A Portable Micro-Nano Oscillation Imaging Detection Device and Method Based on SPR

Abstract

The present invention discloses a portable micro-nano oscillating imaging detection device and method based on SPR. The device includes an SPR optical imaging module, a signal detection and control module, a micro-sample flow path control module, an electric field modulation module, and an integrated packaging module. In the SPR optical imaging module, a collimated polarized light source is coupled with a micro prism at a specific SPR angle, and the reflected light is collected by a micro or lensless CMOS image sensor for large-field-of-view and high-sensitivity imaging. The signal detection and control module is used for temperature control and obtaining the timestamps of the image sensor and the electric field modulation module. The micro-sample flow path control module is used for the flow path control and switching of trace samples to increase the detection stability. The electric field modulation module is used for applying oscillating power to micro-nano oscillators of different materials, shapes and sizes to amplify signals and enhance the detection sensitivity. The integrated packaging module is used for miniaturizing the system packaging, reducing the interference caused by environmental temperature changes, external stray light, mechanical vibration and air flow disturbance, and increasing the system stability.

Description

Technical Field

[0001] This application relates to the technical field of biological and chemical detection equipment, and particularly to a portable micro-nano oscillation imaging detection device and method based on SPR. Background Art

[0002] In this post-genomic era, analyzing the interactions between molecules is crucial for understanding the mechanisms of various physiological activities. However, the currently commonly used detection technologies have disadvantages such as time-consuming and complex operations, and often require large equipment platforms in a laboratory environment. But rapid on-site analysis of the interactions between molecules remains a challenge. Therefore, it is necessary to develop rapid and portable biological detection technologies to achieve the analysis of molecular interactions.

[0003] Traditional surface plasma resonance (SPR) technology has been commercialized, and SPR imaging technology has also developed rapidly in recent years. However, conventional SPR imaging requires a complex optical path system and independent detection components, the system is bulky, and requires professional operation, so its popularization is greatly limited. The traditional dropwise injection method has cumbersome operations, takes a long time, and easily affects the structural stability of the imaging system. In addition, changes in external conditions such as temperature, light, and air flow will also cause interference to the detection.

[0004] Currently, there are few highly integrated portable systems based on SPR imaging for biological detection and molecular interaction analysis on the market. The development of a lab-on-a-chip system that integrates all steps of microbial diagnosis into a miniaturized device has become a trend, which will greatly reduce costs and improve portability. However, the miniaturization of the device will inevitably reduce the detection sensitivity and stability, so portable detection remains a challenge.

[0005] Since micro-nano oscillators with different sizes, materials, and shapes carry different charges in solution, after being connected to the imaging chip through flexible biopolymers and applying an alternating electric field modulation to make the micro-nano oscillators oscillate, oscillation parameters such as their amplitude and phase can be collected for signal amplification. Therefore, combining micro-nano oscillator technology with SPR technology is expected to develop a portable detection device and method. Summary of the Invention

[0006] The purpose of the embodiments of this application is to provide a portable micro-nano oscillation imaging detection device and method based on SPR, so as to solve problems such as the complexity, large volume, high cost, susceptibility to interference, and low throughput of traditional SPR imaging systems, realize the miniaturization and integration of the detection device, and achieve portable and rapid molecular interaction analysis on the basis of ensuring sensitivity and stability.

[0007] According to the first aspect of the embodiments of the present application, a portable micro-nano oscillation imaging detection device based on SPR is provided, which includes an SPR optical imaging module, a signal detection and control module, a micro-sample flow path control module, an electric field modulation module, and an integrated packaging module;

[0008] The SPR optical imaging module includes a collimated polarized light source, a micro prism, an imaging chip, and an image sensor. The collimated polarized light source is coupled to the micro prism at a specific SPR angle. The imaging chip is disposed above the optical prism. The image sensor is integrated with the micro prism for reflected light collection, and the image sensor uses a micro-lens or lensless CMOS image sensor;

[0009] The signal detection and control module is used for temperature control and obtaining the timestamps of the image sensor and the electric field modulation module to achieve phase synchronization;

[0010] The micro-sample flow path control module includes a microfluidic chip, which is disposed above the imaging chip for controlling and switching the flow path of trace samples to increase the detection stability;

[0011] The electric field modulation module is electrically connected to the imaging chip and the micro-sample flow path control module, and is used for applying oscillation power to micro-nano oscillators of different materials, shapes and sizes to amplify signals and enhance detection sensitivity;

[0012] The integrated packaging module includes a matte airtight housing, an imaging base, and a base. The matte airtight housing is used for packaging the device. The base is disposed at the bottom inside the matte airtight housing. The imaging base is disposed on the base. A V-shaped card slot is provided in the imaging base, and the micro prism is disposed in the V-shaped card slot;

[0013] Wherein the material of the micro-nano oscillator is a charged particle, the shape of the micro-nano oscillator is spherical, rod-shaped or sheet-shaped, and the diameter size range of the micro-nano oscillator is 500 nm to 5 μm.

[0014] Further, in the SPR optical imaging module, the imaging chip is a micro-nano oscillator array functionalized imaging chip, and a micro-nano oscillator array is constructed by a biomolecular self-assembly mode. The SPR imaging spatial resolution is 5 to 10 μm, and the time resolution is 10 ms.

[0015] Further, the signal detection and control module includes a temperature controller and a data acquisition card. The temperature controller is used to regulate the ambient temperature, and the data acquisition card is used to obtain the timestamps of the image sensor and the electric field modulation module to achieve phase synchronization.

[0016] Further, the micro-sample flow path control module further includes a sampling pump and a switching valve. The microfluidic chip includes a sampling port, an electrode hole, an electrode notch, a reaction chamber, a sample outlet, a chip card slot, and a fixing hole. The chip card slot is used for fixing the imaging chip. There are 3 to 5 sampling ports, which are connected to the sampling pump and the switching valve to realize the transportation and switching of different samples. A reaction chamber is provided for the reaction of the sample, and the volume of the reaction chamber is 50 to 300 μL. The electrode hole is used to connect the counter electrode to the sample, the electrode notch is used to connect the working electrode to the imaging chip, the fixing hole is used for fixed connection with the base, and the sampling pump controls the sampling flow rate at 50 to 500 μL / min.

