Method for multiplexed detection of biomarkers based on multi-label encoding microvibrator electrically driven plasmonic imaging

Abstract

This invention discloses a method for multiplex biomarker detection based on electro-driven plasmonic imaging using multi-tag-encoded microoscillators. First, a micro / nano oscillator array is constructed on the surface of a metal thin-film chip using a self-assembly method. A tag-encoding strategy is introduced, and different specific probes are functionalized on the surface of the micro / nano oscillators with different tags, serving as sensing elements for the simultaneous detection of multiple biomarkers. Since the oscillation amplitude of the micro / nano oscillators is related to their effective surface charge, surface plasmonic resonance imaging (SPRi) is used to track changes in the oscillation amplitude of individual oscillators. Combined with a tag decoding strategy, the dynamics of micro / nano oscillators with different tags are analyzed, and the affinity and quantitative information of different targets are resolved. This allows for highly sensitive and specific multiplex detection of multiple biomarkers such as nucleic acids, proteins, and small molecules in a single analysis. This invention significantly improves detection efficiency and provides a powerful platform for precision diagnosis, clinical biomarker screening, and longitudinal disease tracking.

Description

Technical Field

[0001] This invention relates to the field of biological and chemical detection methods, specifically to a method for multiple detection of biomarkers based on multi-label encoded microoscillator electro-driven plasma imaging. Background Technology

[0002] Biomarker testing has been widely used in the early diagnosis, prognostic monitoring, and treatment evaluation of various major diseases, including Alzheimer's disease, cardiovascular disease, and cancer. However, single biomarker testing is insufficient to provide adequate disease-specific information, potentially leading to misdiagnosis or missed diagnosis. Therefore, multiplex testing is necessary to meet the needs of precise clinical diagnosis. For example, in ovarian cancer diagnosis, simultaneous detection of eight miRNAs, including miR202-3p, demonstrates significantly better diagnostic performance than CA125 for stage I high-grade serous ovarian cancer. In lung cancer diagnosis, combined detection of miRNA expression levels, such as miR-106a, has been proven effective in differentiating major lung cancer subtypes, increasing diagnostic accuracy to 94%. Therefore, multiplex biomarker testing provides a powerful tool for improving diagnostic accuracy, enabling more effective molecular targeted therapy, and advancing personalized medicine.

[0003] In recent years, numerous multiplex detection technologies for biomarkers have emerged, including fluorescent labeling, electrochemical detection, and probe-labeled Raman scattering spectroscopy. Among these, fluorescent labeling has been extensively studied due to its sensitivity and versatility; the representative SiMoA technology has achieved femtomolar sensitivity in the detection of various biomarkers and has been successfully commercialized. Despite these advances, fluorescence-based multi-tag strategies still face challenges such as photobleaching and spectral overlap limiting the number of tags. Furthermore, labeling-based methods may affect the detection of small molecules.

[0004] Surface plasmon resonance (SPR) is a typical label-free, mass-sensitive technique. When combined with optical imaging systems, SPR imaging technology can provide spatial resolution, leading to the development of multiplex detection techniques based on spatial resolution strategies, exemplified by microfluidic systems. However, SPR sensitivity depends on the relative molecular mass of the analyte, limiting its application in small molecule detection; furthermore, the complex microfluidic structure restricts its clinical translation capabilities.

[0005] These limitations highlight the need for a new generation of SPR-based biosensing platforms, requiring enhanced sensitivity, simplified multiplexing strategies, and robust performance in complex clinical sample testing. Based on surface plasmon resonance imaging (SPR), the use of multi-size encoded microspheres as sensing units enables highly sensitive detection of a variety of biomarkers and holds promise for quantitative applications. The diversity of ligands allows for easy expansion of the number and types of targets detected. This platform demonstrates significant application potential in clinical settings. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention provides a method for multiplex biomarker detection based on electro-driven plasma imaging using multi-tag-coded microoscillators. This method constructs a multi-tag micro / nano oscillator array on a chip surface via self-assembly, with different tags modifying specific probes. The oscillation amplitude of the oscillator is related to its surface charge. SPRi is used to track the oscillation changes of a single oscillator particle, and kinetic analysis is performed combined with tag decoding. This allows for the simultaneous resolution of affinity and quantitative information for multiple biomarkers, including nucleic acids, proteins, and small molecules. This method achieves highly sensitive and specific analysis of multiple targets in a single detection, significantly improving detection efficiency.

