Multichannel microfluidic label-free pathogen detection system and method with internal reference calibration

Abstract

A multi-channel microfluidic label-free pathogen detection system and method with internal reference correction is disclosed, relating to the field of pathogen detection. The method includes: acquiring the initial interference spectrum of each functional detection region before the reaction; injecting the sample to be tested and distributing it to multiple parallel microfluidic channels according to a preset flow path; specific binding of the target pathogen to the aptamer to form an aptamer-pathogen complex layer; acquiring the post-reaction interference spectrum of each functional detection region, comparing the post-reaction interference spectrum with the initial interference spectrum before the reaction, and calculating the change in optical path difference of each functional detection region; calculating the target detection signal and comparing it with a standard curve to obtain the concentration of the target pathogen in the sample. This invention achieves continuous, accurate, and repeatable spectral acquisition of multiple functional detection regions, without the need for nucleic acid amplification or labeling, realizing multi-channel parallel, automated acquisition, internal reference correction, and quantitative analysis of pathogen concentration, thus improving the accuracy and reliability of the detection results.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology, specifically to a multi-channel microfluidic label-free pathogen detection system and method with internal reference calibration. Background Technology

[0002] Pathogenic microorganism detection is widely used in clinical diagnosis, food safety, environmental monitoring, water quality testing, public health control, and biosafety. The presence of pathogens such as bacteria, fungi, and viruses in large quantities in samples can lead to the spread of infectious diseases, food contamination, environmental safety risks, and hospital-acquired infections. Therefore, how to quickly and accurately determine the presence of target pathogens in samples and further obtain pathogen concentration information is a crucial technical challenge in the field of pathogenic microorganism detection.

[0003] Existing pathogen detection systems mainly include culture detection systems, nucleic acid amplification detection systems, immunoassay systems, and optical detection systems. Culture detection systems typically require sample inoculation, culture, colony observation, and identification. While they can reflect viable bacteria, the detection cycle is long, making them unsuitable for rapid screening. Nucleic acid amplification detection systems usually involve sample lysis, nucleic acid extraction, amplification reaction, and amplification product detection. Although highly sensitive, they primarily detect pathogen nucleic acids and cannot directly reflect the capture of intact pathogen particles in the functional detection region. Furthermore, the amplification process can be affected by primer design, template purity, amplification efficiency, non-specific amplification, and contamination. Immunoassay systems typically utilize antibodies to recognize target pathogens or related antigens, but their performance is significantly affected by antibody affinity, specificity, stability, and batch-to-batch consistency. Some immunoassays also require labeling agents such as fluorescence, enzymes, colloidal gold, or nanoparticles, increasing the detection steps and reagent costs.

[0004] Label-free optical detection is an important development direction in biosensors in recent years. These detection systems do not rely on fluorescent, enzyme, or electrochemical labeling, but directly detect optical changes caused by target binding, such as changes in reflection intensity, interference spectrum, refractive index, optical path length, or scattering. Label-free optical detection systems based on thin-film interference or reflection interference can detect the target binding process by detecting changes in interference spectrum caused by variations in the thickness, mass, or refractive index of the biological layer on the chip surface, making them suitable for constructing label-free biosensor systems. However, label-free optical detection systems based on thin-film interference or reflection interference directly read changes in the optical thickness, refractive index, or optical path length of the chip surface. Therefore, their detection results are sensitive to non-specific adsorption, local differences in chip film thickness, sample refractive index fluctuations, fluid background changes, and optical path stability. In the detection of complex samples, relying solely on spectral changes in a single target detection area as the basis for judgment may lead to misinterpretations of non-target adhesion, sample residue, background refractive index changes, or instrument drift as target pathogen binding signals. Therefore, in multi-channel label-free optical inspection systems based on thin-film interference or reflection interference, it is necessary to set up a negative control area, a blank reference area, and a quality control calibration area that are in the same or similar detection environment as the target detection area, and improve the reliability of the detection results through internal parameter correction methods.

[0005] The literature (Rapid, Highly Sensitive, and Label-Free Pathogen Assay System Using a Solid-Phase Self-Interference Recombinase Polymerase Amplification Chip and Hyperspectral Interferometry, authors Xiangyu Jin et al., published in Analytical Chemistry, Vol. 94, pp. 2926–2933, February 2, 2022) discloses a scheme for detecting target nucleic acid amplification products by immobilizing nucleic acid capture probes on the surface of a silicon / silica chip and combining a solid-phase self-interference recombinase polymerase amplification reaction with a label-free interference spectroscopy detection system. This method involves immobilizing nucleic acid capture probes on the surface of a silicon / silica chip, placing the chip in an RPA amplification reaction system, and amplifying the target DNA in both liquid and solid phases, capturing the target amplification products on the silicon / silica chip surface. Subsequently, a label-free interference spectroscopy detection system is used to acquire the reflectance spectrum of the silicon / silica chip, and the presence of the target nucleic acid is determined based on the increase in optical length on the silicon / silica chip surface. While this method can reflect the accumulation of amplification products on the silicon / silica chip surface using spectral changes, it detects pathogen nucleic acids, not the intact pathogen itself. Samples typically still require lysis and nucleic acid extraction to obtain DNA before amplification and detection. This method cannot directly reflect the capture of intact pathogen particles on the silicon / silica chip surface and is not suitable for directly detecting the concentration of the pathogen itself. Furthermore, this method relies on the RPA amplification reaction and requires enzymes, primers, buffer solutions, magnesium ions, and temperature control. The amplification reaction can also be affected by factors such as primer design, template purity, reaction time, non-specific amplification, and primer dimers. In summary, this scheme is difficult to achieve direct, amplification-free detection of intact pathogen particles.

[0006] The literature (Highly-Sensitive, Label-Free Detection of Microorganisms and Viruses via Interferometric Reflectance Imaging Sensor, authors Monireh Bakhshpour-Yucel et al., published in Micromachines, Vol. 14, No. 2, 2023, publicly published on January 21, 2023) discloses a method to functionalize the surface of a silicon / silica chip by immobilizing antibodies, then covalently immobilizing specific antibodies to capture intact microorganisms or viruses onto the silicon / silica chip surface through antigen-antibody reactions. Finally, multi-wavelength reflection interference imaging is used to read the reflection interference changes or single-particle images, achieving label-free, whole-body pathogen detection. This approach enables label-free detection of intact pathogens, but its recognition elements typically rely on antibodies. The affinity, cross-reactivity, stability, and batch-to-batch consistency of these antibodies can affect the detection results. Furthermore, in scenarios involving multiple samples, multiple targets, or multiple concentration gradients, the lack of multi-channel microfluidic structures and automated positioning scanning structures makes it susceptible to factors such as manual chip movement, positioning errors in functional detection areas, and inconsistent reaction conditions across different functional detection areas. Additionally, while this approach can read label-free optical signals after binding to intact microorganisms or viruses, in complex sample scenarios with multiple channels or multi-functional detection areas, the absence of negative control, blank reference, and quality control calibration areas within the same channel or detection unit can lead to the accumulation of proteins, debris, and impurities in complex samples. These impurities may adhere to the silicon / silica chip, creating background signals. This makes it difficult to simultaneously subtract non-specific adsorption, local chip thickness differences, sample refractive index fluctuations, fluid background changes, and optical path drift, thus affecting the accuracy of pathogen concentration quantification.