[0017] Further, the electric field modulation module includes a circuit board and a power supply. The power supply supplies power to the SPR optical imaging module, the signal detection and control module, the micro-sample flow path control module, and the electric field modulation module. The circuit board is used to output sinusoidal and other waveform alternating current modulation to the sample through the microfluidic chip. The adjustable range of the peak-to-peak voltage of the alternating current modulation voltage is -2 to 2 Vpp, and the adjustable range of the frequency is 0.1 to 20 Hz.

[0018] Further, vertical brackets are respectively arranged at both ends of the V-shaped card slot. Inclined plane card slots are arranged on the inner sides of the vertical brackets. Micro prism picking and placing notches are arranged on the brackets to facilitate the picking and placing of the micro prism. The depths of the inclined plane card slot and the V-shaped card slot are both 2 to 4 mm.

[0019] Further, the base includes a height adjustment hole, an imaging base fixing hole, a microfluidic chip fixing hole, a drain tank, an imaging base card slot, a base fixing hole, and an H-shaped groove. The imaging base card slot is used to place and directionally adjust the imaging base. The height adjustment hole is used to adjust the height of the imaging base. The imaging base fixing hole and the microfluidic chip fixing hole are respectively used to fix the imaging base and the microfluidic chip. A drain tank is arranged below the base for draining water. The H-shaped groove is used to integrate the collimated polarized light source and the image sensor. The base fixing hole is provided for fixing the base.

[0020] Further, the matte airtight housing is integrally treated with black matte, and includes an upper cover plate, an LED controller, a side plate, a base, a handle, and a rubber soft pad. The side plate is arranged on the base. The upper cover plate is connected to the side plate through a plug-in structure to form a flip structure. The LED controller is connected to the signal detection and control module, the micro-sample flow path control module, and the electric field modulation module. The rubber soft pad is arranged below the base. The handle is arranged on the upper surface of the upper cover plate.

[0021] According to the second aspect of the embodiments of the present application, a portable micro-nano oscillation imaging detection method based on SPR is provided, including the following steps:

[0022] SPR imaging optical path setup: Turn on the power supply, open the matte airtight housing, turn on the collimated polarized light source, turn on the image sensor, and adjust the power and angle of the collimated polarized light source, as well as the sensitivity and exposure time of the image sensor respectively according to the imaging effect; Place the micro prism on the imaging base, snap the imaging base into the pedestal, adjust to the appropriate imaging height and tighten the screws to fix it, slowly add the mirror oil, snap the micro-nano oscillator functionalized imaging chip into the chip slot of the microfluidic chip, seal it with a two-component adhesive, slowly place it on the micro prism, and tighten the screws to fix it;

[0023] Electric field modulation connection: Connect the electrodes of the circuit board to the imaging chip and the sample through the microfluidic chip in sequence. The working electrode is connected to the imaging chip at the electrode notch, the counter electrode contacts the liquid sample at the electrode hole, and the reaction chamber is sealed by coating the electrodes with a flexible material. Test and adjust the electrical parameters;

[0024] Control module setup: Connect the injection pump, switching valve and the injection port of the microfluidic chip with pipelines, connect the outlet hole to the waste liquid bottle, turn on the temperature controller, and close the matte airtight housing;

[0025] Signal acquisition and detection: Turn on the electric field modulation and image acquisition, obtain the synchronous signals of the circuit and imaging by using a data acquisition card, and analyze the acquired data by using image processing software.

[0026] Furthermore, the preparation process of the micro-nano oscillator array functionalized imaging chip includes the following steps:

[0027] Imaging chip preparation and treatment: Select 18×18 - 22×22 mm BK7 glass slides, place them in anhydrous acetone, anhydrous ethanol, and ultrapure water solutions respectively for ultrasonic treatment for 15 - 30 min to remove impurities; Use a magnetron sputtering device to physically vapor deposit 2 - 3 nm of Cr for 10 - 15 s and 46 - 49 nm of Au for 45 - 50 s, and polish the gold surface with a hydrogen flame; Use oxygen plasma to process according to the parameters of 50 - 100 W and 3 - 15 min;

[0028] Monolayer long-chain self-assembly: Dilute the SH-functionalized bioflexible molecular long chain and the SH-functionalized bioflexible molecular spacer to 1-5 mM respectively using ultrapure water or alcohol, mix them according to a volume ratio of 1:200-1:1000 to construct different-density monolayer arrays for connecting micro-nano oscillators of different diameter sizes, inject them into the reaction chamber area, and leave them static at room temperature in a moist, sealed and dark box. After filling with nitrogen, incubate for 12-36 h to form a monolayer array composed of the bioflexible molecular long chain and the bioflexible molecular spacer. Among them, the bioflexible molecular long chain is longer than the bioflexible molecular spacer. The bioflexible molecular long chain refers to a bioflexible molecule with an active reaction group relative to the subsequent connected micro-nano oscillator, used for biological specific recognition. The spacer refers to a bioflexible molecule without an active reaction group relative to the subsequent connected micro-nano oscillator, used to prevent non-specific adsorption;

[0029] Micro-nano oscillator connection: Slowly inject ultrapure water into the reaction chamber area for 1-2 min to wash the unassembled bioflexible molecules. Dilute the micro-nano oscillator to 1-10 μg / mL using 0.01×PBS solution, and perform ultrasonic treatment for 2-10 min to disperse the aggregated micro-nano oscillators. Inject them into the reaction chamber area and incubate for 0.5-3 h. Slowly rinse several times with 0.01×PBS solution to remove non-specific adsorption interference. Each time, slowly aspirate 2 / 3 of the sample, and always keep liquid in the reaction chamber area to avoid the micro-nano oscillators sticking due to chip drying.

[0030] The technical solutions provided by the embodiments of the present application may include the following beneficial effects:

[0031] As can be seen from the above embodiments, the present application has developed a portable imaging device based on SPR, integrating functions such as SPR optical imaging, signal detection and control, micro-sample flow path control, and electric field modulation. The dispersed functions and components are integrated into a portable integrated device through the structural design of an integrated packaging module, and are expected to play a role in the field of rapid analysis of molecular interactions; based on the use of a micro-lens or lensless CMOS as an image sensor, portable large-field imaging can be achieved; by controlling the detection environment and the flow path system, the anti-interference ability and detection stability are improved; based on SPR, oscillation regulation of micro-nano oscillators of different materials, sizes and shapes is carried out to amplify the signal and improve the sensitivity of imaging detection; the integrated packaging module integrates each module, reduces the interference caused by environmental temperature changes, external stray light, mechanical vibration and air flow disturbance, increases the system stability and realizes the miniaturization of the device.

