Microfluidic chip and use thereof

Abstract

An integrated multifunctional microfluidic chip and a use thereof, relating to the technical field of biological detection. A novel microfluidic chip is designed, and the microfluidic chip covers the whole process of droplet generation, mixing, amplification, magnetic separation, and high-throughput SERS detection, so that high-precision detection of miRNA can be realized. The chip has excellent specificity, high stability, and significant reproducibility, providing a powerful tool for the research and clinical application. In practical application, a microfluidic platform can be used for testing dual-target miRNA indexes of an IPF patient, including miR-21 and miR-155. The present invention provides a basis for analysis of early diagnosis, intraoperative monitoring, and prognostic improvement of IPF.

Description

A microfluidic chip and its application Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a microfluidic chip and applications thereof. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung disease that affects millions of patients worldwide. Almost half of all deaths in the United States are attributable to pulmonary fibrosis. Pulmonary fibrosis is often a progressive disease with severely limited treatment options, a long and painful course, and an extremely poor prognosis. The diagnosis of pulmonary fibrosis relies heavily on invasive procedures. Tissue biopsies can confirm the presence of abnormal ECM deposition and tissue architectural disruption. However, tissue biopsies have significant limitations for diagnosing pulmonary fibrosis. For example, small biopsy specimens often fail to reflect the extent of fibrosis throughout the entire organ, often leading to sampling bias. Furthermore, histological assessment of pulmonary fibrosis is often semiquantitative, subjective, and inaccurate. Furthermore, tissue biopsies are not only invasive to patients but also often increase the risk of complications. Previous studies have demonstrated the involvement of microRNAs (miRNAs) in the development and progression of IPF. MiRNAs are non-coding RNAs of 17-25 nucleotides that regulate genes by binding to target genes and inhibiting translation. In recent years, numerous studies have reported that miRNAs (miRNAs) influence the progression of IPF by regulating signaling pathways, such as cell proliferation and differentiation, tissue repair, and subsequent fibrosis. miR-21 and miR-101 are significantly upregulated in IPF mice and the serum of IPF patients, and promote epithelial-mesenchymal transition (EMT) through TGF-β. Furthermore, miR-21 and miR-101 are positively correlated with the rate of IPF progression. An increasing number of studies have demonstrated that microRNAs are potential biomarkers for the diagnosis of IPF. However, traditional miRNA detection techniques, including quantitative real-time polymerase chain reaction (qRT-PCR), Northern blotting, fluorescence, and electrochemistry, have the disadvantages of requiring expensive and complex instrumentation, complex procedures, and specialized technicians, resulting in time-consuming and low sensitivity. Furthermore, due to the low abundance and high sequence homology of miRNAs in IPF, achieving specific detection remains challenging. Therefore, there is an urgent need to develop new, less invasive methods for the accurate diagnosis and disease monitoring of patients with pulmonary fibrosis.

[0003] To date, a variety of detection technologies have been developed for miRNA detection. Reverse transcription polymerase chain reaction (RT-PCR) has been established as the gold standard for miRNA detection, enabling highly sensitive RNA quantification. However, its time-consuming nature leads to a high incidence of false-positive signals. While Northern blotting can simultaneously detect multiple miRNAs, it is limited by low sensitivity, resulting in inaccurate detection of low-abundance miRNAs. To achieve highly specific miRNA detection, rolling cycle amplification (RCA) has become an attractive approach. As an isothermal amplification technique, RCA is based on a DNA cascade reaction, mimicking the natural rolling cycle replication process of circular DNA to achieve exponential template amplification. By designing specific padlock probes to recognize target miRNAs and constructing circular templates for RCA, single-nucleotide variant detection can be achieved with high precision. Surface-enhanced Raman scattering (SERS), an optical detection technique, has been widely used in bioanalysis for the detection of various biomarkers, including nucleic acids, proteins, small molecules, and pathogens, due to its narrow spectral peaks, minimal optical drift, and strong multiplexing capabilities. Due to the advantages of RCA's highly specific recognition, combining RCA with SERS has become an attractive research area. In particular, for miRNA motifs with high sequence homology and extremely low abundance, the high sensitivity of SERS allows for efficient detection within a small number of amplification cycles, thereby shortening detection time. However, the intensity of the SERS signal is affected by various factors, including nanoparticle size, shape, concentration, and batch preparation. Furthermore, the SERS signal primarily originates from molecules located in tiny "hotspot" regions, which are highly confined, making the SERS signal more susceptible to subtle variations and affecting SERS reproducibility. Given the complex pretreatment steps of RCA and the reproducibility issues of SERS detection, further development of rapid, convenient, and suitable miRNA detection methods for clinical trace samples is needed.