[0007] The technical solution adopted in this invention is as follows: A method for multiplex detection of biomarkers based on electro-driven plasma imaging using multi-tag coded micro-oscillator chips is disclosed. The method employs a biomarker sensor based on multi-tag micro / nano oscillator chips. The multiplex detection method includes simultaneous detection of multiple targets within the same type, simultaneous detection of multiple targets within the same species, and simultaneous detection of heterogeneous targets from different species. Specifically, it includes the following steps: S1. Constructing an SPR imaging system: A prism-coupled surface plasmon resonance imaging system is adopted, with p-polarized light as the light source. The light signal after total internal reflection from the surface of the metal thin film is acquired by the camera to obtain the optical signal. S2. Surface functionalization: On the multi-tag micro / nano oscillator chip, surface functionalization is achieved by a single recognition molecule fixed on the surface of each tag micro / nano oscillator; S3. Sample loading and binding: The sample containing the biomarker to be tested is introduced into the flow cell of the imaging system, so that the target molecule specifically binds to the corresponding recognition molecule. S4. Electrically driven modulation: By applying a periodically varying modulation electrical signal, a regular electric field is applied to the micro / nano oscillator in the solution. S5. Acquisition of time-series optical signals: During the potential modulation process, optical signals on the surface of the metal thin film are continuously acquired to obtain the modulation electrical signals of the characteristic parabolic diffraction spots of each micro / nano oscillator. S6. Signal Extraction: Process the time-series optical signal and extract the signal feature parameters corresponding to the modulation frequency; S7. Displacement calculation: Based on the physical correlation between the signal characteristic parameters and the spatial distance, obtain the amplitude or displacement information of the micro / nano oscillator; S8. Target determination: Based on the coding characteristics of different tag micro-nano oscillators and the changes in the amplitude or displacement information, determine the presence or abundance of different target biomarkers in the sample to be tested.

[0008] Specifically, the multi-tag micro / nano oscillator chip includes: a metal thin film chip substrate, and multi-tag encoded micro / nano oscillators formed on its surface by a self-assembly method. The multi-tags include particles of different materials, shapes and sizes, and the surfaces of the micro / nano oscillators with different tags are modified with different types of biological probes.

[0009] Furthermore, the biomarker sensor is prepared as follows: A metal thin film with a plasma resonance effect and an adhesive layer are sequentially deposited on the surface of a substrate, wherein the substrate is a transparent glass slide, the metal thin film is selected from gold, silver, aluminum or their alloys, and the adhesive layer is selected from chromium or titanium to obtain a sensor chip. Compounds of different lengths and functional groups are fixed on the surface of the sensor chip, and spacers are introduced to form a self-assembled monolayer; Functionalizing different biomolecular recognition units on the surface of micro-nano particles with different labels enables the specific recognition and binding of various targets; By attaching particles with multiple tags to the surface of the sensing chip, the multi-tag micro / nano oscillator chip based on the self-assembly method is obtained.

[0010] Specifically, the transparent glass sheet is first cleaned before coating to remove impurities.

[0011] Specifically, the step involves fixing compounds of different lengths and functional groups on the surface of the sensing chip and introducing spacers to form a self-assembled monolayer; specifically including: A self-assembled monolayer is formed on the chip surface by using long chains as tethers and short chains as spacers; the long chains and short chains are selected from one or more of micro / nano polymer chains, nucleic acid chains, and micro / nano structures with rigid or three-dimensional structures. The formation process of the self-assembled monolayer is as follows: The short chain is brought into contact with the chip surface and a first assembly incubation is performed. The chip surface is washed, and then the long chain is brought into contact with the chip surface for a second assembly incubation. The chip surface is washed again, and the short chains are brought into contact with the chip surface again for a third assembly incubation.

[0012] The process of functionalizing different biomolecular recognition units on the surface of micro / nano particles with different labels is as follows: After the surface of the single-label microspheres is modified to specifically bind to the receptor, they are mixed with liganded biomolecular recognition units and incubated in a dispersion medium. The incubated mixture was subjected to solid-liquid separation, the supernatant was removed and a washing medium was added to redisperse the micro-nano particles. Repeat the separation and dispersion process described above at least once to remove unbound biomolecular recognition units.