[0032] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. Description of the Drawings

[0033] The accompanying drawings here are incorporated into the specification and form a part of this specification, showing embodiments in line with this application, and are used together with the specification to explain the principles of this application.

[0034] Figure 1 It is a schematic diagram of the appearance of a portable micro-nano oscillation imaging detection device.

[0035] Figure 2 It is a schematic diagram of the working principle of a portable micro-nano oscillation imaging detection device.

[0036] Figure 3 It is a schematic diagram of the internal partial assembly structure of a portable micro-nano oscillation imaging detection device.

[0037] Figure 4 It is an exploded schematic diagram of the internal partial assembly structure of a portable micro-nano oscillation imaging detection device.

[0038] Figure 5 It is the first perspective of the three-dimensional schematic diagram of the imaging base.

[0039] Figure 6 It is the second perspective of the three-dimensional schematic diagram of the imaging base.

[0040] Figure 7 It is the three-view schematic diagram of the imaging base.

[0041] Figure 8 It is the three-dimensional top-down view schematic diagram of the microfluidic chip.

[0042] Figure 9 It is the three-dimensional bottom-up view schematic diagram of the microfluidic chip.

[0043] Figure 10 It is the cross-sectional schematic diagram of the sample inlet and outlet holes of the microfluidic chip

[0044] Figure 11 It is the top-down, bottom-up, and front-view schematic diagrams of the microfluidic chip.

[0045] Figure 12 It is the first perspective of the three-dimensional schematic diagram of the base.

[0046] Figure 13 It is the second perspective of the three-dimensional schematic diagram of the base.

[0047] Figure 14 It is the three-view schematic diagram of the base.

[0048] Figure 15 It is the schematic diagram of the structure of the partial assembly drawing of the base.

[0049] Figure 16 It is the exploded schematic diagram of the structure of the partial assembly drawing of the base.

[0050] Figure 17Schematic diagram of the assembly of a portable micro-nano oscillation imaging detection device based on SPR.

[0051] Figure 18 Schematic diagram of the reflectivity simulation of the portable micro-nano oscillation imaging detection device at different light source wavelengths.

[0052] Figure 19 Schematic diagram of the portable micro-nano oscillation imaging detection device detecting the oscillation change of the micro-nano oscillator under different voltages under the same frequency condition.

[0053] Figure 20 Schematic diagram of the portable micro-nano oscillation imaging detection device detecting the oscillation change of the micro-nano oscillator under different frequencies under the same voltage condition.

[0054] Reference numerals: 1, matte airtight housing; 11, upper cover plate; 12, LED controller; 13, enclosing plate; 14, base; 15, handle; 16, rubber soft pad; 2, microfluidic chip; 21, sample inlet; 22, electrode hole; 23, electrode notch; 24, reaction chamber; 25, sample outlet; 26, fixing hole; 27, chip card slot; 3, imaging base; 31, inclined plane card slot; 32, micro prism picking and placing notch; 33, hollow drainage groove; 34, V-shaped card slot; 35, base card slot; 4, base; 41, height adjustment hole; 42, imaging base fixing hole; 43, microfluidic chip fixing hole; 44, drainage trough; 45, imaging base card slot; 46, base fixing hole; 47, H-shaped groove; 5, collimated polarized light source; 6, micro prism; 7, image sensor; 8, imaging chip; 9, screw. Detailed implementation mode

[0055] Here, the exemplary embodiments will be described in detail, and the examples are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the present application.

[0056] The terms used in this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms "a", "the" and "said" used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0057] It should be understood that although terms such as first, second, and third may be used in this application to describe various information, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from each other. For example, without departing from the scope of this application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "when" or "while" or "in response to a determination".

[0058] As Figures 1-4 shown, a portable micro-nano oscillation imaging detection device based on SPR provided by this application may include an SPR optical imaging module, a signal detection and control module, a micro-sample flow path control module, an electric field modulation module, and an integrated packaging module; the SPR optical imaging module includes a collimated polarized light source 5, a micro prism 6, an imaging chip 8, and an image sensor 7. The collimated polarized light source 5 is coupled to the micro prism 6 at a specific SPR angle. The imaging chip 8 is disposed above the micro prism 6. The image sensor 7 is integrated with the micro prism 6 at a specific SPR angle for reflected light acquisition. The image sensor 7 uses a micro or lensless CMOS image sensor 7; the signal detection and control module is used for temperature control and obtaining timestamps of the image sensor 7 and the electric field modulation module; the micro-sample flow path control module includes a microfluidic chip 2. The microfluidic chip 2 is disposed above the imaging chip 8 for flow path control and switching of trace samples and increasing detection stability; the electric field modulation module is electrically connected to the imaging chip 8 and the micro-sample flow path control module for applying oscillation power to micro-nano oscillators of different materials, shapes, and sizes to amplify signals and enhance detection sensitivity; the matte airtight housing 1 is used for device packaging. The base 4 is disposed at the bottom inside the matte airtight housing 1. The imaging base 3 is disposed on the base 4. A V-shaped card slot 34 is provided in the imaging base 3. The micro prism 6 is disposed in the V-shaped card slot 34; wherein the material of the micro-nano oscillator is a charged particle, the shape of the micro-nano oscillator is spherical, rod-shaped, or sheet-shaped, and the diameter size range of the micro-nano oscillator is 500 nm to 5 μm.

[0059] As can be seen from the above embodiments, this application develops a portable imaging device based on SPR, integrating functions such as SPR optical imaging, signal detection and control, micro-sample flow path control, and electric field modulation. The scattered functions and components are integrated into a portable integrated device through the structural design of the integrated packaging module, and it is expected to play a role in the field of rapid analysis of molecular interactions; based on using a lens or lensless CMOS as the image sensor 7, portable large-field imaging can be achieved; controlling the detection environment and the flow path system improves the anti-interference ability and stability; based on SPR, oscillation regulation of micro-nano oscillators is performed to amplify signals and improve the sensitivity of imaging detection.