[0004] The microfluidic chip detection platform, known as the "lab on a chip," can provide a comprehensive platform to overcome the limitations of RCA-based SERS detection. The application of microfluidics technology has endowed SERS detection with excellent repeatability and sensitivity, significantly improving the yield and efficiency of detection. In addition, microfluidics-based microdroplet technology allows each microdroplet to act as an independent microreactor, achieving uniform mixing, fusion, separation, and screening of microdroplets within the chip. Integrating SERS analysis into microfluidic chips automates the complex amplification and detection processes, further improving the repeatability and accuracy of SERS detection, thereby achieving rapid and precise molecular detection and quantitative analysis. At the same time, microfluidics technology facilitates the rapid detection of trace samples (microliter level), which enables microfluidic chips to meet clinical requirements for point-of-care testing (POCT).

[0005] Therefore, given the advantages of microfluidic technology, providing a microfluidic platform that can rapidly process, automate, perform high-throughput and small sample analysis to perform SERS analysis of miR-21 and miR-155 to achieve accurate diagnosis of IPF is an urgent problem to be solved by the present invention. Summary of the Invention

[0006] The present invention provides a microfluidic chip and its application. Addressing the reproducibility issues of surface-enhanced Raman scattering (SERS) measurements and the complex pretreatment steps of rolling circle amplification (RCA) in the prior art, the present invention aims to provide a fully automatic, highly sensitive, and highly specific SERS-RCA microfluidic sensor platform to accurately and reliably detect miR-21 and miR-155 in patients with interstitial pneumonia (IPF).

[0007] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0008] In the first aspect, the present invention discloses a microfluidic chip, comprising a first sample loading area, a second sample loading area, a third sample loading area, a first heating area, a second heating area and a third heating area, each sample loading area is provided with an inlet and a mixing channel that are interconnected, each heating area is provided with a heating channel, and the third heating area is provided with a first heating channel, a second heating channel and a third heating channel that are arranged in parallel with each other, wherein the discharge port of the mixing channel in the first sample loading area is connected to the feed port of the first heating channel in the third heating area, the discharge port of the first heating channel in the third heating area is connected to the feed port of the mixing channel in the second sample loading area, the discharge port of the mixing channel in the second sample loading area is then connected in sequence to the feed ports of the second heating channels in the first and third heating areas and the feed port of the mixing channel in the third sample loading area, and the discharge port of the mixing channel in the third sample loading area is then connected in sequence to the feed port of the third heating channel in the third heating area and the second heating area.

[0009] In a preferred embodiment of the present invention, the injection port of the first sample loading area includes a Y-shaped injection port and an end injection port provided at one end thereof.

[0010] In a preferred embodiment of the present invention, the temperatures of the first heating zone, the second heating zone and the third heating zone are 45° C., 65° C. and 95° C. respectively.

[0011] In a preferred embodiment of the present invention, the microfluidic chip also includes a fourth chamber separation zone and a fifth chamber identification zone, and the separation zone is configured as a Y-shaped channel, wherein the confluence portion of the Y-shaped channel is connected to the discharge port of the heating channel on the second heating zone, and one side of the Y-shaped channel is connected to the feed port of the identification zone, and the other side merges with the discharge port of the identification zone and is connected to the channel for external discharge.

[0012] In a preferred embodiment of the present invention, the length ratio of the first heating channel to the third heating channel in the third heating zone is 3:1.

[0013] In a preferred embodiment of the present invention, the mixing channel of the first sample loading zone is equal in length to the heating channel of the second heating zone.

[0014] In a second aspect, the present invention discloses a use of the microfluidic chip as described in the first aspect in preparing a microfluidic sensor.

[0015] In a third aspect, the present invention discloses a method for preparing a microfluidic sensor using the microfluidic chip according to the first aspect, wherein the microfluidic sensor is a SERS-RCA microfluidic sensor, comprising the following steps:

[0016] (1) DNA ligase and uncirculated padlock probe are added to the first loading zone, and a sample containing the target miRNA is introduced at the other end, mixed, and then the aqueous phase is divided into droplets by an immiscible carrier oil under shear force, and then fully mixed in the mixing channel, and then enter the first heating channel of the third heating zone for enzyme denaturation to terminate the reaction, obtain a circular padlock probe, and enter the second loading zone;

[0017] (2) MBs@zip and polymerase are added to the second loading zone, mixed with the circularized padlock probe, and then enter the first heating zone. MBs@Zip specifically recognizes the zipper region of the padlock probe and initiates the RCA reaction in the presence of polymerase. After the reaction is completed, the mixture flows into the second heating channel of the third heating zone for polymerase denaturation and inactivation to obtain the RCA reaction product, which then enters the third loading zone.

[0018] (3) Adding a SERS probe to the third loading zone and mixing it with the RCA reaction product, the mixture enters the third heating channel of the third heating zone, unfolds the secondary structure formed by the RCA reaction product, promotes binding with the SERS probe, and enters the second heating zone;

[0019] (4) In the second heating zone, complementary base pairing occurs between the p region in the RCA reaction product that forms the secondary structure and the pDNA in the SERS probe to form a SERS-RCA probe, thereby preparing a SERS-RCA microfluidic sensor.

[0020] In a fourth aspect, the present invention discloses a use of a SERS-RCA microfluidic sensor in detecting miR-21 or miR-155 in patients with interstitial pneumonia.