[0007] Aptamers are recognition molecules that can specifically bind to target pathogens through a specific spatial conformation. Compared to antibodies, aptamers typically offer advantages such as chemical synthesis, ease of modification, good batch-to-batch consistency, good stability, and lower cost. By immobilizing aptamers on the surface of an interferometric detection chip, target pathogens in the sample can be captured by the aptamers as they flow through the functional detection area. The binding of the pathogens alters the equivalent optical thickness, refractive index, or interfacial optical properties of the chip surface, thereby causing changes in the interference spectrum. Therefore, combining aptamer-immobilized chips with label-free interferometric detection systems facilitates the direct detection of intact pathogens.

[0008] Microfluidic chip technology enables sample delivery, reaction, incubation, cleaning, and detection within micrometer-level channels. Compared to traditional manual addition or single-point reaction methods, multi-channel microfluidic structures help reduce sample and reagent consumption, improve reaction condition consistency, and achieve parallel detection of multiple samples or control groups through multiple independent channels. However, if a multi-channel microfluidic structure is only used for sample delivery without being combined with label-free optical detection pathways, functional detection region positioning structures, and automatic positioning scanning structures, it remains difficult to achieve continuous, accurate, and repeatable optical acquisition of multiple functional detection regions.

[0009] Existing pathogen detection technologies utilize label-free optical detection, aptamer or antibody recognition, microfluidic sample introduction, and multifunctional detection region reading to achieve pathogen-related detection. However, when directly detecting intact pathogen particles or performing parallel detection of multiple samples, multiple concentration gradients, or multifunctional detection regions, existing pathogen detection systems still suffer from insufficient coordination between the target organism, fluid reaction, functional detection region localization, and spectral acquisition, thus affecting detection throughput, automation level, and result consistency. In summary, existing pathogen detection technologies mainly suffer from the following shortcomings:

[0010] First, existing nucleic acid amplification-based detection systems mainly detect pathogen nucleic acids, which usually require sample lysis, nucleic acid extraction, amplification reaction, and detection of amplification products. The detection process is lengthy, and the detection results mainly reflect the nucleic acid information of the pathogen, making it difficult to directly reflect the capture of intact pathogen particles in the target detection area.

[0011] Second, although existing immunoassay systems can detect pathogens as a whole, they usually rely on antibodies, markers or single-point detection structures, and still have problems such as many sample processing steps, limited detection throughput, large differences in manual operation, and difficulty in continuously and stably collecting samples from multiple functional detection areas.

[0012] Third, although existing chip-based optical detection systems can achieve optical detection of a single functional detection area or a small number of functional detection areas, when multiple samples or control groups are detected in parallel or when multiple functional detection areas are read, the sample loading, reaction, and cleaning processes are prone to relying on manual chip movement, fixed single-point detection, or manual alignment point by point. It is difficult to achieve automatic positioning and continuous scanning of the target detection area within multiple microfluidic channels, resulting in differences in reaction time, cleaning conditions, and operation processes between different functional detection areas. This not only leads to low detection efficiency but also easily introduces detection position errors, affecting the consistency of detection results between different microfluidic channels.

[0013] Fourth, existing label-free optical detection systems primarily rely on changes in a single spectral signal within the target detection area for judgment. However, in the detection of complex samples, spectral changes in the target detection area may originate from the specific binding of the target pathogen, and may also be affected by factors such as non-specific adsorption, local film thickness differences on the chip, sample refractive index fluctuations, fluid background changes, and optical path drift. If the detection chip lacks a negative control area, a blank reference area, and a quality control calibration area that are in the same fluid environment and under similar optical acquisition conditions as the target detection area, it becomes difficult to distinguish between the actual pathogen capture signal and the background interference signal. It is also difficult to simultaneously eliminate the influence of non-specific adsorption, local film thickness differences on the chip, sample refractive index fluctuations, fluid background changes, and optical path drift on the detection results, affecting the accuracy of quantitative detection of pathogen concentration and the consistency between channels. Especially in label-free optical detection systems based on thin-film interference or reflection interference, if only a single functional detection area or manual chip movement is used for detection, it is difficult to simultaneously meet multiple requirements such as direct capture of intact pathogens, parallel reaction of multiple samples or multiple targets, consistent processing of multiple functional detection areas, background signal subtraction and quality control calibration in the same channel, and automated optical scanning acquisition.

[0014] Therefore, there is still a need in the existing technology for a pathogen detection system and method that can integrate specific capture of target pathogens, multi-channel fluid reaction, internal reference calibration in the same channel, automatic positioning and scanning of multiple functional detection areas, and label-free optical detection based on thin-film interference or reflection interference. Summary of the Invention

[0015] To address the problems existing in the prior art, this invention provides a multi-channel microfluidic label-free pathogen detection system and method with internal reference calibration.

[0016] This invention systematically integrates aptamer biosensing technology, a multi-channel microfluidic module with internal reference correction, a chip precision displacement module, and a beam shaping and focusing module to form a complete detection process of "sample introduction baseline acquisition—pathogen capture—channel reaction—cleaning—displacement scanning—spectral acquisition—internal reference correction—signal analysis." This enables the specific capture of target pathogens in multiple target detection areas. By automatically scanning and acquiring the interference spectral signal changes of each functional detection area, and performing internal reference correction for the same channel based on the interference spectral signal changes, the influence of non-specific adsorption, chip local film thickness differences, sample refractive index fluctuations, fluid background changes, and optical path drift on the detection results is eliminated. At the same time, the chip precision displacement module allows each functional detection area to sequentially enter the optical detection position, thereby completing continuous, accurate, and repeatable spectral acquisition of multiple functional detection areas. Ultimately, it achieves nucleic acid amplification-free, label-free, multi-channel parallel, automated acquisition, internal reference correction for the same channel, and quantitative analysis of pathogen concentration, improving the accuracy of detection results for complex samples.

[0017] The technical solution adopted by this invention to solve the technical problem is as follows:

[0018] This invention provides a multi-channel microfluidic label-free pathogen detection system with internal reference calibration, mainly comprising:

[0019] A broadband light source module outputs broadband light as the incident light for interferometric detection;

[0020] The beam shaping and focusing module collimates, splits, focuses, and collects the broadband light output from the broadband light source module;

[0021] The biochip module receives broadband light focused by the beam shaping and focusing module and reflects it back to the beam shaping and focusing module for collection;

[0022] The chip precision displacement module is used to create a relative displacement between the biochip module and the optical detection spot, so that each functional detection area enters the optical detection position in sequence.

[0023] The multi-channel microfluidic module integrated in the biochip module includes multiple independent microfluidic channel structures, each of which includes multiple functional detection regions;

[0024] The signal acquisition module receives the interference light signal reflected by the biochip module and collected by the beam shaping and focusing module, including the periodic oscillation curve of reflectivity as a function of wavelength before the reaction and the periodic oscillation curve of reflectivity as a function of wavelength after the reaction.

[0025] The control and analysis module receives and processes the periodic oscillation curves of reflectance before and after the reaction as a function of wavelength, output by the signal acquisition module. It calculates the spectral peak position shift, phase change, or optical path difference change of each functional detection area. Combined with the changes in the interference spectral signals of each functional detection area, it performs internal parameter correction for the same channel to eliminate the influence of non-specific adsorption, chip local film thickness differences, sample refractive index fluctuations, fluid background changes, and optical path drift on the detection results.

