluid distribution within the microchannel, thereby ensuring the stable advancement of the plasma sample into the reaction zone for thorough mixing. This dual-inlet configuration optimizes the flow rate and the matching between methanol and plasma, effectively eliminating the hydrodynamic interferences common in single-inlet designs. After homogenization, the mixed fluid passes through a 500 µm wide serpentine culture channel, providing sufficient residence time before entering the ultra-long metal oxide nanowire array filtration and SERS detection zone to ensure complete reaction and mixing.

[0030] Figure 1 The diagram further illustrates the core working principle of the integrated SERS microfluidic sensing platform constructed in this invention. This platform cleverly integrates microfluidic chip technology and SERS detection technology, aiming to achieve highly sensitive and rapid detection of trace small molecule drugs in complex biological samples. As shown in the figure, the entire system mainly consists of a sample inlet, a microfluidic channel, a SERS activity detection area, and a signal output module.

[0031] At the macroscopic level, the sample to be tested is injected into the meandering channel within the PDMS microfluidic layer through the inlet and flows towards the outlet under capillary force or external drive. During this process, the sample flows through a SERS functional component located on the surface of the silicon substrate. This core component consists of a patterned array of ultralong ZnO@Ag nanowires. Its dense, vertically arranged structure not only greatly increases the active surface area but also forms abundant "hot spot" regions, providing an ideal location for subsequent Raman signal enhancement. When a 785 nm laser irradiates the detection area, the Raman scattering signal generated by the target molecules is collected and converted into a SERS spectrum, thereby enabling qualitative or quantitative analysis of the sample.

[0032] Further examination of its underlying mechanisms reveals that this platform achieves highly efficient purification and enrichment of plasma samples primarily through the synergistic effect of a dual mechanism: chemical pretreatment and physical filtration. In terms of chemical pretreatment, the methanol solvent introduced during sample introduction effectively disrupts the hydration layer of plasma proteins and alters their charge distribution, promoting the aggregation and precipitation of large protein molecules, thereby removing the biomolecular background that might obscure the target signal. Simultaneously, at the physical filtration level, the ultralong nanowire array, with its unique size exclusion effect, further blocks larger, incompletely precipitated protein particles, allowing only smaller target analytes (such as imatinib, IMT) to pass through and remain within the nanowire gaps. This cascaded design of "chemical destabilization followed by physical sieving" not only significantly reduces interference from complex matrices but also effectively increases the local concentration of the target analyte, thereby greatly improving the sensitivity and accuracy of detection.

[0033] like Figure 2 As shown, the second aspect of the specific embodiments of the present invention provides a method for preparing the above-mentioned SERS microfluidic detection platform, including the following steps: SERS active substrate preparation steps: Patterned ultralong nanowire arrays are prepared on silicon substrates by mask-assisted growth method, and the nanowire arrays are modified with noble metal nanoparticles. Microfluidic mold fabrication steps: Using a customized photolithography mask, a negative mold of the microfluidic structure is fabricated through standard photolithography processes; PDMS microfluidic layer molding and bonding steps: PDMS is cast onto the negative mold and cured to form a PDMS microfluidic layer. Then, the PDMS microfluidic layer is aligned with the SERS active substrate, and the two are sealed together by plasma bonding process to obtain the SERS microfluidic detection platform.

[0034] In specific operations, the mask-assisted growth method in the SERS active substrate preparation steps includes: The seed solution is cyclically dipped into the silicon substrate surface and subjected to high-temperature heat treatment, which is repeated multiple times to form a uniform seed layer. Patterned growth regions are defined on the seed layer using a custom metal mask; The ultra-long nanowire array was grown in the mask area using a hydrothermal method, and the nanowires were oriented perpendicular to the silicon substrate surface.

[0035] In specific operations, the standard photolithography process in the microfluidic mold fabrication step includes: PDMS is mixed with a curing agent and poured onto a negative mold. After curing, it is peeled off to form a microgroove structure on the PDMS surface that corresponds to the pattern of the SERS active substrate, thus obtaining the PDMS microfluidic layer. In the PDMS microfluidic layer forming and bonding steps, irreversible sealing bonding is achieved through oxygen plasma surface treatment, which makes the PDMS microfluidic layer tightly adhere to the silicon substrate.