[0021] Furthermore, miR-21 or miR-155 was specifically bound to the SERS-RCA probe on the SERS-RCA microfluidic sensor to observe the SERS signal.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Highly integrated: The present invention integrates all processes such as temperature control, droplet generation, mixing, amplification, magnetic separation and high-throughput SERS detection into an automated microfluidic chip, avoiding errors that may be caused by manual operation and improving detection accuracy.

[0024] High sensitivity and specificity: By using surface-enhanced Raman scattering technology, the present invention achieves high sensitivity and high specificity detection of low-concentration biomolecules (such as miRNA).

[0025] Broad application prospects: The SERS-RCA microfluidic platform proposed in this invention can be successfully applied to detect dual-target miRNA indicators in IPF patients, providing a reliable analytical tool and research basis for early diagnosis, intraoperative monitoring and improvement of IPF prognosis.

[0026] Easy to operate and highly repeatable: The integrated microfluidic system of the present invention has a simple design, is easy to operate, and has good repeatability, which helps to improve the reliability of experimental results in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of the structure of the microfluidic chip provided by the present invention. 1 is the mixing channel; 2 is the first heating zone; 3 is the second heating zone; and 4 is the third heating zone.

[0028] Figure 2 provides a comprehensive overview and functional diagram of the microfluidic system. a) A composite image shows the physical version and layered schematic of the microfluidic chip, including the 3D-printed nylon substrate, ceramic heating layer, glass slide, and PDMS layer, which together provide the structural integrity of the device. b) A graphic illustration of a droplet sensor for target miRNA detection. The chip contains five specialized functional zones and three thermally controlled regions, each with unique properties: i) a zone for droplet formation and assembly for ligation at ambient temperature; ii) a dedicated 45°C zone for component interaction and integration for RCA reactions; iii) a 95°C zone for component assembly and subsequent initial development of RCA products, as well as complementary pairing with SERS probes in a 65°C environment. The 95°C zone also serves as a location for enzyme denaturation and inactivation, thereby terminating the reaction; iv) a zone for magnetic rod-mediated isolation of the MB-SERS-RCA complex; and v) a space for SERS analysis of the supernatant. c) A schematic diagram of the process illustrates the entire process within the streamlined channel. Key parameters for each zone are clearly labeled, including reactant addition, relevant reaction temperatures, droplet transit through different temperature zones, and zone functionality. d) Microscopic capture of different stages within the microfluidic device, demonstrating i) droplet origin, ii) primary droplet fusion, iii) secondary droplet fusion, and iv) magnetic separation of droplets within their respective zones.

[0029] Figure 3 is a core flow chart for detection using the microfluidic chip of the present invention. a) Principle of the SERS-RCA-microfluidic biosensor for detecting IPF-related miRNAs. b) Microfluidic chip design: ii: RCA priming, introducing streptavidin-coated MBs@Zip, DNA polymerase, DTT, dNTPs, and buffer; iv: SERS signal detection zone, where a magnet is placed to capture the MB-RCA-Au NPs complex and separate the supernatant.

[0030] Figure 4 shows the specificity, reproducibility, and stability of the SERS-RCA microdroplet sensor for miRNA detection. a) Selectivity of the SERS-RCA microfluidic platform for different targets: blank, primers with a single base mismatch to miR-21, random primers, and detection of miR-155 and miR-21; blank, primers with a single base mismatch to miR-155, random primers, and detection of miR-155 and miR-21. NS: Not statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001. b) 1614 cm -1(b) Reproducibility analysis within the microfluidic chip: Comparison of the relative standard deviation (RSD) of the Raman intensity at 1614 cm-1 measured off-chip (miR-21: 13.59%, miR-155: 15.26%) and on-chip (miR-21: 3.69%, miR-155: 6.56%). (c) The stability of the SERS probe was assessed by comparing the Raman intensity at 1614 cm-1 of a probe stored at 4°C for 30 days with that of a freshly synthesized probe.

[0031] Figure 5. a) and c) SERS spectra of MGITC at 1614 cm⁻¹, increasing with increasing concentration of miRNA-21 and miRNA-155, respectively. b) and d) Calibration curves for miRNA-21 and miRNA-155, respectively, were generated by plotting the corresponding SERS intensities versus concentration. Error bars represent the standard deviation (SD) of five replicates. The limit of detection (LOD) was determined to be 0.29 fM for miRNA-21 and 0.37 fM for miRNA-155. e) Comparison of the LOD and detection range between the proposed SERS-RCA droplet sensor and qRT-PCR. The red area represents the result region where no PCR data was obtained. Red dots represent the results of the SERS sensor, and blue dots represent the results of the RT-PCR. f) Bland-Altman analysis of the correlation between the two detection methods, showing the 95% agreement deviation and limit of agreement. DETAILED DESCRIPTION

[0032] The technical solution provided in this application is described in detail below with reference to specific embodiments and the accompanying drawings.