[0060] In a specific implementation, the integrated packaging module includes a matte airtight housing 1, an imaging base 3, and a base 4 for miniaturized packaging of the device. The matte airtight housing 1 provides a closed environment to integrate each module, which can reduce the interference caused by changes in ambient temperature, external stray light, mechanical vibration, and air flow disturbance. The inside is treated with black matte to avoid internal stray light interference, and rubber cushions 16 are provided at the bottom to reduce the impact of mechanical vibration. The matte airtight housing 1 is treated with black matte to reduce the influence of internal stray light reflection on detection, and at the same time provides an airtight darkroom to avoid external influence. Its size is not larger than 100×150×100 mm. The housing is divided into an upper cover plate 11, an LED controller 12, a surrounding plate 13, a base 14, a handle 15, and rubber cushions 16. The surrounding plate 13 is arranged on the base 14, and the upper cover plate 11 is connected to the surrounding plate 13 through a pin structure, and the flip design is convenient for operation. The entire matte airtight housing 1 is treated with black matte to reduce the influence of stray light reflection on detection. The matte airtight housing 1 creates an airtight darkroom at the same time, eliminating the influence of external light changes and air flow disturbance on detection, and cooperating with the temperature controller to eliminate the influence of temperature changes on detection. An LED controller 12 is arranged on the front of the matte airtight housing 1 to connect to the temperature controller, the injection pump, the switching valve, and the circuit board to control electrical parameters such as detection temperature, voltage, frequency, waveform, etc. and the sample injection and sampling. Four buffer rubber cushions 16 are arranged at the bottom of the matte airtight housing 1 to reduce the influence of external vibration on detection. A handle 15 is arranged on the upper cover plate 11 for convenient hand carrying.

[0061] Specifically, a V-shaped card slot 34 is arranged on the imaging base 3. Vertical brackets are respectively arranged at both ends of the V-shaped card slot 34. Inclined surface card slots 31 are arranged on the inner sides of the vertical brackets. The depths of both the inclined surface card slot 31 and the V-shaped card slot 34 are 2 - 4 mm. A micro prism picking and placing notch 32 is arranged on the bracket to facilitate the picking and placing of the micro prism 6. In a specific implementation, as Figure 5 、 6 、shown in Figure 7, the imaging base 3 adopts a "V" shape design to integrate the collimated polarized light source 5 and the image sensor 7 to achieve miniaturization. A V-shaped card slot 34 is arranged in the middle of the imaging base 3 to increase the optical path area. Inclined surface card slots 31 are arranged on both side brackets to place the micro prism 6. Mirror oil is dropped above the micro prism 6 to connect to the imaging chip 8. The depths of the inclined surface card slot 31 and the V-shaped card slot 34 are 2 - 4 mm, which can clamp the micro prism 6 while increasing the optical path area. A micro prism picking and placing notch 32 is arranged at the upper part of the bracket for picking and placing the micro prism 6. A hollow drainage groove 33 is arranged in the base 14 to drain water and avoid accumulation of impurities at the bottom from scratching the micro prism 6.

[0062] Specifically, the image sensor 7 uses a micro or lensless CMOS image sensor 7, which can reconstruct images using algorithms to achieve large field of view imaging. At the same time, the small size of the sensor enables more compact integration. The pixel size of the image sensor is 2 - 7μm, and the sampling frequency is ≤100FPS.

[0063] Specifically, a polarizer is arranged in front of the collimated polarized light source 5 to modulate it into P-polarized light. The wavelength range of the light source is 400 - 800nm, enhancing the SPR imaging effect. The micro prism 6 adopts a Kretschman micro prism coupling structure. The incident angle of the collimated polarized light source 5 relative to the micro prism 6 can be finely adjusted to adapt to the imaging conditions of different imaging chips 8.

[0064] Specifically, the SPR optical imaging module adopts a miniaturized SPR structure, without complex optical paths, and simplifies the imaging module using the fixed incident angle mode. The imaging chip 8 is a micro-nano oscillator array functionalized imaging chip, and a micro-nano oscillator array is constructed using the biomolecular self-assembly mode. The SPR imaging spatial resolution is 5 - 10μm, and the time resolution is 10ms. In one embodiment, the imaging chip 8 uses magnetron sputtering to physically vapor deposit 2 - 3nm Cr (10 - 15s) and 46 - 49nm Au (45 - 50s) on a BK7 glass slide. The preparation process of the micro-nano oscillator array functionalized imaging chip includes the following steps:

[0065] Imaging chip preparation and processing: Select an 18×18 - 22×22mm BK7 glass slide, and place it in anhydrous acetone, anhydrous ethanol, and ultrapure water solutions in sequence for ultrasonic treatment for 15 - 30min to remove impurities; use a magnetron sputtering device to physically vapor deposit 2 - 3nm Cr for 10 - 15s and 46 - 49nm Au for 45 - 50s, and polish the gold surface using a hydrogen flame; use oxygen plasma to process according to the parameters of 50 - 100W and 3 - 15min;

[0066] Self-assembly of monolayer arrays: Dilute the SH-functionalized long chains of bioflexible molecules and SH-functionalized bioflexible molecule spacers to 1-5 mM respectively using ultrapure water or alcohol, mix them evenly at a volume ratio of 1:200-1:1000 to construct monolayer arrays with different densities for connecting micro-nano oscillators of different diameters, inject them into the reaction chamber area, and let them stand at room temperature in a moist, sealed and dark box. After filling with nitrogen, incubate for 12-36 h so that the long chains of bioflexible molecules and bioflexible molecule spacers form a monolayer array. Among them, the long chains of bioflexible molecules are longer than the bioflexible molecule spacers. The long chains of bioflexible molecules refer to bioflexible molecules with reactive groups relative to the subsequent connected micro-nano oscillators for bio-specific recognition. The spacers refer to bioflexible molecules without reactive groups relative to the subsequent connected micro-nano oscillators to prevent non-specific adsorption. In the self-assembly of monolayer arrays, the density of long chains in the array is controlled by the volume ratio between the long chains and the spacers. The long chains separated by a predetermined distance can connect micro-nano oscillators in the next step, thereby connecting the micro-nano oscillators to the array. The separated distance is determined by the size of the oscillators.

[0067] Connection of micro-nano oscillators: Slowly inject ultrapure water into the reaction chamber area for 1-2 min to wash the unassembled bioflexible molecules. Dilute the micro-nano oscillators to 1-10 μg / mL with 0.01×PBS solution, and ultrasonically treat for 2-10 min to disperse the aggregated micro-nano oscillators. Inject them into the reaction chamber area and incubate for 0.5-3 h. Slowly rinse several times with 0.01×PBS solution to remove non-specific adsorption interference. Slowly aspirate 2 / 3 of the sample each time, and always keep liquid in the reaction chamber area to avoid the micro-nano oscillators sticking due to chip drying.