[0013] Specifically, the recognition molecule in step S1 is selected from one or more of nucleic acid complementary probes, antibodies, aptamers, peptides, receptor proteins, or molecularly imprinted polymers.

[0014] Furthermore, the oscillation amplitude of the micro / nano oscillator is obtained in the following way: The chip is placed on a near-field optical sensing or imaging system, and a regular potential modulation is applied to the micro-nano oscillator in the reaction cavity. The time-series optical signal of the micro-nano oscillator oscillating with the modulation signal is collected using a photoelectric detection device. The time-series optical signal is processed to extract the signal change features corresponding to the modulation frequency, and the mechanical displacement of the micro / nano oscillator is determined based on the correlation between the optical signal intensity and the spatial position of the particles.

[0015] Furthermore, the decoupling of the micro / nano oscillator tag is achieved in the following way: Extract the characteristic parameters corresponding to the time-series optical signal or mechanical displacement; The feature parameters are processed using a data analysis model to classify and decode different micro / nano oscillator tags.

[0016] Furthermore, the simultaneous detection of the aforementioned multiple nucleic acid biomarkers is achieved through the following methods: The target response signal of the detection system after the introduction of the sample to be tested is obtained, or the change in the target response signal before and after the introduction of the sample to be tested is obtained; based on the target response signal or the change in signal, combined with the pre-established calibration relationship or quantitative analysis model, the concentration or abundance of the corresponding biomarker in the sample to be tested is determined.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a dynamic SPRi system based on electrical modulation by coupling an electrical modulation and an optical imaging system. As a charge-sensitive detection platform, it is fundamentally different from the mass-sensitive sensing strategy and breaks through the limitation of traditional SPR detection in terms of mass sensitivity. It can perform highly sensitive detection of nucleic acids and small molecule biomarkers.

[0018] The biomarker detection sensor of the present invention can control the number of chains connected to particles by optimizing the ratio of the chip self-assembly layer chains, thereby improving detection sensitivity and facilitating subsequent high-sensitivity detection of biomarkers.

[0019] This invention tracks the motion of a single micro-nano oscillator using a prism-based surface plasmon resonance imaging system. More than 150 micro-nano oscillators can be tracked simultaneously in the same field of view, meeting the requirements of high-throughput detection.

[0020] This invention introduces a micro / nano particle size encoding system and develops a corresponding decoupling method to construct a simplified multiple detection strategy, enabling the simultaneous quantitative detection of multiple biomarkers.

[0021] By using the method of this invention, the motion state changes of micro-nano oscillators encoded with different sizes due to the binding or dissociation of analytes with micro-nano particles can be tracked and binding dynamics analysis can be performed. This allows for the resolution of affinity and quantitative information of different biomarkers, thereby achieving highly specific detection of multiple biomarkers and potentially enabling the direct detection of multiple biomarkers in real complex biological samples. Attached Figure Description

[0022] Figure 1 A schematic diagram of a multi-tag micro / nano oscillator chip is shown; Figure 2 A schematic diagram of an objective-type surface plasmon resonance imaging system is shown. Figure 3 The regular motion curves of the micro / nano oscillator under electric field modulation are shown; Figure 4 The FFT plots and hybrid tag micro / nano oscillator array diagrams based on surface plasmon resonance are shown for three types of tag micro / nano oscillators. Figure 5 A schematic diagram of three tag-mixed micro / nano oscillator arrays exposed to three miRNA mixture samples and the amplitude variation curve over time are shown. Figure 6 A schematic diagram and amplitude variation curves over time are shown for three types of tag-hybrid micro / nano oscillator arrays exposed to a mixture of nucleic acids, proteins and small molecules. Detailed Implementation

[0023] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0024] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0025] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0026] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0027] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0029] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0031] Example 1: This embodiment provides a method for fabricating a biomarker sensor based on multi-tag encoded micro / nano oscillators, the specific steps of which are as follows: 1) Fabrication and processing of metal thin-film chips In this embodiment, a 3 nm chromium (Cr) film was first deposited on a BK7 optical glass slide using magnetron sputtering, followed by a 47 nm gold (Au) film, resulting in a 50 nm thick gold film glass slide used as a highly sensitive SPR sensing chip. The gold film glass slide was then rinsed sequentially with anhydrous ethanol and pure water, and dried with nitrogen gas to remove surface impurities.