[0026] Furthermore, the beam shaping and focusing module includes a collimating lens, a beam splitter, an objective lens, a converging lens, an aperture, and a receiving optical fiber; the broadband light output from the broadband light source module is collimated by the collimating lens, guided by the beam splitter, and transmitted to the biochip module by the objective lens in sequence. The interference light signal formed by the reflection of the biochip module is then collected by the objective lens, reflected internally by the beam splitter, and focused by the converging lens in sequence, and finally transmitted to the signal acquisition module through the aperture and the receiving optical fiber.

[0027] Furthermore, the biochip module includes a silicon substrate, a silicon dioxide thin film layer, and a transparent cover sheet arranged sequentially, and the multi-channel microfluidic module is integrated on the silicon dioxide thin film layer; the basic optical path difference caused by the silicon dioxide thin film layer is expressed as: OPD0=2nd; OPD0 is the basic optical path difference corresponding to the silicon dioxide thin film layer before the reaction, n is the refractive index of the silicon dioxide thin film layer, and d is the physical thickness of the silicon dioxide thin film layer.

[0028] Furthermore, each microfluidic channel structure also includes: multiple positioning markers in the non-reactive area located on the outside of the microfluidic channel structure, multiple parallel sample inlets, multiple parallel microfluidic channels, and multiple parallel waste outlets; the sample inlets are connected to the inlet of the microfluidic channel, and the waste outlets are connected to the outlet of the microfluidic channel; the microfluidic channels are used to introduce test samples, standard solutions, quality control solutions, and cleaning solutions; multiple functional detection areas include a target detection area, a negative control area, a blank reference area, and a quality control calibration area.

[0029] Furthermore, the target detection area is immobilized with an aptamer that specifically binds to the target pathogen, used to specifically capture the target pathogen and obtain the target binding signal; the negative control area is immobilized with random sequences, mismatch sequences, or non-specific recognition molecules, used to reflect non-specific adsorption, impurity adhesion, and sample matrix interference signals; the blank reference area is only provided with a sealing layer without immobilizing the aptamer, used to reflect local thickness differences of the biochip module, sample refractive index fluctuations, fluid background changes, and optical path drift; the quality control calibration area is provided with a standard film layer, standard particles, a film layer of known thickness, or a positive quality control reaction layer, used to calibrate the differences in optical response between different channels, different detection batches, or different scanning positions.

[0030] Furthermore, before the detection begins, the control and analysis module scans these positioning marks and establishes a chip coordinate system, converting each functional detection area into corresponding scanning coordinates. The chip precision displacement module corrects the positional deviation of the biochip module based on the positioning marks and scanning coordinates, and then moves along a preset path. The signal acquisition module sequentially acquires the periodic oscillation curves of the pre-reaction reflectivity of each functional detection area as a baseline signal. The control and analysis module controls an external microfluidic pump valve system to deliver the sample to be tested to multiple microfluidic channels, allowing the target pathogen to be captured by the aptamer within the target detection area. After the detection is completed, the control and analysis module controls the external microfluidic pump valve system to introduce cleaning fluid into the corresponding microfluidic channels, allowing the cleaning fluid to flow along the sample injection direction through each functional detection area and then be discharged from the waste outlet. After cleaning, the chip precision displacement module drives the biochip module back to the scanning coordinates corresponding to the baseline signal acquisition, and the signal acquisition module again acquires the periodic oscillation curves of the post-reaction reflectivity of each functional detection area as a baseline signal.

[0031] Furthermore, it also includes a result display module connected to the control and analysis module, which is used to display the test results, microfluidic channel number, target pathogen species, positive / negative judgment, concentration quantification results, quality control status, and test report.

[0032] This invention provides a multi-channel microfluidic label-free pathogen detection method with internal reference calibration, which mainly includes the following steps:

[0033] Step S1: Fix the biochip module onto the chip precision displacement module;

[0034] Step S2: The signal acquisition module sequentially acquires the periodic oscillation curves of the pre-reaction reflectance of each functional detection area before sample introduction as a baseline signal.

[0035] Step S3: The control and analysis module controls the external microfluidic pump valve system to inject the sample to be tested into the multi-channel microfluidic module and distribute it to multiple parallel microfluidic channels according to the preset flow path, so that the sample to be tested flows through each functional detection area respectively.

[0036] Step S4: The target pathogen in the sample to be tested specifically binds to the aptamer, forming an aptamer-pathogen complex layer on the surface of the target detection area;

[0037] Step S5: Inject cleaning solution to remove unbound pathogens, nonspecific adsorbents, sample impurities, and residual samples;

[0038] Step S6: The signal acquisition module acquires the periodic oscillation curve of the reflectance after the formation of the aptamer-pathogen complex layer in each functional detection area as a function of wavelength. The control and analysis module compares the periodic oscillation curve of the reflectance after the reaction as a function of wavelength with the periodic oscillation curve of the reflectance before the reaction as a function of wavelength, and calculates the spectral peak position shift, phase change or optical path difference change in each functional detection area.

[0039] Step S7: The control and analysis module calculates the target detection signal and compares it with a pre-established standard curve of a standard sample with a known concentration of target pathogen to obtain the concentration of target pathogen in the sample to be tested. The final detection conclusion is displayed through the structure display module.

[0040] Furthermore, the formula for calculating the periodic oscillation curve of the pre-reaction reflectance as a function of wavelength is as follows:

[0041] ;

[0042] in, This is a periodic oscillation curve showing the change in reflectivity as a function of wavelength before the reaction. Where λ is the wavelength, U1 and U2 are the amplitudes of the two reflected beams, U is the amplitude of the incident light, n is the refractive index of the silicon dioxide thin film, and d is the physical thickness of the silicon dioxide thin film.

[0043] The formula for calculating the periodic oscillation curve of reflectivity as a function of wavelength after the reaction is as follows:

[0044] ;

[0045] in, This is a periodic oscillation curve showing the change in reflectivity with wavelength after the reaction. The change in equivalent optical path difference caused by the binding of the target pathogen, n bio σ is the equivalent refractive index of the aptamer-pathogen complex layer, and σ is the equivalent optical thickness increase relative to the baseline state before the reaction.

[0046] The periodic oscillation curve of reflectivity as a function of wavelength before the reaction and the periodic oscillation curve of reflectivity as a function of wavelength after the reaction. A Fast Fourier Transform (FFT) is performed to convert the signal from the wavelength domain to the frequency domain. In the frequency domain, the main peak of the interference spectrum corresponds to the inherent optical thickness of the silicon dioxide thin film. The position *m* of the main peak represents the frequency component corresponding to this inherent optical thickness. The phase value corresponding to the main peak position *m* is found in the complex result generated by the FFT. This phase value reflects the phase state of the reflected interference spectrum at the main peak frequency component. The phase difference before and after the reaction is used to characterize the change in the equivalent optical thickness or optical path difference of the detection area. Based on the phase value, the phases of the two interference spectrum signals before and after the reaction are calculated. The change in optical thickness is determined by the difference between these two phases, and the calculation formula is as follows:

[0047] ;

[0048] in, The phase difference between the reflection interference spectra before and after the reaction at the main peak position m is denoted as m.

[0049] Furthermore, based on the periodic oscillation curves of reflectance versus wavelength after the reaction and before the reaction, the following data were calculated: phase change ΔφA in the target detection area, phase change ΔφB in the negative control area, phase change ΔφC in the blank reference area, and response change ΔφD in the quality control calibration area. Then, based on these data, the target detection signal Δφ after internal reference correction was calculated. 校正 The calculation formula is as follows:

[0050] .