[0033] This invention utilizes a newly reported microwave-assisted strategy to prepare cross-linked DNA-modified gold nanoparticles (Au NPs), ensuring uniform hotspot distribution. Furthermore, a specific RCA padlock probe is designed to enhance detection specificity and enable identification of single nucleotide variants (SNVs). Subsequently, all processes—temperature control, droplet generation, mixing, amplification, magnetic separation, and high-throughput SERS detection—are integrated into an automated microfluidic chip, demonstrating excellent specificity, high stability, and remarkable reproducibility. In practical applications, the SERS-RCA microfluidic platform has been successfully used to detect dual-target miRNA markers in IPF patients. Results indicate that simultaneous detection of miR-21 and miR-155 is highly correlated with IPF patients at all stages. Therefore, the detection method provided by this invention provides a reliable analytical tool and research foundation for the early diagnosis, intraoperative monitoring, and improved prognosis of IPF.

[0034] The following describes it in detail through specific embodiments.

[0035] Example 1 Structure of the microfluidic chip

[0036] A microfluidic chip as shown in Figure 1 includes a first sample loading area i, a second sample loading area ii, a third sample loading area iii, a first heating area 2, a second heating area 3 and a third heating area 4, each sample loading area is provided with an interconnected sample inlet and a mixing channel 1, each heating area is provided with a heating channel, and the third heating area is provided with a first heating channel, a second heating channel and a third heating channel arranged in parallel with each other, wherein the discharge port of the mixing channel in the first sample loading area is connected to the feed port of the first heating channel in the third heating area, the discharge port of the first heating channel in the third heating area is connected to the feed port of the mixing channel in the second sample loading area, the discharge port of the mixing channel in the second sample loading area is then connected in sequence to the feed ports of the second heating channels in the first and third heating areas and the feed port of the mixing channel in the third sample loading area, and the discharge port of the mixing channel in the third sample loading area is then connected in sequence to the feed port of the third heating channel in the third heating area and the second heating area.

[0037] The inlet for the first sample loading zone includes a Y-shaped inlet and an end inlet located at one end. The temperatures of the first, second, and third heating zones are 45°C, 65°C, and 95°C, respectively. The length ratio of the first heating channel to the third heating channel in the third heating zone is 3:1. The mixing channel in the first sample loading zone is equal in length to the heating channel in the second heating zone.

[0038] The microfluidic system is designed with multiple compartments. Through varying temperature variations, a series of reaction processes are implemented within the channel, ultimately facilitating accurate detection of target miRNAs. In the first loading zone (i), DNA ligase and a non-circular padlock probe are introduced, while at the other end, a sample containing the target miRNA is introduced. The two streams mix in a Y-shaped channel, where the aqueous phase is divided into droplets by shear forces from a water-insoluble carrier oil (Figure 2d). After thorough mixing, the padlock probe circularizes in the mixing channel at room temperature. Enzyme denaturation is then performed at 95°C in the third heating zone to terminate the reaction. In the second loading zone (ii), the circularized padlock probe mixes with incoming streptavidin-labeled magnetic beads capturing DNA (MBs@zip) and polymerase in the winding channel. The DNA then enters the 45°C heating zone, where MBs@Zip specifically recognizes the zipper region of the padlock probe and initiates the RCA reaction in the presence of the polymerase. After the reaction, the mixture flows into the third heating zone at 95°C for denaturation and inactivation of the polymerase. Furthermore, in the third loading zone iii, the RCA product is fully mixed with the introduced SERS probe, and the mixture passes through the 95 °C high temperature zone of the third heating zone to unfold the secondary structure formed by the RCA product and promote the binding with the SERS probe.

[0039] Upon reaching the second heating zone, i.e., 65°C, complementary base pairing occurs between the p region in the RCA product and the pDNA in the SERS probe, forming a SERS-RCA probe. Notably, guided by prior off-chip optimization, the channel lengths for padlock probe circularization, RCA, and SERS probe recognition are equal: the mixing channel in the first sample loading zone is equal in length to the heating channel in the second heating zone. The ratio of the channel lengths for enzyme denaturation and RCA product expansion is 3:1, meaning the length ratio of the first heating channel to the third heating channel in the third heating zone is also 3:1.

[0040] The microfluidic chip of the present invention also includes a fourth separation zone iv and a fifth identification zone v. The separation zone is configured as a Y-shaped channel, wherein the confluence of the Y-shaped channel is connected to the outlet of the heating channel on the second heating zone. One side of the Y-shaped channel is connected to the feed inlet of the identification zone, while the other side merges with the outlet of the identification zone to form an outward discharge channel. At the junction of the Y-shaped channels in the separation zone, as shown in Figure 2d, the original microdroplet containing the magnetic SERS-RCA complex and the supernatant are separated into two smaller sub-droplets: a small droplet containing the MB-SERS-RCA product (formed by amplification on MBs@zip to form the MBs@zip@RCA product and then binding to the SERS probe) flows through the narrow channel due to magnetic attraction, while a larger droplet containing the unreacted SERS probe in the supernatant flows through the wider channel. Larger droplets containing unreacted SERS probes from the supernatant enter the fifth chamber, the identification zone, where they perform SERS analysis on the continuous droplets in the supernatant. The identified sample then merges with the MB-SERS-RCA product on the other side of the Y-shaped channel and is then discharged through an external channel. By focusing the laser beam at the center of the detection channel, the Raman signal originating from the supernatant droplets is detected, enabling accurate assessment of miR-21 and miR-155 levels.