[0068] In one embodiment, the method for preparing the imaging chip includes the following steps:

[0069] a. Preparation and treatment of the imaging chip 8:

[0070] Select a 22×22 mm BK7 glass slide and place it in anhydrous acetone, anhydrous ethanol, and ultrapure water solutions in turn for ultrasonic treatment for 30 min. Using a magnetron sputtering device, physically vapor deposit 2 nm Cr (10 s) and 47 nm Au (47 s). Then quickly polish the gold surface with a hydrogen flame (75 L / H) (10 s). Treat it with oxygen plasma according to the parameters of 80 W and 5 min.

[0071] b. Self-assembly of monolayer arrays:

[0072] Dilute the SH-PEG-Biotin (10KD) long chain and the SH-PEG-OH (2KD) spacer to 1 mM respectively using ultrapure water, mix them evenly at a volume ratio of 1:500, inject them into the reaction chamber area, and let them stand at room temperature in a moist, sealed and dark box. After introducing nitrogen, incubate for 24 h.

[0073] c. Micro-nano oscillator connection:

[0074] Slowly inject ultrapure water into the reaction chamber area for 1 min. Dilute the micro-nano oscillator coated with streptavidin to 2 μg / mL using 0.01×PBS solution, inject it into the reaction chamber area and incubate for 2 h. Then slowly rinse 3 times with 0.01×PBS solution to remove non-specific adsorption interference. Slowly aspirate 2 / 3 of the sample each time, and keep the liquid in the reaction chamber area to avoid the bonding of micro-nano oscillators caused by chip drying.

[0075] It should be noted that the above process is an embodiment of the imaging chip preparation method, and the parameters therein are all examples, which do not mean that only the above parameters can be adopted in specific implementations.

[0076] Specifically, the signal detection and control module includes a temperature controller and a data acquisition card. The temperature controller includes a temperature sensor, an electric heating component for heating, and a semiconductor refrigeration sheet for cooling. The temperature is detected in real time by the temperature sensor and fed back to the heating and cooling components to achieve temperature control, and then the temperature of the sample solution to be measured is adjusted and controlled. Reduce the influence of temperature changes generated by the operation of internal devices and external environmental temperature changes on the detection results, and improve the anti-interference ability of the device. The data acquisition card obtains the timestamps of the image sensor 7 and the oscillation module, realizes the synchronous signal of the circuit and imaging, and is used for phase calibration to accurately detect the amplitude and phase of the micro-nano oscillator oscillation.

[0077] Specifically, the micro-sample flow path control module further includes a sampling pump and a switching valve. The microfluidic chip 2 includes a sampling port 21, an electrode hole 22, an electrode notch 23, a reaction chamber 24, a sampling hole 25, a fixing hole 26, and a chip slot 27. The chip slot 27 is used for fixing the imaging chip 8. The sampling port 21 is provided with 3 to 5, which are connected to the sampling pump and the switching valve to realize the transportation and switching of multiple samples. The reaction chamber 24 is provided for the reaction of the sample. The electrode hole 22 is used to connect the reference electrode and the counter electrode to the sample, and the electrode notch 23 is used to connect the working electrode to the imaging chip 8.

[0078] In a specific implementation, the microfluidic chip 2 includes a sample inlet 21, an electrode hole 22, an electrode notch 23, a reaction chamber 24, a sample outlet 25, a fixing hole 26, and a chip card slot 27. The microfluidic chip 2 is made of a transparent material for observing the sample situation in real time. The microfluidic chip 2 is provided with 3 to 5 sample inlets 21 and 1 sample outlet 25, which can be used in conjunction with a sample injection pump and a switching valve to achieve the transportation and rapid switching of multiple samples. The sample inlet 21 is 1 to 2 mm higher than the sample outlet 25 to prevent the sample from flowing back. A reaction chamber 24 is provided for sample reaction. An electrode hole 22 is provided to connect the reference electrode and the counter electrode to the sample, and an electrode notch 23 is provided to connect the working electrode to the imaging chip 8. A chip card slot 27 is provided for fixing the imaging chip 8, and four fixing holes 26 are fixedly connected to the microfluidic chip fixing holes 43 on the base 4 to prevent errors caused by the slippage of the imaging chip 8.

[0079] In one embodiment, as shown in conjunction with Figure 8 , 9 , 10, and 11, the microfluidic chip 2 includes a sample inlet 21, an electrode hole 22, an electrode notch 23, a reaction chamber 24, a sample outlet 25, a fixing hole 26, and a chip card slot 27. The microfluidic chip 2 is made of transparent PMMA material, which is convenient for observing the sample situation in the reaction chamber 24 in real time. A two-component adhesive is used to achieve the encapsulation with the imaging chip 8. The microfluidic chip 2 has three sample inlets 21 and one sample outlet 25, and can be combined with a sample injection pump and a switching valve to achieve rapid sample switching. The bottom of the sample inlet 21 is higher than the sample outlet 25 to prevent the sample from flowing back. A chip card slot 27 is provided for fixing the imaging chip 8, and four fixing holes 26 are further fixed to the base 4.

[0080] Specifically, the base 4 includes a height adjustment hole 41, an imaging base fixing hole 42, a microfluidic chip fixing hole 43, a drain tank 44, an imaging base card slot 45, a base fixing hole 46, and an H-shaped groove 47. The imaging base card slot 45 is used to place and directionally adjust the imaging base 3. The height adjustment hole 41 can adjust the height of the imaging base 3 to meet the optical path requirements of different samples. The imaging base fixing hole 42 and the microfluidic chip fixing hole 43 are respectively used to fix the imaging base 3 and the microfluidic chip 2. A drain tank 44 is provided below the base 4 for draining water. The H-shaped groove 47 is used to integrate the collimated polarized light source 5 and the image sensor 7. The base fixing hole 46 is provided to fix the base 4. In a specific implementation, the base 4 includes a height adjustment hole 41, an imaging base fixing hole 42, a microfluidic chip fixing hole 43, a drain tank 44, an imaging base card slot 45, a base fixing hole 46, and an H-shaped groove 47. The imaging base card slot 45 is used to place and directionally adjust the imaging base 3. One or two height adjustment holes 41 can adjust the height of the imaging base 3 to meet the optical path requirements of different samples. Two to four imaging base fixing holes 42 and two to four microfluidic chip fixing holes 43 are respectively used to fix the imaging base 3 and the microfluidic chip 2 to increase the anti-interference ability of the system. A drain tank 44 is provided below the base 4 for draining water. The H-shaped groove 47 is provided to integrate the collimated polarized light source 5 and the image sensor 7 to achieve miniaturization. The base fixing hole 46 is provided to fix the base 4.