[0051] The beneficial effects of this invention are:

[0052] This invention provides a multi-channel microfluidic label-free pathogen detection system with internal reference correction. An aptamer is used for the specific capture of the target pathogen. A multi-channel microfluidic module is used for sample and washing solution delivery. The microfluidic channel structure is used to improve detection throughput. Each microfluidic channel structure includes a target detection area, a negative control area, a blank reference area, and a quality control calibration area, used to obtain target binding signals, non-specific adsorption background, fluid and optical path background, and quality control calibration signals, respectively. A chip precision displacement module is used to move the biochip module along a preset path. A signal acquisition module is used to acquire the interference spectral signals of each functional detection area. After the pathogen is captured by the aptamer, it changes the equivalent optical thickness, refractive index, or optical path difference of the target detection area surface, causing changes in the interference spectrum. By establishing a standard curve between the corrected optical signal change and the pathogen concentration, the concentration of the target pathogen in the sample can be detected.

[0053] This invention realizes a complete detection process from sample introduction, pathogen capture, channel reaction, cleaning treatment to automatic scanning acquisition and signal analysis. Compared with existing technologies, it has the following advantages:

[0054] 1. The target pathogen recognition element uses an aptamer. The aptamer is fixed in the target detection area to directly capture the target pathogen without the need for nucleic acid amplification or labeling, thus achieving complete pathogen detection.

[0055] 2. Multiple independent microfluidic channel structures are designed on the biochip module. Each microfluidic channel includes a target detection area, a negative control area, a blank reference area, and a quality control calibration area. The effects of non-specific adsorption, sample refractive index fluctuation, chip thickness difference, and optical path drift on the detection results are automatically eliminated through internal reference correction, thereby improving the detection accuracy.

[0056] Compared with label-free detection schemes that rely solely on changes in a single signal in the target detection area, this invention can correct for the effects of non-specific adsorption, sample refractive index fluctuations, chip thickness differences, and optical path drift on the detection results by using the same channel's internal parameters. This makes the corrected target detection signal closer to the true optical response generated by the specific capture of the target pathogen, thereby improving the accuracy and consistency between channels in complex sample detection and multi-channel quantitative analysis.

[0057] 3. This invention uses a chip precision displacement module to move the biochip module along a preset path, allowing the functional detection areas within each microfluidic channel to sequentially enter the optical detection position. This enables continuous, accurate, and repeatable spectral acquisition across multiple channels and regions, improving system automation and throughput. Compared to manual chip movement or single-point fixed detection methods, this invention reduces detection position errors and human error, increasing throughput and automation in multi-sample detection. Attached Figure Description

[0058] Figure 1 The present invention provides a structural block diagram of a multi-channel microfluidic label-free pathogen detection system with internal reference calibration.

[0059] Figure 2 This is the optical path diagram of the beam shaping and focusing module.

[0060] Figure 3 The schematic diagram illustrates the implementation principle of a multi-channel microfluidic label-free pathogen detection system with internal reference calibration provided by this invention.

[0061] Figure 4 This is a schematic diagram of a multi-channel microfluidic module.

[0062] Figure 5 This is a simulation of the interference spectrum effect before and after the reaction.

[0063] Figure 6 The flowchart of a multi-channel microfluidic label-free pathogen detection method with internal reference calibration provided by the present invention is shown. Detailed Implementation

[0064] In a first aspect, the present invention provides a multi-channel microfluidic label-free pathogen detection system with internal reference calibration.

[0065] like Figures 1 to 4 As shown, the present invention provides a multi-channel microfluidic label-free pathogen detection system with internal reference calibration, which mainly includes:

[0066] The module consists of: a broadband light source module 1, a beam shaping and focusing module 2, a biochip module 3, a chip precision displacement module 4, a multi-channel microfluidic module 5, a signal acquisition module 6, a control and analysis module 7, and a result display module 8.

[0067] According to the present invention, the broadband light source module 1 is mainly used to provide broadband detection light. The broadband light output by the broadband light source module 1 is used as the incident light for interferometric detection. Preferably, a halogen lamp with a spectral range of 350nm-1000nm is used. The halogen lamp can provide continuous and uniform broadband white light, covering the visible light to near-infrared region, which is suitable for interferometric detection.

[0068] According to the present invention, the beam shaping and focusing module 2 is disposed between the broadband light source module 1 and the biochip module 3, and is mainly used for collimating, splitting, focusing and collecting the broadband light output from the broadband light source module 1. Specifically, the broadband light output from the broadband light source module 1 is collimated, split, focused and collected by the beam shaping and focusing module 2 and then irradiates the functional detection area of ​​the biochip module 3. The interference light reflected from the functional detection area is then collected by the same beam shaping and focusing module 2 and transmitted to the signal acquisition module 6.

[0069] Specifically, such as Figure 2 As shown, the beam shaping and focusing module 2 mainly includes optical components such as a collimating lens 201, a beam splitter 202, an objective lens 203, a converging lens 204, an aperture 205, and a receiving fiber 206. The collimating lens 201 collimates the broadband light from the broadband light source module 1 into parallel light. The beam splitter 202 guides the collimated broadband light to the objective lens 203 and redirects the interference light reflected from the biochip module 3 to the converging lens 204. The reflected interference light is collected by the objective lens 203 and transmitted to the beam splitter 202, where it is reflected internally. Finally, it is transmitted sequentially through the converging lens 204, the aperture 205, and the receiving fiber 206 to the signal acquisition module 6. The objective lens 203 is mainly used to focus the broadband light onto the functional detection area of ​​the biochip module 3 and collect the interference light signal reflected from the functional detection area of ​​the biochip module 3.

[0070] According to the present invention, the biochip module 3 preferably employs a silicon / silicon dioxide thin-film interference chip. For example... Figure 4 As shown, the biochip module 3 mainly includes a silicon substrate 301, a silicon dioxide thin film layer 302, and a transparent cover plate 303 (preferably a quartz cover plate). The silicon substrate 301, the silicon dioxide thin film layer 302, and the transparent cover plate 303 are arranged in sequence. The multi-channel microfluidic module 5 is integrated on the biochip module 3.

[0071] The detection principle of this invention is based on the thin-film interference effect. Thin-film interference is mainly formed by the optical path difference between the reflected light from the upper interface (air / silicon dioxide interface) and the reflected light from the lower interface (silicon / silicon dioxide interface) of the biochip module 3. Specifically, as... Figure 5 As shown, the biochip module 3 uses a silicon substrate 301 as the bottom layer and a silicon dioxide thin film layer 302 as the interference layer, with a multi-channel microfluidic module 5 integrated on the surface of the silicon dioxide thin film layer 302. During detection, broadband light is focused by the beam shaping and focusing module 2 onto the functional detection area within the microfluidic channel 506. Since the functional detection area is in the sample liquid and cleaning liquid environment, the upper interface (air / silica interface) of the functional detection area of ​​the biochip module 3 can be approximated as the equivalent optical interface formed between the liquid medium within the microfluidic channel 506, the surface biorecognition layer, and the silicon dioxide thin film layer 302, while the lower interface (silicon / silica interface) is the interface between the silicon dioxide thin film layer 302 and the silicon substrate 301. Incident light is reflected at the above different interfaces, and interference spectra are formed between the different reflected light due to the optical path difference. When the target pathogen 10 is captured by this aptamer 9, the equivalent optical thickness, refractive index, or optical path difference of the surface of each functional detection area of ​​the biochip module 3 will change, thereby causing changes in the interference spectrum signal.