[0041] Example 2 Preparation of microfluidic chip

[0042] (1) Silicon-based template preparation

[0043] Initially, the silicon wafer was cleaned with anhydrous ethanol and dried in a preheated oven at 75°C for 10 minutes before cooling to room temperature. A spin coater was then used with parameters set to three stages: 500 rpm for 20 seconds, 800 rpm for 20 seconds, and 20,000 rpm for 45 seconds. The wafer was placed in the spin coater, coated with photoresist, and treated accordingly. Subsequently, the evenly coated wafer underwent a two-stage soft bake in the oven: 75°C for 10 minutes and 115°C for 30 minutes. After cooling, the wafer was placed in the photolithography machine, which was preheated for 10 minutes after nitrogen activation. The printed mask was cleaned with anhydrous ethanol and secured to the machine using the mask vacuum function. The wafer was then placed in the machine and secured using the sample vacuum function, with the distance adjusted to ensure close contact between the mask and wafer. A shutter function initiated the photolithography process, and upon completion, a nitrogen purge function separated the mask and wafer. The sample vacuum was deactivated, and the wafer was placed on a baker for a two-stage post-bake: 75°C for 5 minutes and 115°C for 5 minutes. The wafer was then cooled to room temperature. Finally, the wafer was soaked in developer for 10 minutes, cleaned with acetone, and rinsed three times with deionized water. Residual photoresist was removed using isopropyl alcohol, and the wafer was rinsed again with deionized water. The wafer was then placed in a well-ventilated area to air dry.

[0044] (2) Microfluidic chip fabrication

[0045] PDMS and curing agent were mixed in a 10:1 weight ratio, degassed in a vacuum desiccator, and poured into a clean vacuum tray. The mixture was then placed on a photolithographically patterned silicon wafer, degassed again to eliminate any bubbles, and then heated at 80°C for 2 hours. The PDMS layer was carefully peeled from the silicon wafer, covered with tape for precise cutting, and then perforated using a hole punch. The PDMS layer and glass slide were cleaned sequentially with a detergent solution, deionized water, anhydrous ethanol, and isopropyl alcohol for 5 minutes each and dried on a temperature-controlled platform at 80°C for 10 minutes. After cooling, the PDMS layer was exposed to oxygen plasma in a plasma cleaner for 4 minutes and gently bonded, concave side down, to a glass slide. The bonded PDMS chip was baked at 110°C for 30 minutes and, after cooling to room temperature, examined under a microscope to ensure clear channels. The channels were pretreated with a 2% trichloro-(1H,1H,2H,2H-perfluorooctyl)silane solution to form a hydrophobic layer. The chip is then connected to syringe pumps, and the appropriate flow rate is set for each pump to ensure accurate liquid delivery according to the experimental requirements.

[0046] (3) Extraction of total RNA from serum

[0047] Circulating total RNA was extracted using the Plasma / serum exosome and free circulating RNA extraction kit (NGB-59500) purchased from NorgenBiotek (Thorold, Ontario, Canada) according to the manufacturer's instructions. Serum samples used in this study were obtained from patients with specific diseases (e.g., pulmonary fibrosis). After centrifugation, the serum samples were added to appropriate reagents and processed according to the following protocol:

[0048] Purification from 1 mL of cell-free serum: Add 3 mL of nuclease-free water to 1 mL of serum, followed by 100 μL of ExoC buffer to create a mixture. Add 200 μL of slurry E to this mixture, mix thoroughly by vortexing for 10 seconds, and incubate at room temperature for 5 minutes. Vortex for 10 seconds to mix thoroughly. Centrifuge at 2,000 RPM for 2 minutes. Discard the supernatant to create the first slurry pellet. Apply 200 μL of ExoR buffer to the first slurry pellet and mix thoroughly by vortexing for 10 seconds to create the second slurry pellet. Resuspend the second slurry pellet in 200 μL of ExoR buffer and incubate at room temperature for 5 minutes. After incubation, mix thoroughly by vortexing for 10 seconds, then centrifuge at 500 RPM for 2 minutes. Transfer the supernatant to a mini filter column assembled in an elution tube and centrifuge at 6,000 RPM for 1 minute to create the third slurry pellet.