[0081] Combined with Figure 12 、 13 As shown in FIGS. 14, the base 4 includes a height adjustment hole 41, an imaging base fixing hole 42, a microfluidic chip fixing hole 43, a drain tank 44, an imaging base card slot 45, a base fixing hole 46, and an H-shaped groove 47. Two height adjustment holes 41 are used to adjust the height of the imaging base 3. Four imaging base fixing holes 42 and four microfluidic chip fixing holes 43 are respectively used to fix the imaging base 3 and the microfluidic chip 2. A drain tank 44 is designed in the middle and lower part of the base 4. The H-shaped groove 47 is provided to increase the optical path area and facilitate the integration of other components.

[0082] Specifically, the electric field modulation module includes a circuit board and a power supply. The power supply supplies power to the SPR optical imaging module, the signal detection and control module, the micro-sample flow path control module, the electric field modulation module, and the integrated packaging module. The circuit board is used to output sinusoidal and other waveform alternating current modulation to the sample through the microfluidic chip 2. The circuit board is used to apply alternating current with different parameters. The power supply is used to supply power to the SPR optical imaging module, the signal detection and control module, the micro-sample flow path control module, and the electric field modulation module. In a specific implementation, the circuit board is small in size and can use a two-electrode or three-electrode to output sinusoidal and other waveform alternating current modulation to the sample through the microfluidic chip 2. The electrodes can be made of materials such as platinum and gold. The working electrode is connected to the imaging chip 8 at the electrode notch 23, and the reference electrode and the counter electrode (if any) use electrodes coated with a flexible material to connect to the sample at the electrode hole 22 and seal the hole. The power supply can provide power for the entire device to achieve outdoor integrated portable detection.

[0083] Combined with Figure 15 、 16 As shown, it is the overall assembly schematic diagram and structure diagram of the base 4. After assembly, each component can be flexibly disassembled, with certain adjustability and structural stability, which can improve the detection performance of the entire device.

[0084] The working principle of the above-mentioned SPR-based portable micro-nano oscillation imaging detection device is as follows:

[0085] A polarizer is arranged in front of the collimated polarized light source 5 to modulate the light source into P-polarized light. At a specific SPR angle, the electrons of the imaging chip 8 itself interact with the P-polarized incident light to couple and generate vibrations, thereby generating a propagable SPR signal at the interface. Detecting subtle refractive index changes in the sample will generate obvious SPR signals, and the SPR signals in the reflected light of the imaging chip 8 can be collected by the image sensor 7. The image sensor 7 uses a micro or lensless CMOS image sensor 7, and uses an algorithm to reconstruct the image to achieve large field of view imaging. At the same time, the small size of the sensor can achieve more compact integration. On the basis of the original SPR signal, the micro-nano oscillator array is functionalized on the imaging chip 8, and the movement form of the micro-nano oscillator is regulated by applying different electric fields to achieve signal amplification. The data acquisition card is used to obtain the timestamps of the image sensor 7 and the oscillation module, realize the synchronous signal of the circuit and imaging, and is used for phase calibration to achieve accurate detection of the amplitude and phase of the micro-nano oscillator oscillation.

[0086] As Figure 17As shown in the figure, the portable micro-nano oscillation imaging detection device is integrated in the matte airtight housing 1. The housing is divided into an upper cover plate 11, an LED controller 12, a surrounding plate 13, a base 14, a handle 15 and a rubber cushion 16. The upper cover plate 11 is connected to the surrounding plate 13 through a pin structure, and the flip design is convenient for operation. The entire matte airtight housing 1 is subjected to black matte treatment to reduce the influence of stray light reflection on detection. The matte airtight housing 1 also creates an airtight darkroom to eliminate the influence of external light changes on detection. The LED controller 12 is arranged on the front of the matte airtight housing 1 to connect to the temperature controller, the injection pump, the switching valve and the circuit board, and control the electrical parameters such as detection temperature, voltage, frequency, waveform, etc. and the sample injection and sampling. Four buffer rubber cushions 16 are arranged at the bottom of the matte airtight housing 1 to reduce the influence of external vibration on detection. The handle 15 is arranged on the upper cover plate 11 for convenient hand-held carrying.

[0087] The microfluidic chip 2 includes a sample injection port 21, an electrode hole 22, an electrode notch 23, a reaction chamber 24, a sample outlet hole 25, a fixing hole 26, and a chip card slot 27. The microfluidic chip 2 uses a transparent PMMA material to facilitate real-time observation of the sample situation in the reaction chamber 24. A two-component adhesive is used for encapsulation with the imaging chip 8. The microfluidic chip 2 has three sample injection ports 21 and one sample outlet hole 25, and the sample can be quickly switched by combining the injection pump and the switching valve. The bottom of the sample injection port 21 is higher than the sample outlet hole 25 to prevent sample backflow. The chip card slot 27 is provided for fixing the imaging chip 8, and the four fixing holes 26 are further fixed to the base 4.

[0088] The imaging base 3 adopts a V-shaped design. Inclined slots 31 are provided on each of the two side brackets, and a V-shaped slot 34 is provided in the middle to stably fix the micro prism 6 and increase the optical path passing area as much as possible. A micro prism access notch 32 is provided at the upper part of the bracket for convenient access of the micro prism 6. A hollow drainage groove 33 is provided below the base 14 to prevent impurities from accumulating and scratching the micro prism 6. The base slot 35 is provided to facilitate the directional height adjustment of the base 14.

[0089] The base 4 includes height adjustment holes 41, imaging base fixing holes 42, microfluidic chip fixing holes 43, a drainage groove 44, an imaging base slot 35, a base fixing hole 46, and an H-shaped groove 47. The two height adjustment holes 41 are used to adjust the height of the imaging base 3, and the four imaging base fixing holes 42 and the four microfluidic chip fixing holes 43 are used to fix the imaging base 3 and the microfluidic chip 2 respectively. A drainage groove 44 is designed in the middle and lower part of the base 4. The H-shaped groove 47 is provided to increase the optical path area and facilitate the integration of other components.