[0072] Specifically, under the condition of approximately perpendicular incidence of broadband light, the basic optical path difference caused by the silicon dioxide thin film layer 302 can be expressed as: OPD0 = 2nd; where OPD0 is the basic optical path difference corresponding to the silicon dioxide thin film layer 302 before the reaction, n is the refractive index of the silicon dioxide thin film layer 302 (approximately 1.47), and d is the physical thickness of the silicon dioxide thin film layer 302.

[0073] According to the present invention, the biochip module 3 is fixed on the chip precision displacement module 4. The chip precision displacement module 4 is mainly used to generate a relative displacement between the biochip module 3 and the optical detection spot, so that the target detection area 507, negative control area 508, blank reference area 509 and quality control calibration area 510 in different microfluidic channels 506 can sequentially enter the optical detection position.

[0074] Specifically, the chip precision displacement module 4 can employ an electric displacement stage, a piezoelectric displacement stage, a multi-axis precision displacement platform with a Z-axis focusing mechanism, an optical scanning mirror, a MEMS scanner, a movable fiber optic probe, or a multi-path switching structure. When the chip precision displacement module 4 uses a multi-axis precision displacement platform with a Z-axis focusing mechanism, the multi-axis precision displacement platform is used to generate relative displacement in the X and Y directions between the biochip module 3 and the optical detection spot, and the Z-axis focusing mechanism is used to adjust the distance between the functional detection area and the objective lens 203. During detection, the chip precision displacement module 4 sequentially moves the target detection area 507, the negative control area 508, the blank reference area 509, and the quality control calibration area 510 into the optical detection position according to a preset serpentine or matrix path, thereby achieving continuous, accurate, and repeatable spectral acquisition of multiple microfluidic channels 506 and multiple functional detection areas.

[0075] According to the present invention, the multi-channel microfluidic module 5 is integrated in the biochip module 3, which includes multiple independent microfluidic channel structures. The microfluidic channel structures are prepared by the following method: using negative photoresist SU-8 and UV-curable optical adhesive NOA, a transparent microfluidic channel structure is formed on the silicon dioxide thin film layer 302 through photolithography. The bottom surface of the channel in the functional detection area is still the complete silicon dioxide thin film layer 302, which does not affect the occurrence of thin film interference. By covering the microfluidic channel structure with a transparent cover plate 303 with low reflection and high transmittance to form a closed structure, the occurrence of liquid spillage, contamination and other problems can be effectively prevented.

[0076] Specifically, each microfluidic channel structure includes: positioning marker 1 501, positioning marker 2 502, positioning marker 3 503, positioning marker 4 504, multiple parallel injection ports 505, multiple parallel microfluidic channels 506, target detection area 507, negative control area 508, blank reference area 509, quality control calibration area 510, and multiple parallel waste ports 511. Positioning markers 1 501, 2 502, 3 503, and 4 504 are all located in the non-reactive region outside the microfluidic channel structure. The number of sample inlets 505, microfluidic channels 506, and waste outlets 511 are all the same. For example, four parallel sample inlets 505, four parallel microfluidic channels 506, and four parallel waste outlets 511 can be set. The sample inlets 505 are connected to the inlets of the microfluidic channels 506, and the outlets of the microfluidic channels 506 are connected to the waste outlets 511. The first three of the four parallel microfluidic channels 506 are used to introduce different samples, while the fourth microfluidic channel 506 is used to introduce standard solutions or quality control solutions. On the other hand, cleaning... The liquid can share the same inlet 505 as the sample, and the sample liquid path and the cleaning liquid path can be switched through an external microfluidic pump valve system. After the sample reaction is completed, the control and analysis module 7 controls the microfluidic pump valve system to introduce the cleaning liquid into the corresponding microfluidic channel 506, so that the cleaning liquid flows along the sample injection direction through the target detection area 507, the negative control area 508, the blank reference area 509 and the quality control calibration area 510, and is finally discharged from the waste liquid outlet 511, thereby removing unbound pathogens, non-specific adsorbents, sample impurities and residual samples.

[0077] Specifically, the microfluidic channel 506 is not limited to a linear structure, but can also be a serpentine channel, a bifurcation channel, a ring channel, a gradient dilution channel, a droplet microfluidic channel, or a composite microfluidic structure that includes functional regions for mixing, incubation, rinsing, and enrichment.

[0078] Specifically, the target detection area 507 is equipped with an aptamer 9 that specifically binds to the target pathogen, used to specifically capture the target pathogen 10 and obtain the target binding signal. It is the main functional detection area for finally determining the pathogen concentration. The negative control area 508 is equipped with random sequences, mismatched sequences, or non-specific recognition molecules, used to reflect background signals such as non-specific adsorption, impurity adhesion, and sample matrix interference. The blank reference area 509 is equipped with only a sealing layer without aptamer 9, used to reflect local thickness differences of the biochip module 3, sample refractive index fluctuations, fluid background changes, and optical path drift. The quality control calibration area 510 is equipped with a standard membrane layer, standard particles, a membrane layer of known thickness, or a positive quality control reaction layer, used to calibrate the differences in optical response between different channels, different detection batches, or different scanning positions. The aptamer 9 is fixed in the target detection area 507. When the target pathogen 10 in the sample flows through the target detection area 507 via the microfluidic channel 506, it is captured by the aptamer 9. When the sample flows through each functional detection area within the same microfluidic channel 506, the system collects the interference spectra of each functional detection area before and after the reaction. Based on the changes in the interference spectrum signals of the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510, the system performs internal parameter correction of the same microfluidic channel 506 to eliminate the influence of non-specific adsorption, chip local film thickness differences, sample refractive index fluctuations, fluid background changes, and optical path drift on the detection results.

[0079] Before the detection begins, the control and analysis module 7 scans or identifies these positioning markers (positioning marker 1 501, positioning marker 2 502, positioning marker 3 503, positioning marker 4 504), then establishes a chip coordinate system, and converts the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510 in each microfluidic channel 506 into corresponding scanning coordinates. The chip precision displacement module 4 corrects the translational deviation, rotational deviation, and installation deviation of the biochip module 3 according to the positioning markers and scanning coordinates, and then moves according to a preset path (serpentine path or matrix path). The signal acquisition module 6 sequentially acquires the periodic oscillation curve of the pre-reaction reflectivity of each functional detection area in each microfluidic channel 506 as a baseline signal. Subsequently, the control and analysis module 7 drives the microfluidic pump valve system to deliver the sample to be tested to multiple microfluidic channels 506, allowing the target pathogen 10 to be captured by the aptamer 9 within the target detection area 507. After detection, cleaning solution is injected into each microfluidic channel 506 to remove unbound pathogens 10, non-specific adsorbates, sample impurities, and residual samples. After cleaning, the chip precision displacement module 4 drives the biochip module 3 back to the scanning coordinates corresponding to the baseline signal acquisition, and the signal acquisition module 6 again acquires the periodic oscillation curves of the reflectivity of each functional detection area after reaction versus wavelength.