[0036] Example 1 This embodiment provides a SERS microfluidic detection platform, which is prepared by the following method: 1. Preparation of SERS-active substrates (mask-assisted growth method) Silicon substrate pretreatment and seed layer preparation: The monocrystalline silicon wafer was ultrasonically cleaned sequentially with acetone, anhydrous ethanol, and deionized water for 15 minutes, and then dried with nitrogen. Seed layer solution preparation: Zinc acetate dihydrate (Zn(OAc)₂·2H₂O) was dissolved in a mixed solvent of 2-methoxyethanol and monoethanolamine, with a Zn(OAc)₂ concentration of 0.1 M and a molar ratio of monoethanolamine to Zn(OAc)₂ of 1:1. The clean silicon wafer was vertically immersed in the seed solution and slowly pulled up at a speed of 5 mm / s, followed by heat treatment on a 300°C hot plate for 10 minutes. This dip-coating-heat treatment process was repeated three times to form a uniform and dense ZnO seed layer on the silicon wafer surface.

[0037] Patterned Mask and Nanowire Growth: A stainless steel metal mask with a 5mm x 15mm rectangular opening was designed and customized. A silicon wafer with a seed layer was tightly aligned and fixed to the metal mask to expose the growth area. A hydrothermal growth solution was prepared: 25mM zinc nitrate hexahydrate (Zn(NO3)2·6H2O), 12.5mM hexamethylenetetramine (HMTA), 5mM polyethyleneimine (PEI), and 0.35mM ammonia (NH4OH). PEI was mixed with Zn... 2+ Coordination inhibits lateral growth and promotes the directional extension of nanowires along the c-axis. A silicon wafer (growth side down) was immersed in 80 mL of growth solution and reacted in an 88°C oven for 12–24 hours. After the reaction, the sample was removed, rinsed with deionized water, and dried at room temperature, resulting in a vertically aligned array of ultralong ZnO nanowires with a height of approximately 37 ± 3 μm within the mask region.

[0038] Noble metal nanoparticle modification: Ion sputtering deposition was performed using a JS-1600 model at a vacuum degree of 5x10⁻⁶. -1 Silver nanoparticles (Ag NPs) were deposited on the surface of ZnO nanowires under conditions of Pa and 20 W power. The sputtering time was strictly controlled at 75 seconds, at which point the average particle size of the Ag NPs was approximately 11.81 nm, uniformly distributed on the nanowire surface and at the intersection nodes, forming abundant SERS electromagnetic hot spots. Electromagnetic simulations showed that the electric field intensity at the gaps between the nanowires was significantly enhanced after Ag modification. Finally, the metal mask was removed to obtain a patterned SERS-active substrate.

[0039] 2. Packaging of the SERS microfluidic detection platform (SU-8 standard photolithography and bonding) Microfluidic mold preparation: The master mold was prepared using SU-8 photoresist and standard photolithography process.

[0040] PDMS microfluidic layer molding: PDMS and curing agent were mixed and poured into a master substrate, which was then cured in a 60℃ oven for 1 hour. After curing, the PDMS layer was carefully peeled off, and holes were punched at the corresponding positions using a 0.8mm punch to serve as the sample inlet and outlet. The resulting PDMS microfluidic layer was approximately 4mm thick, and its inner surface replicated the microgroove structure on the master substrate, with a channel height of approximately 40μm.

[0041] Plasma bonding: The PDMS microfluidic layer and the bonding surfaces of the aforementioned SERS active substrate are simultaneously placed in an oxygen plasma cleaner for 60 seconds. Immediately after removal, the two are aligned and bonded together, and slight pressure is applied to form an irreversible covalent bond at room temperature, thus obtaining the SERS microfluidic detection platform.

[0042] Figure 3 The image shows the relevant detection results for ultra-long ZnO@Ag nanowires. Figure 3 a, II shows that the silver nanoparticles are uniformly distributed with an average diameter of 11.81 nm, forming abundant three-dimensional electromagnetic hotspots at the nanowire connections and sidewalls. Large-area SEM images ( Figure 3 (a, III-V) confirmed the formation of a highly ordered, densely covered array, generating extensive SERS-active regions. Furthermore, Figure 3 The illustrations in a, III show that the average top clearance size is 0.67 µm. Meanwhile, Figure 3 EDS spectra in b and c show the spatial co-location of Zn, O, and silver. Figure 3 Obvious diffraction peaks were observed at 2θ = 31.8°, 36.3°, 47.6°, 56.7°, and 67.9°, corresponding to the (100), (101), (102), (110), and (112) planes of hexagonal fine-grained zincite ZnO, respectively. No impurity phases were detected. After Ag nanoparticle deposition, additional peaks were observed at approximately 2θ ≈ 38.1° and 44.2°, which could be assigned to the (111) and (200) planes of face-centered cubic Ag, confirming the coexistence of ZnO and metallic Ag without altering the crystal structure of ZnO. Further XPS analysis was performed to examine its surface composition and chemical state. Figure 3 As shown in Figure e, the investigated spectra clearly reveal the presence of Zn, O, Ag, and C, with the c1s peak at ~284 eV used as the binding energy reference. High-resolution o1s spectra ( Figure 3f) can be divided into two parts: lattice oxygen (O_L, 530.1 eV) and chemisorbed oxygen (O_H, 531.87 eV). The prominent O_H signal indicates that the surface has abundant hydroxyl groups, which are known to promote charge transfer and contribute to the chemical enhancement of SERS. Figure 3 g shows Zn 2p peaks at 1022.11 and 1044.08 eV, characterized by Zn 2+ .at the same time, Figure 3 h shows that the three-dimensional repetition peaks of Ag are located at 368.01 and 373.98 eV, attributable to metallic Ag. The slight blue shift relative to bulk Ag indicates that the electronic interaction between Ag nanoparticles and ZnO leads to charge redistribution, thereby promoting the synergistic enhancement of electromagnetic and chemical effects in SERS.