[0032] 2) Preparation of the reaction chamber In this embodiment, polydimethylsiloxane (PDMS) is used to prepare the reaction chamber. First, PDMS monomer and crosslinking agent (DOWCORNING) are mixed in a 10:1 ratio, stirred evenly, and allowed to stand to remove air bubbles. Then, the PDMS adhesive is poured into a smooth plastic petri dish and baked on a heating plate at 75°C for 1.5 hours until it solidifies. After peeling off the PDMS film, it is cut into a certain shape, and then holes are punched in the PDMS film to create the reaction chamber. The prepared detection chamber is placed on a cleaned chip.

[0033] 3) Preparation of surface self-assembled monolayers A self-assembled monolayer is formed on the chip surface using long chains as tethers and short chains as spacers. The long and short chains are selected from 63nm biotin-polyethylene glycol-thiol and 3nm methyl-polyethylene glycol-thiol, respectively. This type of assembly structure provides structural stability and precise flexibility control (effective elastic constant). This ensures a purely electrically driven response of the oscillator in a fluid environment, shielding it from unwanted thermal noise interference. The formation process of the self-assembled monolayer is as follows: (1) After diluting the short chain, inject it into the reaction chamber and incubate it in a drying oven for at least one hour; (2) Clean the reaction chamber and chip surface, add the diluted long chain into the reaction chamber, and place it in a drying oven for overnight incubation; (3) Clean the reaction chamber and chip surface again, add the short chain back into the reaction chamber, and incubate in a drying oven for at least one hour to finally form a self-assembled monolayer.

[0034] 4) Surface functionalization of micro / nanoparticles Different biomolecular recognition units are functionalized on the surface of micro / nano particles with different labels. The specific process is as follows: Single-tagged micro / nanoparticles with specific binding receptors on their surface were mixed with liganded biomolecular recognition units and incubated at room temperature using a shaker for at least two hours. The mixture was then centrifuged at a speed of at least 4000 rpm for at least 5 minutes to achieve solid-liquid separation. The supernatant was aspirated using a pipette and solvent was added, followed by shaking until the particles were completely dispersed. This process was repeated three times. The micro / nanoparticles were made of materials including silica, gold, and glass, and their shapes included spheres, rods, and tetrahedrons, with a size range of 0.05–5 micrometers in diameter and more than three coded elements.

[0035] Through the above operations, different biomolecular recognition units (selected from one or more of nucleic acid complementary probes, antibodies, and aptamers) are functionalized on the surface of micro-nano particles with different labels by utilizing bioaffinity or covalent binding, thereby achieving specific recognition and binding of a variety of targets.

[0036] 5) Construction of multi-tag micro / nano oscillator arrays Multiple tagged particles are mixed and attached to the surface of the sensor chip via bioaffinity or covalent bonding. Specifically, different functionalized particles are mixed and added to the reaction chamber of a self-assembled monolayer sensor chip. The mixture is allowed to stand for at least ten minutes, and then excess particles are gently washed away using a buffer solution (PBS solution diluted 5 to 100 times with water). This yields a multi-tagged micro / nano oscillator chip based on a self-assembly method (e.g., ...). Figure 1 (As shown).

[0037] Example 2: A method for multiplex detection of biomarkers based on electro-driven plasmon resonance (SPR) imaging using multi-size coded microoscillators. This embodiment provides a method for multiplex detection of biomarkers based on a multi-tag micro / nano oscillator chip prepared in Example 1, including simultaneous detection of multiple targets within the same type, detection of multiple targets within the same species, and simultaneous detection of heterogeneous targets of different species. The specific preparation steps are as follows: S1. Construct an electrically driven SPR imaging system This embodiment employs a prism-coupled surface plasmon resonance imaging system (such as...). Figure 2(As shown). Its working principle is as follows: When a beam of light enters an optically less dense medium from an optically denser medium, if the incident angle is greater than the critical angle, total internal reflection will occur, generating an evanescent wave. When the incident angle equals the resonance angle, the surface plasmon wave in the metal thin film resonates with the evanescent wave, and part of the incident light energy is transferred to the surface plasmon, causing the reflected light energy to drop sharply, resulting in a resonance peak (dark band) in the reflection spectrum. The position of the resonance peak changes with the refractive index of the metal thin film surface. The SPR is very sensitive to the refractive index of the medium attached to the surface of the metal thin film, so surface bonding events can be detected by detecting changes in the resonance angle.