[0080] Specifically, the internal reference calibration structure is not limited to a fixed combination of four areas: target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510. Two or three functional detection areas can be set according to the detection requirements, or multiple repeated target detection areas, multiple repeated negative control areas, multiple repeated blank reference areas, or multiple repeated quality control calibration areas can be set to achieve background subtraction, channel normalization, or inter-batch calibration.

[0081] According to the present invention, the signal acquisition module 6 is mainly used to receive the interference light signal reflected by the biochip module 3 and transmitted by the beam shaping and focusing module 2, and convert it into spectral data that can be processed by the control and analysis module 7.

[0082] Specifically, the signal acquisition module 6 can be a spectrometer or a photodetector, used to acquire the interference spectra of each functional detection area before and after the reaction.

[0083] Specifically, the detection process can be set up as a two-round scan:

[0084] First round: Baseline scan before reaction. The chip precision displacement module 4 drives the biochip module 3 to scan the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510 of all microfluidic channels 506 in sequence, and collect the periodic oscillation curve of reflectivity as a function of wavelength before reaction.

[0085] Second round: Post-reaction detection scanning. After the sample reaction and cleaning are completed, the chip precision displacement module 4 drives the biochip module 3 back to the same position coordinates, and scans the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510 of all microfluidic channels 506 again, and collects the periodic oscillation curve of reflectivity changing with wavelength after the reaction.

[0086] According to the present invention, the control and analysis module 7 is mainly used to uniformly control and process data of various functional modules of the system. On the one hand, the control and analysis module 7 controls the working state of the broadband light source module 1, the sample injection and cleaning process of the multi-channel microfluidic module 5, the scanning path of the chip precision displacement module 4, and the acquisition timing of the signal acquisition module 6. On the other hand, the control and analysis module 7 processes the acquired interference spectra before and after the reaction, calculates the spectral peak position shift, phase change, or optical path difference change of the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510, and performs internal parameter correction of the same channel in combination with the interference spectral signal changes of the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510 to deduct the influence of non-specific adsorption, chip local film thickness difference, sample refractive index fluctuation, fluid background change, and optical path drift on the detection results, and finally obtains the target detection signal related to the pathogen concentration. The target detection signal after internal reference calibration can be compared with the pre-established standard curve of the target pathogen standard sample with known concentration to obtain the concentration of the target pathogen in the sample to be tested. Combined with the position number of different microfluidic channels 506 or functional detection areas, the detection results of multiple samples, multiple targets or multiple concentration gradients are output. Finally, the detection conclusion is displayed through the structure display module 8.

[0087] According to the present invention, the result display module 8 is connected to the control and analysis module 7 and is used to display the detection results, microfluidic channel number, target pathogen species, positive / negative judgment, concentration quantification results, quality control status, and detection report. For multi-microfluidic channel detection results, the result display module 8 can display the detection conclusions according to the microfluidic channel number, sample number, or target number.

[0088] According to the present invention, the target pathogen recognition element is preferably an aptamer. The aptamer can be a DNA aptamer, RNA aptamer, modified nucleic acid aptamer, peptide aptamer, or a combination thereof, and can be set in the target detection region 507 by covalent bonding, affinity interaction, physical adsorption, or other immobilization methods. Aptamer capture does not require lysis and nucleic acid extraction, nor does it require enzymes and amplification systems, thus avoiding the risk of contamination. The aptamer has advantages such as being chemically synthesizable, having good batch-to-batch consistency, being easy to modify, having good stability, and being low in cost.

[0089] Secondly, the present invention provides a multi-channel microfluidic label-free pathogen detection method with internal reference calibration.

[0090] The present invention provides a multi-channel microfluidic label-free pathogen detection method with internal reference calibration. The overall process includes: baseline acquisition before sample injection, injection of the sample to be tested, capture of the target pathogen, microfluidic channel reaction, cleaning treatment, displacement scanning, spectral acquisition after reaction, internal reference calibration and signal analysis; each step is uniformly and coordinated by the control and analysis module 7.

[0091] This invention provides a multi-channel microfluidic label-free pathogen detection method with internal reference correction. The detection principle is based on the thin-film interference effect. After broadband detection light illuminates the functional detection area of ​​the biochip module 3, reflected light is generated at different optical interfaces. Multiple reflected beams form an interference spectrum due to optical path differences. An aptamer capable of specifically recognizing the target pathogen is pre-fixed on the surface of the target detection area 507. When the sample flows through the target detection area 507, the target pathogen specifically binds to the aptamer, forming an aptamer-pathogen composite layer on the surface of the target detection area 507. This aptamer-pathogen composite layer alters the equivalent optical thickness, refractive index, or optical path difference of the target detection area 507 surface, thereby causing changes in the peak position, phase, or optical path difference of the interference spectrum.

[0092] like Figure 6 As shown, the present invention provides a multi-channel microfluidic label-free pathogen detection method with internal reference calibration, the specific implementation process of which is as follows:

[0093] Step S1: Installation and positioning of biochip module 3;

[0094] The biochip module 3 is fixed on the chip precision displacement module 4. The position coordinates of each functional detection area are determined by visual positioning or mechanical limiting, and the correspondence between each functional detection area and the beam shaping and focusing module 2 is established.

[0095] Step S2: Collect the periodic oscillation curves of the pre-reaction reflectivity of each functional detection area as a function of wavelength;

[0096] The broadband light source module 1 emits broadband detection light, which is collimated, split, focused, and collected by the beam shaping and focusing module 2 and then irradiated onto each functional detection area of ​​the biochip module 3. The interference light reflected from the functional detection areas is then collected by the same beam shaping and focusing module 2 and transmitted to the signal acquisition module 6. The signal acquisition module 6 sequentially acquires the periodic oscillation curve of the pre-reaction reflectivity of each functional detection area before sample introduction as a function of wavelength, which serves as the baseline signal for subsequent detection.

[0097] For a specific wavelength Its thin-film interference effect can be described by the following formula:

[0098] ;

[0099] in, The figure shows a periodic oscillation curve of reflectivity as a function of wavelength before the reaction. U1 and U2 are the amplitudes of the two reflected beams, respectively, U is the amplitude of the incident light, n is the refractive index of the silicon dioxide thin film layer 302 (approximately 1.47), and d is the physical thickness of the silicon dioxide thin film layer 302. This formula describes the periodic oscillation curve of reflectivity as a function of wavelength, which is the observed interference spectrum.

[0100] Step S3: Inject the sample to be tested into multiple parallel microfluidic channels 506;

[0101] The control and analysis module 7 drives the microfluidic pump valve system to inject the sample to be tested into the multi-channel microfluidic module 5 and distribute it to multiple parallel microfluidic channels 506 according to the preset flow path, so that the sample to be tested flows through each functional detection area.

[0102] Specifically, the precision displacement module 4 of the control chip moves the biochip module 3 according to a preset path (serpentine path or matrix path), so that the target detection area 507, negative control area 508, blank reference area 509 and quality control calibration area 510 enter the optical detection position in sequence, realizing automatic sequential scanning of multiple functional detection areas on the same biochip module 3.