[0049] Isolation of Free-Circulating RNA: Add 300 μL of Lysis Buffer A to the third slurry pellet, mix thoroughly by vortexing for 10 seconds, and then centrifuge at 500 RPM for 2 minutes. Transfer 300 μL of the supernatant from Lysis Buffer A to a 1.5 mL tube, then add 300 μL of 96-100% ethanol and vortex for 10 seconds to mix thoroughly to obtain a mixture. Transfer 600 μL of this mixture to a new Mini Spin Column. Centrifuge at 6,000 RPM for 1 minute. Discard the flow-through and reassemble the spin column and its collection tube. Apply 600 μL of Wash Buffer to the column and centrifuge at 6,000 RPM for 30 seconds. Discard the flow-through and reassemble the spin column and collection tube. Perform two washes in total. Spin the empty column at 14,000 RPM for 1 minute. Discard the collection tube. Transfer the spin column to a new 1.7 mL elution tube. Apply 50 μL of the eluate to the column and centrifuge at 2,000 RPM for 1 minute, then at 8,000 RPM for 2 minutes. To maximize recovery, transfer the elution buffer back to the column and allow it to stand at room temperature for 2 minutes. Centrifuge at 2,000 RPM for 1 minute, then at 8,000 RPM for 2 minutes.

[0050] The components of lysis buffer A are:

[0051] Tris-HCl, pH 7.4: 50 mM

[0052] EDTA: 1 mM

[0053] Sodium Chloride (NaCl): 140 mM

[0054] 1% Triton X-100

[0055] RNase inhibitor: 1 U / mL

[0056] Note: All buffer components should be RNase-free to avoid RNA degradation.

[0057] The concentration and quality of the extracted RNA were assessed using a multi-function microplate reader to ensure its suitability for subsequent experimental analysis. The isolated RNA was stored at -80°C for future experimental use.

[0058] (4) Preparation of detection-related nanomaterials

[0059] Preparation of SERS probes: Mix 400 μL of a 1 nM AuNP solution with 20 μL of 100 μM SH-DNA or DNA in a 5 mL glass vial. Heat the vial in a household microwave oven (microwave input power 1150 W, microwave output power 700 W) on high for 3 minutes. Resuspend the pellet in PBS (0.1 M NaCl, pH 7.4). Wash the pellet with 0.01 M phosphate buffer (0.3 M NaCl, pH 7.4) and centrifuge at 7500 rpm for 20 minutes at 15°C. Finally, resuspend the pellet in PBS buffer. The resulting DNA-AuNP conjugate, i.e., the SERS probe, can be stored at 4°C in the dark or used in subsequent experiments.

[0060] DNA capture with streptavidin-labeled magnetic beads (MBs@zip): Resuspend the streptavidin beads and transfer 20 μL to a 1.5 mL centrifuge tube. Magnetic separation for 1 minute and remove the supernatant. Add 1X TBS to a final volume of approximately 0.5 mL and resuspend the streptavidin beads. Magnetic separation for 10 seconds and remove the supernatant to complete one wash step. Wash twice more. Resuspend the beads in Binding & Washing Buffer I (2X) at 2 times the original volume of beads. Add an equal volume of biotinylated nucleic acid sample (biotin-modified ZIP region chains), vortex thoroughly to mix, and incubate at room temperature for 1 hour. Magnetic separation for 1 minute and remove the supernatant. Add 1 mL of Binding & Washing Buffer I (1X) to resuspend the beads.

[0061] (5) Chip preparation

[0062] To address the challenges of tedious manual sample handling and precise control of detection conditions, an integrated microfluidic system was developed to automate droplet generation, mixing, reaction, RCA, magnetic separation, and SERS detection for miRNA detection. A novel device was designed to support the microfluidic chip, consisting of a nylon 3D-printed base, a temperature-controlled heating plate, and a temperature sensor (Figure 2(a)). This design allows for more precise and controllable chip operation, improving experimental reproducibility and accuracy.

[0063] The overall design of the microfluidic chip, depicted in Figure 2b, highlights five functionally distinct regions within the chip. Each of these regions plays a unique role in a series of temperature-controlled reactions, enabling rapid and highly sensitive detection of miRNAs. Figures 2c and 2d clearly illustrate the functions of these compartments and the detailed mixing and separation steps involved in the chip's reactions.

[0064] Specifically, in the 'i' region, the coordinated reaction begins with the introduction of DNA ligase, a non-circularized padlock probe, and a miRNA sample. An immiscible carrier oil facilitates droplet segmentation. The mixing process and padlock probe circularization are completed at room temperature, and the reaction is terminated by enzyme denaturation at 95°C.

[0065] After entering the 'ii' region, the circularized padlock probe fuses with the incoming streptavidin-labeled magnetic bead capture DNA and polymerase. Once the streptavidin-labeled magnetic bead capture DNA recognizes the zipper region of the padlock probe, the RCA reaction begins at 45°C.

[0066] In region 'iii', the RCA products are fully integrated with the SERS probes. These secondary structures are denatured at 95°C, promoting their binding to the SERS probes. When the temperature reaches 65°C, hybridization begins, generating SERS-RCA probes.

[0067] In the final stage, as shown in Figure 2c, the 'iv' region begins magnetically separating the MB-SERS-RCA complex from the supernatant within the droplet, as vividly captured in Figure 2d (iv). This critical process extracts the target molecules from the reaction mixture, simplifying downstream analysis. To enable SERS identification within the supernatant droplet, a unique 'v' region was designed that accurately quantified miRNA-21 and miRNA-155 levels.