[0038] Specifically, the light source uses p-polarized light from a light-emitting diode (LED) with a power of 15 mW and a wavelength of approximately 680 nm. The light signal, after total internal reflection from the metal thin film surface, is captured by a CMOS camera to obtain an SPR image, with a maximum imaging field of view of 640×480 pixels. The CMOS camera can detect changes in brightness within the field of view on the chip surface. The grayscale value of the SPR image is the SPR signal value, and the change in refractive index of the corresponding area can be inferred by calculating the change in grayscale value in different areas.

[0039] S2, Surface Functionalization On the multi-tag micro / nano oscillator chip prepared in Example 1, the micro / nano oscillator array contains at least two types of tagged micro / nano oscillators. Each tagged micro / nano oscillator fixes a single recognition molecule (different optical signals caused by the size, shape, and material properties of the oscillator itself will construct the tag). The recognition molecule is selected from one or more of nucleic acid complementary probes, antibodies, and aptamers.

[0040] S3. Sample loading and binding: The sample containing the biomarker to be tested is introduced into the flow cell (i.e., reaction chamber) of the SPR imaging system, so that the target molecule specifically binds to the corresponding recognition molecule.

[0041] S4, Electric Drive Modulation A regular electric field modulation is applied to the micro / nano oscillator in the solution by applying a modulated electrical signal containing an AC component. In this embodiment, the voltage amplitude range is 1.3 V and the frequency is 5 Hz.

[0042] S5. Acquire time-series SPR images During the potential modulation process, SPR images of the metal thin film surface were continuously acquired using a CMOS camera (with a frame rate of 23 fps) to obtain time-series images of the characteristic parabolic diffraction spots of each micro / nano oscillator. The acquisition time included a baseline of 30 seconds, binding of 60 seconds, and dissociation of 60 seconds, for a total of 150 seconds.

[0043] S6, Signal Extraction Frequency domain signal processing techniques (such as Fast Fourier Transform, FFT) are used to extract the signal change amplitude corresponding to the modulation frequency from the time series image.

[0044] The motion modes of the micro / nano oscillator are analyzed as follows: When an electric field is applied to a micro / nano oscillator, its oscillation equation is as follows: ; in, For quality, For amplitude, The damping coefficient is... The number of tethering ropes connecting the micro / nano oscillator. For effective charge, For gravity, For buoyancy, The effective elastic constant of the polyethylene glycol chain, Let be the applied alternating electric field. By estimating the values ​​of each term in the formula, compared with the electric field force, the drag, gravity, and buoyancy experienced by the micro / nano oscillator can be neglected. Therefore, the formula can be simplified to: ; In this invention, the circuit model is simplified to the Randle circuit model because of the electric field strength The following formula can be used for calculation: ; Therefore, the final oscillation amplitude of the micro / nano oscillator The following formula can be used for calculation: ； in, For the resistance of the solution, Polarization resistor, It is a double-layer capacitor. The frequency at which the sine wave is applied.

[0045] S7, Displacement Calculation Each micro / nano oscillator in the solution scatters plasma waves, producing a parabolic pattern. Since the evanescent field associated with the plasma waves decays exponentially from the metal film surface into the solution, the scattering intensity is extremely sensitive to the distance between the micro / nano oscillator and the surface, and can be given by the following formula: ; Where I0 is a constant, and l is the decay constant of the evanescent field, approximately 200 nm. By measuring the image intensity, the distance between the micro / nano particles and the surface can be determined with an accuracy of ~1 nm. When a potential modulation is applied to the micro / nano oscillator, the distance between the micro / nano oscillator and the chip changes with the modulation due to the negative charge on the surface of the micro / nano oscillator, thus causing a periodic change in its SPR signal intensity.

[0046] In time-series plasma images acquired by a CMOS camera, each micro / nano oscillator exhibits characteristic parabolic diffraction patterns. To separate the SPR image amplitude induced by the oscillator amplitude, a fast Fourier transform analysis was performed on the time-series images to obtain the oscillation amplitude image of the oscillator based on the plasma intensity. Based on the exponential dependence of plasma image intensity on particle-interface distance, the mechanical displacement of the micro / nano particles was determined. Figure 3 ).