[0103] Specifically, such as Figure 4 The solid arrows represent the sample injection direction, and the dashed arrows represent the scanning direction of the biochip module 3. The process involves first identifying the positioning markers → aligning with the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510 of the first microfluidic channel 506 → moving down to the quality control calibration area 510, blank reference area 509, negative control area 508, and target detection area 507 of the second microfluidic channel 506 → moving down to the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510 of the third microfluidic channel 506 → moving down to the quality control calibration area 510, blank reference area 509, negative control area 508, and target detection area 507 of the fourth microfluidic channel 506. The advantage of this serpentine path is that it involves a short movement distance, fast scanning speed, and continuous acquisition of the four functional detection areas of each microfluidic channel 506, reducing time drift.

[0104] Step S4: Specifically bind the target pathogen in the sample to the aptamer in the target detection area 507;

[0105] When the sample to be tested contains the target pathogen 10, the target pathogen 10 specifically binds to the aptamer 9 fixed on the surface of the target detection area 507, forming an aptamer-pathogen composite layer on the surface of the target detection area 507, causing changes in the equivalent optical thickness, refractive index, or optical path difference of the surface of the target detection area 507.

[0106] Step S5: Inject cleaning solution to remove unbound pathogens, nonspecific adsorbents, sample impurities, and residual samples;

[0107] After the sample reaction is completed, the control and analysis module 7 controls the microfluidic pump valve system to inject cleaning solution into each microfluidic channel 506, so that the cleaning solution flows through each functional detection area in sequence, and carries away pathogens, non-specific adsorbents, sample impurities and residual samples that have not specifically bound to the aptamer 9 from the functional detection area and discharges them through the waste liquid port 511, so as to reduce background interference and improve the accuracy of interference spectroscopy detection results.

[0108] Step S6: Collect the periodic oscillation curve of reflectance as a function of wavelength after the reaction, and calculate the spectral peak position shift, phase change, or optical path difference change.

[0109] The signal acquisition module 6 acquires the periodic oscillation curve of the reflectance after the formation of the aptamer-pathogen complex layer in each functional detection area as a function of wavelength. The control and analysis module 7 compares the periodic oscillation curve of the reflectance after the reaction as a function of wavelength with the periodic oscillation curve of the reflectance before the reaction as a function of wavelength, and calculates the spectral peak position shift, phase change or optical path difference change in each functional detection area.

[0110] Specifically, after the target pathogen 10 is captured by the aptamer 9, an aptamer-pathogen complex layer is added to the surface of the functional detection region relative to the baseline state, causing changes in the equivalent optical thickness, refractive index, or optical path difference of the functional detection region. This change can be described by the following formula:

[0111] ;

[0112] in, This is a periodic oscillation curve showing the change in reflectivity with wavelength after the reaction. The change in equivalent optical path difference caused by the binding of the target pathogen, n bio σ is the equivalent refractive index of the aptamer-pathogen complex layer, and σ is the equivalent optical thickness increase relative to the baseline state before the reaction.

[0113] The periodic oscillation curve of reflectivity as a function of wavelength before the reaction and the periodic oscillation curve of reflectivity as a function of wavelength after the reaction. A Fast Fourier Transform (FFT) is performed to transform the signal from the wavelength domain to the frequency domain. In the frequency domain, the main peak of the interference spectrum corresponds to the inherent optical thickness (n*d) of the silicon dioxide thin film layer 302. The position of the main peak (labeled m) represents the frequency component corresponding to this inherent optical thickness. In the complex result generated by the FFT, the phase value corresponding to the main peak position m is found. This phase value directly reflects the precise position of the interference fringes. The phase difference is calculated based on the phase value: the phases of the two interference spectrum signals before the reaction (baseline) and after the reaction (with pathogen capture) are calculated separately. The change in optical thickness is determined by the difference between these two phases, and the specific calculation formula is as follows:

[0114] ;

[0115] in, The phase difference between the reflection interference spectra before and after the reaction at the main peak position m is denoted as m.

[0116] Step S7: Calculate the target detection signal and compare it with a pre-established standard curve of a standard sample with a known concentration of target pathogen to obtain the concentration of target pathogen in the sample to be tested;

[0117] The control and analysis module 7 calculates the target detection signal after internal reference correction in the same microfluidic channel 506 based on the changes in the interference spectrum before and after the reaction in the target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510. It then compares the target detection signal after internal reference correction with a pre-established standard curve of a standard sample of a known concentration of target pathogen to obtain the concentration of target pathogen in the sample to be tested. Combined with the position number of different microfluidic channels 506 or functional detection areas, it outputs the detection results of multiple samples, multiple targets, or multiple concentration gradients. Through the correspondence between the change in optical thickness and the concentration of target pathogen, it performs quantitative and qualitative analysis of the target pathogen. Finally, the detection conclusion can be displayed through the structure display module 8.

[0118] Specifically, based on the above formula, interference spectral signals before and after the reaction are collected for each of the four functional detection areas—target detection area 507, negative control area 508, blank reference area 509, and quality control calibration area 510—in each microfluidic channel 506, and the following data are calculated respectively:

[0119] Phase change ΔφA in target detection area 507;

[0120] Phase change ΔφB in negative control region 508;

[0121] Phase change ΔφC in blank reference area 509;

[0122] Phase change (calibration coefficient) ΔφD in quality control calibration area 510.

[0123] Then, based on the above data, the target detection signal after intrinsic parameter correction is calculated, and the specific calculation formula is as follows:

[0124] ;

[0125] The target detection signal after internal control calibration contains not only the target pathogen binding signal, but may also contain non-specific adsorption, local film thickness differences on the chip, sample refractive index fluctuations, fluid background changes, and optical path drift. The negative control area 508 is used to subtract non-specific adsorption, the blank reference area 509 is used to subtract fluid background changes and optical path drift, and the quality control calibration area 510 is used to normalize the instrument response and inter-batch differences of the biochip module. The target detection signal obtained after internal control calibration is closer to the actual target pathogen capture signal.

[0126] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multi-channel microfluidic label-free pathogen detection system with internal reference calibration, characterized in that, include: A broadband light source module outputs broadband light as the incident light for interferometric detection; The beam shaping and focusing module collimates, splits, focuses, and collects the broadband light output from the broadband light source module; The biochip module receives broadband light focused by the beam shaping and focusing module and reflects it back to the beam shaping and focusing module for collection; The chip precision displacement module is used to create a relative displacement between the biochip module and the optical detection spot, so that each functional detection area enters the optical detection position in sequence. The multi-channel microfluidic module integrated in the biochip module includes multiple independent microfluidic channel structures, each of which includes multiple functional detection regions; The signal acquisition module receives the interference light signal reflected by the biochip module and collected by the beam shaping and focusing module, including the periodic oscillation curve of reflectivity as a function of wavelength before the reaction and the periodic oscillation curve of reflectivity as a function of wavelength after the reaction. The control and analysis module receives and processes the periodic oscillation curves of reflectance before and after the reaction as a function of wavelength, output by the signal acquisition module. It calculates the spectral peak position shift, phase change, or optical path difference change of each functional detection area. Combined with the changes in the interference spectral signals of each functional detection area, it performs internal parameter correction for the same channel to eliminate the influence of non-specific adsorption, chip local film thickness differences, sample refractive index fluctuations, fluid background changes, and optical path drift on the detection results.

2. The multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to claim 1, characterized in that, The beam shaping and focusing module includes a collimating lens, a beam splitter, an objective lens, a converging lens, an aperture, and a receiving optical fiber. The broadband light output from the broadband light source module is collimated by the collimating lens, guided by the beam splitter, and transmitted to the biochip module through the objective lens. The interference light signal formed by the reflection of the biochip module is then collected by the objective lens, reflected internally by the beam splitter, and focused by the converging lens before finally being transmitted to the signal acquisition module through the aperture and the receiving optical fiber.