[0090] This application also provides a portable micro-nano oscillation imaging detection method based on SPR. The method may include the following steps:

[0091] a. SPR imaging optical path setup: Turn on the power supply, open the upper cover plate 11, turn on the collimated polarized light source 5, turn on the image sensor 7, and adjust the power and angle of the collimated polarized light source 5, the sensitivity of the image sensor 7, and the exposure time according to the imaging effect. Place the micro prism 6 on the imaging base 3, slowly add immersion oil, bond the functionalized imaging chip 8 of the micro-nano oscillator array and the chip slot 27 in the microfluidic chip 2 with a two-component adhesive, and slowly place it on the micro prism 6;

[0092] b. Electric field modulation connection: Connect the electrodes of the circuit board to the imaging chip 8 and the sample through the microfluidic chip 2 in sequence. The working electrode is connected to the imaging chip 8 at the electrode notch 23, and the counter electrode and the reference electrode (if any) contact the liquid sample at the electrode hole 22, and the holes are sealed by coating the electrodes with a flexible material. Adjust the electrical parameters, insert the imaging base 3 into the base 4, adjust to the appropriate imaging height, adjust the height adjustment hole 41 to the appropriate imaging height, and tighten the imaging base fixing hole 42 and the microfluidic chip fixing hole 43;

[0093] c. Control module setup: Connect the injection pump, switching valve, and the injection port 21 of the microfluidic chip 2 using pipelines, and connect the outlet hole to a waste liquid bottle. Turn on the temperature controller and close the upper cover plate 11;

[0094] d. Signal acquisition and detection: Turn on the electric field modulation and image acquisition, obtain the synchronous signals of the circuit and imaging using a data acquisition card, and analyze the acquired data using image processing software.

[0095] Combined with Figure 18 As shown, the imaging effects of the imaging detection device with different wavelengths of the collimated polarized light source were simulated. As can be seen from the figure, in the wavelength range of 400 - 800 nm, as the wavelength increases, the slope of the reflectance change with the angle is larger, that is, the imaging is more sensitive. However, the resolution is lower when the wavelength is longer, so it is necessary to select an appropriate wavelength according to specific requirements.

[0096] Combined with Figure 19 As shown, functionalization of the imaging chip: Dilute SH-PEG-Biotin (10KD) and SH-PFG-OH (2KD) to 1 mM with ultrapure water in a ratio of 1:500, and connect 5 μg / mL 5-μm streptavidin-coated polystyrene microspheres. Apply sinusoidal AC electric fields with voltages of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 Vpp at a frequency of 1 Hz. Use the CMOS image sensor 7 to record the real-time change of the gray value of the micro-nano oscillator oscillation. It can be seen that the gray value of the micro-nano oscillator changes periodically at a frequency of 1 Hz, and the change of the gray value of the micro-nano oscillator is larger as the voltage increases.

[0097] Combined with Figure 20As shown, imaging chip functionalization: Dilute SH-PEG-Biotin (10KD) and SH-PFG-OH (2KD) to 1 mM with ultrapure water at a ratio of 1:1000, and connect 5 μg / mL of 2 μm streptavidin-coated silica microspheres. Apply sinusoidal AC electric fields with frequencies of 0, 2, 4, 6, 7, 9, and 11 Hz respectively at a voltage of 0.5 Vpp. Use the CMOS image sensor 7 to record the change in the grayscale value of the micro-nano oscillator oscillation, and perform Fourier transform (FFT) on the collected signals. It can be seen that different frequency-modulated micro-nano oscillators have different maximum FFT amplitude responses and positions.

[0098] After considering the specification and the practice disclosed herein, those skilled in the art will readily conceive of other embodiments of the present application. The present application is intended to cover any variations, uses, or adaptations of the present application, which follow the general principles of the present application and include the common general knowledge or conventional technical means in the technical field not disclosed in the present application.

[0099] It should be understood that the present application is not limited to the exact structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A portable micro-nano oscillation imaging detection device based on SPR, characterized in that, It includes an SPR optical imaging module, a signal detection and control module, a micro-sample flow path control module, an electric field modulation module, and an integrated packaging module; The SPR optical imaging module includes a collimated polarized light source, a micro prism, an imaging chip, and an image sensor. The collimated polarized light source is coupled to the micro prism at a specific SPR angle. The imaging chip is arranged above the micro prism. The image sensor is integrated with the micro prism for reflected light acquisition. The image sensor uses a micro-lens or lensless CMOS image sensor. The imaging chip is a micro-nano oscillator array functionalized imaging chip, and a micro-nano oscillator array is constructed by the self-assembly mode of biomolecules; The signal detection and control module is used for temperature control and obtaining the timestamps of the image sensor and the electric field modulation module to achieve phase synchronization, and realizing the synchronous signal of the circuit and imaging, so as to accurately detect the amplitude and phase of the micro-nano oscillator oscillation; The micro-sample flow path control module includes a microfluidic chip, which is arranged above the imaging chip for the flow path control and switching of trace samples to improve the detection stability; The electric field modulation module is electrically connected to the imaging chip and the micro-sample flow path control module, and outputs sinusoidal and other waveform alternating current modulation, which is used to apply oscillation power to micro-nano oscillators of different materials, shapes and sizes to amplify the signal and enhance the detection sensitivity; The integrated packaging module includes a matte airtight housing, an imaging base, and a pedestal. The matte airtight housing is used for the packaging of the device. The pedestal is arranged at the bottom inside the matte airtight housing. The imaging base is arranged on the pedestal. A V-shaped card slot is arranged in the imaging base, and the micro prism is arranged in the V-shaped card slot; Among them, the material of the micro-nano oscillator is a charged particle, the shape of the micro-nano oscillator is spherical, rod-shaped or sheet-shaped, and the diameter size range of the micro-nano oscillator is 500 nm to 5 μm.

2. The device according to claim 1, wherein In the SPR optical imaging module, the SPR imaging spatial resolution is 5 to 10 μm, and the time resolution is 10 ms.

3. The device according to claim 1, characterized in that The signal detection and control module includes a temperature controller and a data acquisition card. The temperature controller is used to regulate the ambient temperature, and the data acquisition card is used to obtain the timestamps of the image sensor and the electric field modulation module.