[0047] S8, Target Determination Based on the changes in oscillation amplitude, and combined with the spatial position information of the oscillator in the multi-tag encoding and the characteristics of the tags themselves (including the material, shape, and size of different tags), the presence and concentration of different targets are simultaneously determined. Specifically, by fitting and combining kinetic parameters, the change signal of the oscillation amplitude of each tag oscillator is correlated with the standard curve to obtain the concentration of each corresponding marker in the sample to be tested.

[0048] Example 3: Decoupling of Micro / Nano Oscillator Tags This embodiment provides a method for signal decoupling of the above-mentioned multi-tag coding system.

[0049] Based on the principle of evanescent field scattering interference, the intensity of the SPR image recorded by the CCD camera can be expressed as: ; in, For overall reflected light, For the location of micro / nano particles, This represents the relative position of the particles on the image. The background reflected wave of the chip For the vanishing wave, Let ψ be the scattered wave and ψ be the phase shift. This indicates that the SPR image intensity of each micro / nano oscillator is related to its volume in the evanescent field. Since the typical SPR imaging depth l is approximately 200 nm, which is smaller than the radius of the micro / nano particle, the effective volume of the particle in the evanescent field can be expressed as: ; in, It is the volume of micro / nano particles in the vanishing field. Where is the particle radius, The depth of the particle in the evanescent field. By analyzing the changes in the evanescent field during the oscillation of micro / nano oscillators of tags of different sizes, materials, and shapes, a quantitative relationship between single-particle tags and scattering signals is established, thereby achieving precise decoupling of the tags (e.g., ...). Figure 4 (As shown).

[0050] Example 4: Simultaneous detection of multiple targets within the same type (miRNA multiplex detection based on micro / nano oscillators) This embodiment uses the simultaneous detection of three miRNA targets (miRNA-106a, miRNA-21, and miRNA-141) as an example to illustrate the implementation method of simultaneous detection of multiple targets within the same type.

[0051] First, three types of tagged micro / nanoparticles (such as gold micro / nanoparticles of different sizes) were incubated with three biotinylated single-stranded nucleic acid complementary probes for at least one hour. The microspheres were then centrifuged at 4000 rpm or higher for at least 5 minutes to remove the supernatant, resuspend, and the process was repeated at least three times. Finally, the microspheres were resuspended in PBS solution diluted 100 times with water for the detection of miRNA-106a, miRNA-21, and miRNA-141, respectively. Solutions containing the three miRNA targets were then flowed into the reaction chamber. The voltage amplitude range for potentiometric modulation was set to 1.3V, the frequency to 5 Hz, and images were acquired at 23 fps. For each concentration, baseline was acquired for 30 seconds, binding for 60 seconds, and dissociation for 60 seconds, for a total of 150 seconds of SPR time-series images. The buffer consisted of PBS solution diluted 5 to 100 times with water.

[0052] The SPR image was processed using Fast Fourier Transform (FFT) and the individual micro / nano oscillator codes were decoupled to obtain the signal curves of the amplitude changes of individual micro / nano oscillators (e.g., Figure 5 (As shown). Furthermore, when dealing with complex multivariate mixed samples, statistical methods such as Gaussian mixture models can be used to separate and fit overlapping signal peaks from multiple targets. Through fitting, binding kinetic parameters can be obtained, and the concentrations of the three targets can be quantified separately. When constructing the quantitative standard curve, it should be explicitly mentioned that the standard error (SEM) is used to assess the data's volatility and reliability.

[0053] Example 5: Simultaneous detection of different types of heterologous targets (multiplex detection of nucleic acids, proteins and small molecules based on micro / nano oscillators) This embodiment uses the simultaneous detection of three heterologous targets—protein (BSA), nucleic acid, and small molecule (glucose)—as an example to illustrate the implementation method of simultaneous detection of different types of heterologous targets.

[0054] First, the three types of tagged micro-nanoparticles were incubated with biotinylated anti-BSA (for BSA detection), single-stranded nucleic acid complementary probe (for nucleic acid detection), and succinylated malt lectin complementary probe (for glucose detection) for more than two hours. Then, they were centrifuged at a speed of more than 4000 r / min for more than 5 minutes, the supernatant was removed, and the microspheres were resuspended. This process was repeated 3 times. Finally, the microspheres were resuspended in PBS solution diluted 100 times with water.