3. The multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to claim 1, characterized in that, The biochip module includes a silicon substrate, a silicon dioxide thin film layer, and a transparent cover sheet arranged sequentially. The multi-channel microfluidic module is integrated on the silicon dioxide thin film layer. The basic optical path difference caused by the silicon dioxide thin film layer is expressed as: OPD0=2nd; OPD0 is the basic optical path difference corresponding to the silicon dioxide thin film layer before the reaction, n is the refractive index of the silicon dioxide thin film layer, and d is the physical thickness of the silicon dioxide thin film layer.

4. The multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to claim 1, characterized in that, Each microfluidic channel structure also includes: multiple positioning markers in the non-reactive area located on the outside of the microfluidic channel structure, multiple parallel injection ports, multiple parallel microfluidic channels, and multiple parallel waste ports; the injection ports are connected to the inlet of the microfluidic channel, and the waste ports are connected to the outlet of the microfluidic channel; the microfluidic channels are used to introduce test samples, standard solutions, quality control solutions, and cleaning solutions; multiple functional detection areas include a target detection area, a negative control area, a blank reference area, and a quality control calibration area.

5. The multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to claim 4, characterized in that, The target detection area is equipped with an aptamer that specifically binds to the target pathogen, used to specifically capture the target pathogen and obtain the target binding signal; the negative control area is equipped with random sequences, mismatch sequences, or non-specific recognition molecules, used to reflect non-specific adsorption, impurity adhesion, and sample matrix interference signals; the blank reference area is equipped with only a sealing layer without aptamers, used to reflect local thickness differences of the biochip module, sample refractive index fluctuations, fluid background changes, and optical path drift; the quality control calibration area is equipped with a standard film layer, standard particles, a film layer of known thickness, or a positive quality control reaction layer, used to calibrate the differences in optical response between different channels, different detection batches, or different scanning positions.

6. The multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to claim 4, characterized in that, Before the detection begins, the control and analysis module scans these positioning marks and establishes a chip coordinate system, converting each functional detection area into corresponding scanning coordinates. The chip precision displacement module corrects the positional deviation of the biochip module based on the positioning marks and scanning coordinates, and then moves along a preset path. The signal acquisition module sequentially acquires the periodic oscillation curves of the pre-reaction reflectivity of each functional detection area as a baseline signal. The control and analysis module controls an external microfluidic pump valve system to deliver the sample to be tested to multiple microfluidic channels, allowing the target pathogen to be captured by the aptamer within the target detection area. After the detection is completed, the control and analysis module controls the external microfluidic pump valve system to introduce cleaning fluid into the corresponding microfluidic channels, allowing the cleaning fluid to flow along the sample injection direction through each functional detection area and then be discharged from the waste outlet. After cleaning, the chip precision displacement module drives the biochip module back to the scanning coordinates corresponding to the baseline signal acquisition, and the signal acquisition module again acquires the periodic oscillation curves of the post-reaction reflectivity of each functional detection area as a baseline signal.

7. The multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to claim 1, characterized in that, It also includes a results display module connected to the control and analysis module, which is used to display the test results, microfluidic channel number, target pathogen species, positive / negative judgment, concentration quantification results, quality control status, and test report.

8. A multi-channel microfluidic label-free pathogen detection method with internal reference calibration implemented by the multi-channel microfluidic label-free pathogen detection system with internal reference calibration according to any one of claims 1-7, characterized in that, Includes the following steps: Step S1: Fix the biochip module onto the chip precision displacement module; Step S2: The signal acquisition module sequentially acquires the periodic oscillation curves of the pre-reaction reflectance of each functional detection area before sample introduction as a baseline signal. Step S3: The control and analysis module controls the external microfluidic pump valve system to inject the sample to be tested into the multi-channel microfluidic module and distribute it to multiple parallel microfluidic channels according to the preset flow path, so that the sample to be tested flows through each functional detection area respectively. Step S4: The target pathogen in the sample to be tested specifically binds to the aptamer, forming an aptamer-pathogen complex layer on the surface of the target detection area; Step S5: Inject cleaning solution to remove unbound pathogens, nonspecific adsorbents, sample impurities, and residual samples; Step S6: The signal acquisition module acquires the periodic oscillation curve of the reflectance after the formation of the aptamer-pathogen complex layer in each functional detection area as a function of wavelength. The control and analysis module compares the periodic oscillation curve of the reflectance after the reaction as a function of wavelength with the periodic oscillation curve of the reflectance before the reaction as a function of wavelength, and calculates the spectral peak position shift, phase change or optical path difference change in each functional detection area. Step S7: The control and analysis module calculates the target detection signal and compares it with a pre-established standard curve of a standard sample with a known concentration of target pathogen to obtain the concentration of target pathogen in the sample to be tested. The final detection conclusion is displayed through the structure display module.

9. The multi-channel microfluidic label-free pathogen detection method with internal reference calibration according to claim 8, characterized in that, The formula for calculating the periodic oscillation curve of the reflectivity before the reaction as a function of wavelength is as follows: ； in, This is a periodic oscillation curve showing the change in reflectivity as a function of wavelength before the reaction. Where λ is the wavelength, U1 and U2 are the amplitudes of the two reflected beams, U is the amplitude of the incident light, n is the refractive index of the silicon dioxide thin film, and d is the physical thickness of the silicon dioxide thin film. The formula for calculating the periodic oscillation curve of reflectivity as a function of wavelength after the reaction is as follows: ； in, This is a periodic oscillation curve showing the change in reflectivity with wavelength after the reaction. The change in equivalent optical path difference caused by the binding of the target pathogen, n bio σ is the equivalent refractive index of the aptamer-pathogen complex layer, and σ is the equivalent optical thickness increase relative to the baseline state before the reaction. The periodic oscillation curve of reflectivity as a function of wavelength before the reaction and the periodic oscillation curve of reflectivity as a function of wavelength after the reaction. A Fast Fourier Transform (FFT) is performed to convert the signal from the wavelength domain to the frequency domain. In the frequency domain, the main peak of the interference spectrum corresponds to the inherent optical thickness of the silicon dioxide thin film. The position *m* of the main peak represents the frequency component corresponding to this inherent optical thickness. The phase value corresponding to the main peak position *m* is found in the complex result generated by the FFT. This phase value reflects the phase state of the reflected interference spectrum at the main peak frequency component. The phase difference before and after the reaction is used to characterize the change in the equivalent optical thickness or optical path difference of the detection area. Based on the phase value, the phases of the two interference spectrum signals before and after the reaction are calculated. The change in optical thickness is determined by the difference between these two phases, and the calculation formula is as follows: ； in, The phase difference between the reflection interference spectra before and after the reaction at the main peak position m is denoted as m.

10. The multi-channel microfluidic label-free pathogen detection method with internal reference calibration according to claim 8, characterized in that, Based on the periodic oscillation curves of reflectance versus wavelength after the reaction and before the reaction, the following data were calculated: phase change ΔφA in the target detection area, phase change ΔφB in the negative control area, phase change ΔφC in the blank reference area, and response change ΔφD in the quality control calibration area. Then, the target detection signal Δφ after internal parameter correction was calculated using these data. 校正 The calculation formula is as follows: 。

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