[0043] Example 2 This embodiment provides performance verification of the SERS microfluidic detection platform. 1. Validation of size-selective filtration performance A three-stage microfluidic testing device was constructed, in which suspensions of fluorescent polystyrene microspheres with diameters of 500 nm, 1 μm, and 5 μm were introduced into the SERS microfluidic detection platform at a total flow rate of 30 μL / min. Observations were performed using a laser confocal microscope. Figure 4 Fluorescence images obtained from upstream to downstream regions (I–III), from Figure 4 As can be seen, region I exhibits 100% initial fluorescence intensity. Due to the filtering effect of the nanowire array, a large number of microspheres are captured, while a small portion passes through the nanowires and reaches region III. Therefore, the fluorescence intensity of region II is slightly weaker than that of region I, while the fluorescence intensity of region III is significantly reduced because most microspheres remain in region II. These results confirm the effective capture of microspheres along the nanowire array under continuous flow, demonstrating its filtering function. The platform achieves a single-pass filtration efficiency of 52.2% for 500 nm microspheres, confirming its size-selective filtration capability.

[0044] 2. SERS Sensitivity and Detection Limit Test Rhodamine 6G (R6G) was used as the probe molecule. Under optimized conditions (532 nm laser, 15 mW), the detection limit of this substrate for R6G reached 1.0 x 10⁻⁶. -10 M. Based on the formula in the supporting information, the enhancement factor (EF) is calculated to be 1.85 × 10⁻⁶. 6 .

[0045] Since the platform's detection capability is directly related to the SERS performance of ultralong ZnO@Ag NWs, their SERS activity was systematically evaluated. Depositing Ag NPs on ZnO NWs significantly enhanced the SERS performance, mainly due to the excitation of localized surface plasmon resonances (LSPRs) and the formation of dense electromagnetic hotspots within the nanoscale particle interstices. To evaluate the SERS activity of the prepared ultralong ZnO@Ag NWs substrate, R6G was used as a probe molecule for a series of SERS measurements. Figure 5 a shows R6G(10) collected from substrates prepared with different silver sputtering times. -7 The SERS spectrum of M) and 614 cm⁻¹ -1 Intensity comparison of corresponding peak values ​​( Figure 5 (Right panel of a). R6G((10 -7 The characteristic Raman bands of M were clearly resolved in all spectra, with prominent peaks at approximately 614, 772, 1365, 1507, and 1651 cm⁻¹. -1 Specifically, 1507 and 1651cm -1 The peaks at 614 and 772 cm⁻¹ belong to the C-C stretching vibration of the aromatic ring. -1 The peak at 1365 cm is related to the ring bending mode. -1 The peak at point 614 belongs to the tensile vibration of the CC bridge. Furthermore, the effect of silver sputtering time on SERS enhancement was further evaluated by monitoring the intensity changes of the peak at point 614. Figure 5 As shown in the right figure, the SERS signal of R6G gradually increases with increasing sputtering time, which can be attributed to the growth and aggregation of AgNPs. This SERS microfluidic detection platform was used to detect imatinib in human plasma. Within the range of 100 ng / mL to 5000 ng / mL, 1031 cm⁻¹ -1 The Raman characteristic peak intensity at a given location shows a good linear relationship with the concentration (R0). 2 =0.975). Even at concentrations as low as 2.5 ng / mL, the characteristic peaks of IMT remained discernible, establishing a practical limit of detection (LOD) of 2.5 ng / mL.