[0055] Three functionalized micro / nanoparticles were mixed to construct a multi-tag micro / nano oscillator array according to the method in Example 1. A solution containing three targets (BSA, nucleic acid, and glucose) was flowed into the reaction chamber. The voltage amplitude for potential modulation was set to 1.3 V, the frequency to 5 Hz, and images were acquired at 23 fps. Baseline acquisition lasted 30 seconds, target binding lasted 60 seconds, and dissociation lasted 60 seconds, for a total of 150 seconds of SPR time-series images.

[0056] The SPR time series image was processed using Fast Fourier Transform (FFT). Following the method in Example 3, the size encoding of individual micro / nano oscillators was decoupled to determine the detection target type corresponding to each oscillator, resulting in signal curves showing the amplitude changes of individual micro / nano oscillators. By decoupling the size encoding of individual micro / nano oscillators, the detection target type was determined, and its binding dynamic parameters were fitted. The concentrations of three targets (e.g., ...) were then quantified. Figure 6 (As shown).

[0057] Compared to passive micro-motion tracking methods that rely entirely on the Brownian motion of micro / nano particles, lack signal decoupling mechanisms, and can only determine binding by statistically analyzing the attenuation of the overall particle motion amplitude, this invention has significant advantages in simultaneously distinguishing and detecting multiple heterogeneous targets in mixed samples within the same reaction system (e.g., its embodiments can only detect single-stranded DNA or BSA separately in independent reaction chambers). This invention not only endows the sensor with strong external signal modulation and anti-interference capabilities by actively applying AC electric field modulation (e.g., a 5Hz potential), but also innovatively introduces a tag encoding strategy with multiple sizes, shapes, and materials, combined with Fast Fourier Transform (FFT) to extract the oscillation amplitude of specific frequencies. In terms of experimental results, the passive micro-motion tracking method relies on high-frequency imaging of >100Hz and can only achieve qualitative / quantitative analysis of a single target; while this invention can not only work at a lower acquisition frame rate, but also successfully achieves simultaneous detection and quantification of multiple heterogeneous targets such as nucleic acids, proteins and small molecules on the same chip in a single sample injection (high-throughput tracking of >150 oscillators in the same field of view). Through an active encoding strategy, it breaks through the limitations of the passive method and truly realizes a high-specificity biomarker multiplex detection platform.

[0058] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for multiplex detection of biomarkers based on multi-tag coded microoscillator electrodriven plasma imaging, characterized in that, The method employs a multi-tag micro / nano oscillator chip-based biomarker sensor; the biomarker multiplex detection method includes simultaneous detection of multiple targets within the same type, simultaneous detection of multiple targets within the same species, and simultaneous detection of heterogeneous targets from different species, specifically including the following steps: S1. Constructing an SPR imaging system: A prism-coupled surface plasmon resonance imaging system is adopted, with p-polarized light as the light source. The light signal after total internal reflection from the surface of the metal thin film is acquired by the camera to obtain the optical signal. S2. Surface functionalization: On the multi-tag micro / nano oscillator chip, surface functionalization is achieved by a single recognition molecule fixed on the surface of each tag micro / nano oscillator; S3. Sample loading and binding: The sample containing the biomarker to be tested is introduced into the flow cell of the imaging system, so that the target molecule specifically binds to the corresponding recognition molecule. S4. Electrically driven modulation: By applying a periodically varying modulation electrical signal, a regular electric field is applied to the micro / nano oscillator in the solution. S5. Acquisition of time-series optical signals: During the potential modulation process, optical signals on the surface of the metal thin film are continuously acquired to obtain the modulation electrical signals of the characteristic parabolic diffraction spots of each micro / nano oscillator. S6. Signal Extraction: Process the time-series optical signal and extract the signal feature parameters corresponding to the modulation frequency; S7. Displacement calculation: Based on the physical correlation between the signal characteristic parameters and the spatial distance, obtain the amplitude or displacement information of the micro / nano oscillator; S8. Target determination: Based on the coding characteristics of different tag micro-nano oscillators and the changes in the amplitude or displacement information, determine the presence or abundance of different target biomarkers in the sample to be tested.