4. The device according to claim 1, characterized in that, The micro-sample flow path control module further includes a sample injection pump and a switching valve. The microfluidic chip includes a sample injection port, an electrode hole, an electrode notch, a reaction chamber, a sample outlet hole, a chip card slot, and a fixing hole. The chip card slot is used for fixing the imaging chip. 3 to 5 sample injection ports are arranged, which are connected to the sample injection pump and the switching valve to realize the transportation and switching of different samples. A reaction chamber is arranged for the sample to react, and the volume of the reaction chamber is 50 to 300 μL. The electrode hole is used to connect the counter electrode to the sample, the electrode notch is used to connect the working electrode to the imaging chip, the fixing hole is used for fixed connection with the pedestal, and the sample injection pump controls the sample injection flow rate at 50 to 500 μL / min.

5. The device according to claim 1, characterized in that, The electric field modulation module includes a circuit board and a power supply. The power supply supplies power to the SPR optical imaging module, the signal detection and control module, the microfluidic channel control module, and the electric field modulation module. The circuit board is used to output sinusoidal and other waveform alternating current modulation to the sample through a microfluidic chip. The adjustable range of the peak-to-peak voltage of the alternating current modulation voltage is -2~2 Vpp, and the adjustable range of the frequency is 0.1~20 Hz.

6. The device according to claim 1, characterized in that, Vertical brackets are respectively arranged at both ends of the V-shaped card slot. Inclined plane card slots are arranged on the inner sides of the vertical brackets. Micro prism picking and placing notches are arranged on the brackets to facilitate the picking and placing of the micro prism. The depths of the inclined plane card slots and the V-shaped card slot are both 2~4 mm.

7. The device according to claim 1, characterized in that, The base includes height adjustment holes, imaging base fixing holes, microfluidic chip fixing holes, drain tanks, imaging base card slots, base fixing holes, and H-shaped grooves. The imaging base card slot is used to place and directionally adjust the imaging base. The height adjustment holes are used to adjust the height of the imaging base. The imaging base fixing holes and the microfluidic chip fixing holes are respectively used to fix the imaging base and the microfluidic chip. A drain tank is arranged below the base for draining water. The H-shaped groove is used to integrate the collimated polarized light source and the image sensor. Base fixing holes are provided to fix the base.

8. The device according to claim 1, characterized in that, The matte airtight outer shell is integrally treated with black matte, and includes an upper cover plate, an LED controller, a surrounding plate, a base, a handle, and rubber soft pads. The surrounding plate is arranged on the base. The upper cover plate is connected to the surrounding plate through a pin structure to form a flip structure. The LED controller is connected to the signal detection and control module, the microfluidic channel control module, and the electric field modulation module. The rubber soft pads are arranged below the base. The handle is arranged on the upper surface of the upper cover plate.

9. A portable micro-nano oscillation imaging detection method based on SPR, characterized in that, A portable micro-nano oscillation imaging detection device based on SPR according to claim 1, the method comprising the following steps: SPR imaging optical path construction: Turn on the power supply, open the matte airtight outer shell, turn on the collimated polarized light source, turn on the image sensor, and respectively adjust the power and angle of the collimated polarized light source, and the sensitivity and exposure time of the image sensor according to the imaging effect; Place the micro prism on the imaging base, snap the imaging base into the base, adjust to the appropriate imaging height and tighten the screws to fix, slowly add mirror oil, snap the micro-nano oscillator array functionalized imaging chip into the chip card slot of the microfluidic chip, seal it with a two-component adhesive, slowly place it on the micro prism, and tighten the screws to fix; Electric field modulation connection: Connect the electrodes of the circuit board to the imaging chip and the sample through the microfluidic chip in sequence. The working electrode is connected to the imaging chip at the electrode notch, the counter electrode contacts the liquid sample at the electrode hole, and the electrode is coated with a flexible material to seal the reaction chamber; Test and adjust the electrical parameters; Control module construction: Connect the inlet pump, switching valve, and the inlet of the microfluidic chip with pipelines, and connect the outlet hole to a waste liquid bottle. Turn on the temperature controller and close the matte airtight outer shell; Signal acquisition and detection: Turn on the electric field modulation and image acquisition, obtain the synchronous signals of the circuit and imaging by using a data acquisition card, and analyze the collected data by using image processing software.

10. The method according to claim 9, wherein The preparation process of the micro-nano oscillator array functionalized imaging chip includes the following steps: Imaging chip preparation and treatment: Select a 18×18~22×22 mm BK7 glass slide, and place it in anhydrous acetone, anhydrous ethanol, and ultrapure water solution in sequence for ultrasonic treatment for 15~30 min to remove impurities; use a magnetron sputtering device to physically vapor deposit 2~3 nm Cr for 10~15 s and 46~49 nm Au for 45~50 s, and polish the gold surface with a hydrogen flame; use oxygen plasma to process according to the parameters of 50~100 W and 3~15 min; Self-assembly of monolayer arrays: Dilute the SH-functionalized bio-flexible molecular long chain and the SH-functionalized bio-flexible molecular spacer to 1~5 mM respectively using ultrapure water or alcohol, mix them according to a volume ratio of 1:200~1:1000 to construct monolayer arrays with different densities for connecting micro-nano oscillators with different diameter sizes, inject them into the reaction chamber area, and place them at room temperature in a moist, sealed dark box. After filling with nitrogen, incubate for 12~36 h to make the bio-flexible molecular long chain and the bio-flexible molecular spacer form a monolayer array, where the bio-flexible molecular long chain is longer than the bio-flexible molecular spacer chain. The bio-flexible molecular long chain refers to a bio-flexible molecule with an active reaction group relative to the subsequent connected micro-nano oscillator for bio-specific recognition, and the spacer refers to a bio-flexible molecule without an active reaction group relative to the subsequent connected micro-nano oscillator to prevent non-specific adsorption; Connection of micro-nano oscillators: Slowly inject ultrapure water into the reaction chamber area for 1~2 min to wash the unassembled bio-flexible molecules, dilute the micro-nano oscillators to 1~10 μg / mL with 0.01×PBS solution, and perform ultrasonic treatment for 2~10 min to disperse the aggregated micro-nano oscillators. Inject them into the reaction chamber area and incubate for 0.5~3 h. Slowly rinse several times with 0.01×PBS solution to remove non-specific adsorption interference. Slowly aspirate 2 / 3 of the sample each time, and always keep liquid in the reaction chamber area to avoid the micro-nano oscillators sticking due to chip drying.

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