[0046] like Figure 5 As shown in b, the peak intensity decreases with decreasing R6G concentration. Furthermore, from 614 cm⁻¹... -1 Peak intensity plotted Figure 5 The calibration curve in c is compared with the logarithm of the R6G concentration (R 2 =0.968) shows a good linear relationship, confirming reliable quantitative performance. Figure 5 The inserted portion of c further shows 1.0 × 10 -10Despite a six-order-of-magnitude reduction in concentration, the characteristic Raman bands remained clearly visible in the magnified spectra under M, highlighting the excellent SERS enhancement of the substrate. It is important to note that while the ultralong ZnO nanowires provide a large specific surface area and abundant anchoring sites for Ag NPs, resulting in numerous electromagnetic hotspots, the effective enhancement within a single laser spot (2–3 µm) primarily benefits from the uniformly distributed nanowire cluster domains. The micrometer-scale gaps between these clusters scatter and attenuate the signal, partially limiting the overall SERS response. Therefore, under the same conditions, the SERS performance of ultralong nanoarrays is slightly lower than that of shorter nanoarrays (<5 µm). In our experiments, short ZnO nanowires (~2 µm) were prepared, and their SERS performance was evaluated using the same method. The results show that shorter nanowires exhibit stronger Raman enhancement. This trade-off between SERS efficiency and filtering capability forms the fundamental principle behind the design of this platform. Although the intrinsic enhancement is somewhat weakened, the ultralong structure allows for simultaneous sample pretreatment and analyte enrichment, significantly improving the analytical reliability of complex biological matrices. In addition, to assess spatial uniformity, 614 cm⁻¹ was used. -1 The SERS intensity in the band is mapped onto the substrate. A total of 1600 measurement points were collected in the active region of the SERS on a single SERS microfluidic detection platform, and the resulting intensity distribution is as follows: Figure 5 As shown in d. The relative standard deviation (RSD) of the SERS intensity was 9.03%, indicating good spatial homogeneity, suitable for quantitative analysis. Furthermore, to evaluate batch-to-batch reproducibility, SERS spectra of R6G from five independently prepared substrates were randomly collected. Figure 5 e) 614, 1365, and 1651 cm were selected. -1 Three representative peaks, covering the low, medium, and high wavenumber regions, with RSDs of 5.08%, 8.39%, and 10.12%, respectively, indicate that the reproducibility of the preparation process is satisfactory.

[0047] Example 3 This embodiment provides the clinical application of individualized matrix matching calibration. To eliminate differences in plasma matrix among different patients, an individualized calibration strategy was adopted.

[0048] Calibration curve establishment: Blank plasma from a patient before medication was taken, and high-concentration (3000 ng / mL), medium-concentration (1000 ng / mL), and low-concentration (500 ng / mL) imatinib standards were added respectively. The characteristic peak intensity was obtained by instrument detection, and a calibration curve specific to this patient was plotted.

[0049] Specifically, the calibration curve is constructed based on the peak spectrum of each patient, such as... Figure 6As shown in a–f. As expected, significant differences in sensitivity and absolute signal intensity were observed among patients, reflecting the inherent biological variability of human plasma. However, in all cases, 1031 cm⁻¹ -1 The peak IMT intensity at a given location showed a strong linear correlation with its concentration, and the effective quantification range covered the clinically relevant concentration window. Subsequently, patient samples were collected at three time points (1, 2, and 3 months) to quantify their IMT concentrations. Figure 6 As shown in g, the IMT concentration predicted by the SERS microfluidic platform (dashed line, X) P (X represents patient ID) and reference LC-MS / MS measurements (solid line, X) R It is closely related to the time trend. Figure 6 h shows a strong correlation between the predicted value and the reference value (R). 2 =0.961), the data points are closely aligned along the line of identity (y=x), indicating good quantitative accuracy. Figure 6 Bland-Altman analysis is presented to further assess the consistency between the two methods. The red dashed line represents the line of identity, and the blue solid line represents the mean bias; the upper and lower dashed lines correspond to the 95% threshold of consistency. The mean relative difference between SERS and LC-MS / MS results was 6.864%, reflecting a slight negative bias, but no significant systematic bias was observed within the tested concentration range. Importantly, all measurements were within predefined acceptable ranges, further supporting the reliability of the IMT quantification SERS platform. Compared to conventional PLSR-based plasma IMT quantification strategies, the patient-matched calibration method used in this study demonstrates higher accuracy and precision.

[0050] Clinical sample testing: Real plasma samples were collected from 6 patients after medication. No complex pretreatment was required; <40μL of sample was directly injected into the SERS microfluidic detection platform. The SERS microfluidic detection platform automatically completed protein precipitation and filtration, and the detection was completed within 5 minutes.

[0051] Results Comparison: Comparison of SERS test results with the gold standard LC-MS / MS results shows that the two are completely consistent in terms of time trend. Bland-Altman analysis shows that the average relative deviation is only 6.864%, and all data points are within the 95% agreement limit, with no significant systematic bias.