2. The method for multiplex detection of biomarkers according to claim 1, characterized in that, The multi-tag micro / nano oscillator chip includes: a metal thin film chip substrate, and multi-tag encoded micro / nano oscillators formed on its surface by a self-assembly method. The multi-tags include particles of different materials, shapes and sizes, and the surfaces of the micro / nano oscillators with different tags are modified with different types of biological probes.

3. The method for multiplex detection of biomarkers according to claim 1, characterized in that, The biomarker sensor is prepared as follows: A metal thin film with a plasma resonance effect and an adhesive layer are sequentially deposited on the surface of a substrate. The metal thin film is selected from gold, silver, aluminum or their alloys, and the adhesive layer is selected from chromium or titanium to obtain a sensor chip. Compounds of different lengths and functional groups are fixed on the surface of the sensor chip, and spacers are introduced to form a self-assembled monolayer; Functionalizing different biomolecular recognition units on the surface of micro-nano particles with different labels enables the specific recognition and binding of various targets; By attaching particles with multiple tags to the surface of the sensing chip, the multi-tag micro / nano oscillator chip based on the self-assembly method is obtained.

4. The method for multiplex detection of biomarkers according to claim 3, characterized in that, The substrate is first cleaned to remove impurities before plating.

5. The method for multiplex detection of biomarkers according to claim 3, characterized in that, Compounds of different lengths and functional groups are fixed on the surface of the sensor chip, and spacers are introduced to form a self-assembled monolayer. Specifically, it includes: A self-assembled monolayer is formed on the chip surface by using long chains as tethers and short chains as spacers; the long chains and short chains are selected from one or more of micro / nano polymer chains, nucleic acid chains, and micro / nano structures with rigid or three-dimensional structures. The formation process of the self-assembled monolayer is as follows: The short chain is brought into contact with the chip surface and a first assembly incubation is performed. The chip surface is washed, and then the long chain is brought into contact with the chip surface for a second assembly incubation. The chip surface is washed again, and the short chains are brought into contact with the chip surface again for a third assembly incubation.

6. The method for multiplex detection of biomarkers according to claim 3, characterized in that, The specific process of functionalizing different biomolecular recognition units on the surface of micro / nano particles with different labels is as follows: After the surface of the single-label microspheres is modified to specifically bind to the receptor, they are mixed with liganded biomolecular recognition units and incubated in a dispersion medium. The incubated mixture was subjected to solid-liquid separation, the supernatant was removed and a washing medium was added to redisperse the micro-nano particles. Repeat the separation and dispersion process described above at least once to remove unbound biomolecular recognition units.

7. The method for multiplex detection of biomarkers according to claim 1, characterized in that, The recognition molecule in step S1 is selected from one or more of the following: nucleic acid complementary probes, antibodies, aptamers, peptides, receptor proteins, or molecularly imprinted polymers.

8. The method for multiplex detection of biomarkers according to claim 1, characterized in that, The oscillation amplitude of the micro / nano oscillator is obtained through the following method: The chip is placed on a near-field optical sensing or imaging system, and a regular potential modulation is applied to the micro-nano oscillator in the reaction cavity. The time-series optical signal of the micro-nano oscillator oscillating with the modulation signal is collected using a photoelectric detection device. The time-series optical signal is processed to extract the signal change features corresponding to the modulation frequency, and the mechanical displacement of the micro / nano oscillator is determined based on the correlation between the optical signal intensity and the spatial position of the particles.

9. The method for multiplex detection of biomarkers according to claim 8, characterized in that, The decoupling of the micro / nano oscillator tag is achieved in the following way: Extract the characteristic parameters corresponding to the time-series optical signal or mechanical displacement; The feature parameters are processed using a data analysis model to classify and decode different micro / nano oscillator tags.

10. The method for multiplex detection of biomarkers according to claim 8, characterized in that, The simultaneous detection of multiple nucleic acid biomarkers is achieved through the following methods: The target response signal of the detection system after the introduction of the sample to be tested is obtained, or the change in the target response signal before and after the introduction of the sample to be tested is obtained; based on the target response signal or the change in signal, combined with the pre-established calibration relationship or quantitative analysis model, the concentration or abundance of the corresponding biomarker in the sample to be tested is determined.

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