[0068] By integrating various reactions into a single integrated microfluidic system, it provides an efficient solution for miRNA detection. Because the microenvironment within the droplet is controllable, this platform not only reduces manual operations but also ensures higher reproducibility and accuracy.

[0069] Example 3 Specificity of chip detection

[0070] Table 1 List of mismatched gene sequences used in the study

[0071]

[0072] To validate the sequence recognition capability of the RCA-SERS droplet sensor for single-nucleotide variants, a series of miRNA sequences, including those with single-nucleotide variants in miR-21 and miR-155, as well as completely random DNA sequences (Tables 1 and 2), were designed to assess the SERS-RCA sensor's ability to discriminate between highly homologous miRNAs. Figure 4(a) shows that a significant decrease in the SERS signal was observed only when the target miRNA and the specific probe on the sensor were perfectly matched (P < 0.001). In contrast, the presence of single-nucleotide variants in the target sequence did not significantly reduce the SERS signal. This can be attributed to the failure of the padlock probe to circularize due to mismatches at the ligation site, leading to termination of the RCA process and preventing DNA ligase from ligating the probe. These results demonstrate that the designed SERS-RCA droplet sensor exhibits high specificity and can accurately detect miRNAs at the genomic level without being affected by interference from RNA from other species.

[0073] Example 4: Repeatability and stability of the SERS-based microfluidic platform.

[0074] To further evaluate the advantages of the SERS-RCA microfluidic sensor in the reproducibility of miRNA detection, 50 points were selected along a straight line on the capillary outside the chip, and the 1614 cm -1 The relative standard deviation of the SERS intensity at each location was calculated. Subsequently, Raman signals were measured at the detection point within the microfluidic chip. Figure 4b plots the data from 50 Raman signal intensity measurements, each with a 2-second exposure time. The results show that the relative standard deviation (RSD) of the SERS intensity measured off-chip is significantly higher (miR-21: 13.59%, miR-155: 15.26%) than the RSD obtained from the 50 measurements within the chip (miR-21: 3.69%, miR-155: 6.56%). This is primarily due to the uneven distribution of hot spots caused by thermal motion of the gold colloids during a single laser exposure of the external SERS signal intensity. In contrast, for the on-chip measurements, the signal intensity during a single exposure comes from the cumulative signal of the gold colloids within multiple, rapidly and uniformly flowing droplets. Given that the droplet flow rate significantly exceeds the thermal motion of the gold colloids, the reproducibility of the on-chip SERS signal detection is enhanced.

[0075] Furthermore, 50 Raman data measurements were used to compare the SERS probes stored in a 4 °C refrigerator for 30 days with those of freshly synthesized SERS probes at 1614 cm -1 The Raman intensity of the SERS probe decreased slightly (Figure 4c). Although the SERS signal intensity decreased slightly, the change was not significant, with a difference analysis showing P > 0.05. This finding indicates that the SERS probe has good temporal stability.

[0076] Example 5: Working curve

[0077] Table 2 List of gene sequences used in this study

[0078]

[0079] As shown in Figure 5a and c, the SERS signal intensity is negatively correlated with the concentration of the target miRNA. Figure 5b and d show that at 10 -8 M to 10 -15 In the range of M, the SERS signal has a strong linear relationship with the concentration of miR-21 and miR-155, R 2 The detection limits of miR-21 and miR-155 were 0.29 fM and 0.37 fM, respectively.

[0080] Example 5: Verification of the recoverability of detection technology

[0081] Table 32 Recovery rate and relative standard deviation of SERS-RCA microdroplet sensor.

[0082]

[0083] To evaluate the accuracy and precision of the analytical method and instrument, the recovery of miR-21 and miR-155 at five different concentrations was examined. Five replicates were run for each sample, and the recovery of the analytes in the samples was calculated by comparing the concentrations of the standards before and after the spike recovery experiments. Table 3 lists the recovery data for this study. -1 The SERS intensity and working curves at 100 nm showed satisfactory recoveries of the identified miRNAs, ranging from 97% to 110% with an RSD of less than 8%. The high recoveries demonstrate that the SERS-RCA droplet sensor can accurately measure the analytes present in the sample without significant loss or alteration during sample pretreatment. This implies a high degree of reliability and accuracy in the analytical results.

[0084] Table 4 List of sequences used for qPCR detection of miR-21 and miR-155.