[0052] Example 4 This embodiment provides a performance comparison with short nanowire substrates. To verify the advantages of ultralong nanowires in filtration performance, short ZnO@Ag NWs (~2 μm) were prepared as a control. The results showed that although short nanowires exhibited stronger intrinsic SERS enhancement due to their high hotspot density, their filtration efficiency was significantly lower than that of ultralong nanowires. This invention achieves superior on-chip pretreatment capabilities by sacrificing some intrinsic enhancement, thereby realizing more reliable quantitative results in complex plasma matrices.

[0053] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A SERS microfluidic detection platform, characterized in that, include: Silicon substrate; SERS functional components are disposed on the surface of the silicon substrate. The SERS functional components include a patterned array of ultralong metal oxide nanowires and noble metal nanoparticles modified on the surface of the ultralong metal oxide nanowire array. A PDMS microfluidic layer, wherein a microfluidic channel is formed within the PDMS microfluidic layer, and the SERS functional component is exposed within the microfluidic channel; The PDMS microfluidic layer and the silicon substrate are irreversibly sealed together through plasma surface activation treatment.

2. The SERS microfluidic detection platform according to claim 1, characterized in that, The material of the ultralong metal oxide nanowire array is zinc oxide, and the material of the noble metal nanoparticles is silver.

3. The SERS microfluidic detection platform according to claim 1, characterized in that, The height of the microfluidic channel is 30–50 μm, and multiple inlets are provided upstream of the microfluidic channel for introducing samples and auxiliary reagents respectively.

4. A method for preparing a SERS microfluidic detection platform, characterized in that, Includes the following steps: SERS active substrate preparation steps: A patterned ultralong nanowire array is prepared on a silicon substrate by mask-assisted growth, and the nanowire array is modified with noble metal nanoparticles. Microfluidic mold fabrication steps: Combine SU-8 photoresist with standard photolithography process to fabricate the female mold. PDMS microfluidic layer molding and bonding steps: PDMS is cast onto the negative mold and cured to form a PDMS microfluidic layer. Then, the PDMS microfluidic layer is aligned with the SERS active substrate, and the two are sealed together by plasma bonding process to obtain the SERS microfluidic detection platform.

5. The preparation method according to claim 4, characterized in that, In the SERS active substrate preparation step, the mask-assisted growth method includes: The seed solution is cyclically dipped into the silicon substrate surface and subjected to high-temperature heat treatment, which is repeated multiple times to form a uniform seed layer. A patterned growth region is defined on the seed layer using a custom metal mask; The ultra-long nanowire array is grown in the mask area using a hydrothermal method, and the nanowires are oriented perpendicular to the silicon substrate surface.

6. The preparation method according to claim 4, characterized in that, In the microfluidic mold fabrication step, the standard photolithography process includes: SU-8 photoresist was uniformly coated onto the silicon wafer using a spin coater. A customized photomask was then placed over the photoresist surface and subjected to UV exposure. After development with a developer, the negative mold was obtained. PDMS is mixed with a curing agent and poured onto the negative mold. After curing, it is peeled off to form a microgroove structure on the PDMS surface that corresponds to the pattern of the photomask, thus obtaining the PDMS microfluidic layer. In the PDMS microfluidic layer forming and bonding steps, the irreversible sealing bonding is achieved through oxygen plasma surface treatment, so that the PDMS microfluidic layer is tightly attached to the silicon substrate.

7. A method for detecting trace substances using the SERS microfluidic detection platform according to any one of claims 1–3, characterized in that, Includes the following steps: The sample solution containing the analyte is introduced into the microfluidic channel of the SERS microfluidic detection platform; The patterned ultralong metal oxide nanowire array was used to perform size-selective filtration and Raman spectroscopy detection of target analytes in the sample solution. Raman spectral signals were acquired, and the concentration of the analyte was quantitatively analyzed based on the intensity of characteristic peaks.

8. The method according to claim 7, characterized in that, The size-selective filtration is achieved through the gaps in the ultra-long metal oxide nanowire array or the diameter of the ultra-long metal oxide nanowire array, and is used to retain large protein molecules while allowing small molecule analytes to pass through.

9. The method according to claim 7, characterized in that, The trace substance is imatinib in plasma, the detection limit of the detection method is less than 10 ng / mL, and the sample volume required for a single detection is less than 50 μL.

10. The method according to claim 7, characterized in that, The quantitative analysis employs an individualized matrix matching calibration method, using a blank matrix from which the sample to be tested is used to construct a standard curve.

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