[0085]

[0086] To evaluate the clinical feasibility of the proposed method, serum samples from IPF patients and healthy subjects were collected, and standard curves of miR-21 and miR-155 were established by qRT-PCR. The primer sequences are shown in Table 4 and compared with the SERS-RCA microfluidic biosensor. The experimental results showed that the LODs of miR-21 and miR-155 were 5.28 nM and 0.74 nM, respectively, under qRT-PCR detection (Table 5), with a detection range of 10 -8 M to 10 -11 While their sensitivities are already quite high, our SERS-RCA microfluidic biosensor exhibits significantly higher sensitivity. According to Bland-Altman analysis (Figure 5f), there is a certain degree of bias and dispersion between the PCR and SERS-RCA droplet sensors. Specifically, the bias is -1.9059, the standard deviation of the bias is 4.5490, and the limits of agreement for 95% agreement range from -10.822 to 7.0100. Although PCR is considered the "gold standard," SERS-RCA droplet sensors demonstrate considerable reliability in consistency analysis compared to PCR and may be more accurate and reliable in specific application scenarios. For example, in low-concentration or highly noisy samples (Figure 5e: red area), the proposed method exhibits higher sensitivity and resolution, providing more accurate measurements. In these cases, PCR may fail to detect a signal, while the proposed method can. Furthermore, compared to traditional PCR and other techniques, the constructed biosensor offers faster detection times and more straightforward operation (Table 5).

[0087] Table 5 Performance comparison of various sensors

[0088]

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. All equivalent changes and modifications made based on the content of the present invention are included in the patent scope of the present invention.

Claims

1. A microfluidic chip, characterized in that: The invention comprises a first sample loading area, a second sample loading area, a third sample loading area, a first heating area, a second heating area and a third heating area, each sample loading area is provided with an inlet and a mixing channel which are interconnected, each heating area is provided with a heating channel, and the third heating area is provided with a first heating channel, a second heating channel and a third heating channel which are arranged in parallel with each other, wherein the discharge port of the mixing channel of the first sample loading area is connected with the feed port of the first heating channel of the third heating area, the discharge port of the first heating channel of the third heating area is connected with the feed port of the mixing channel of the second sample loading area, the discharge port of the mixing channel of the second sample loading area is connected with the feed ports of the second heating channels of the first heating area and the third heating area and the feed port of the mixing channel of the third sample loading area in sequence, and the discharge port of the mixing channel of the third sample loading area is connected with the feed port of the third heating channel of the third heating area and the second heating area in sequence.

2. The microfluidic chip according to claim 1, characterized in that: The injection port of the first sample loading area includes a Y-shaped injection port and an end injection port arranged at one end thereof.

3. The microfluidic chip according to claim 1, characterized in that: The temperatures of the first heating zone, the second heating zone and the third heating zone are 45° C., 65° C. and 95° C. respectively.

4. The microfluidic chip according to claim 1, characterized in that: It also includes a fourth chamber separation zone and a fifth chamber identification zone, wherein the separation zone is configured as a Y-shaped channel, wherein the confluence portion of the Y-shaped channel is connected to the discharge port of the heating channel on the second heating zone, one side of the Y-shaped channel is connected to the feed port of the identification zone, and the other side merges with the discharge port of the identification zone and is connected to the channel for external discharge.

5. The microfluidic chip according to claim 1, characterized in that: The length ratio of the first heating channel to the third heating channel in the third heating zone is 3:

1.

6. The microfluidic chip according to claim 1, characterized in that: The mixing channel in the first sample loading zone is equal in length to the heating channel in the second heating zone.

7. Use of the microfluidic chip according to any one of claims 1 to 6 in preparing a microfluidic sensor.

8. A method for preparing a microfluidic sensor using the microfluidic chip according to any one of claims 1 to 6, characterized in that: The microfluidic sensor is a SERS-RCA microfluidic sensor, comprising the following steps: (1) DNA ligase and uncirculated padlock probe are added to the first loading zone, and a sample containing the target miRNA is introduced at the other end, mixed, and then the aqueous phase is divided into droplets by the immiscible carrier oil under the action of shear force, and then fully mixed in the mixing channel, and then enter the first heating channel of the third heating zone to perform enzyme denaturation to terminate the reaction, obtain a circularized padlock probe, and enter the second loading zone; (2) MBs@zip and polymerase are added to the second loading zone, mixed with the circularized padlock probe, and then enter the first heating zone. MBs@Zip specifically recognizes the zipper region of the padlock probe and initiates the RCA reaction in the presence of the polymerase. After the reaction is completed, the mixture flows into the second heating channel of the third heating zone to denature and inactivate the polymerase, obtain the RCA reaction product, and enter the third loading zone; (3) Adding a SERS probe to the third loading zone and mixing it with the RCA reaction product, the mixture enters the third heating channel of the third heating zone, unfolds the secondary structure formed by the RCA reaction product, promotes the binding with the SERS probe, and enters the second heating zone; (4) In the second heating zone, complementary base pairing occurs between the p region in the RCA reaction product with a secondary structure and the pDNA in the SERS probe to form a SERS-RCA probe, thereby preparing a SERS-RCA microfluidic sensor.

9. Use of SERS-RCA microfluidic sensor in detecting miR-21 or miR-155 in patients with interstitial pneumonia.

10. The use according to claim 9, characterized in that: miR-21 or miR-155 was specifically bound to the SERS-RCA probe on the SERS-RCA microfluidic sensor, and the SERS signal was observed.

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