Measuring device and its detection method

Abstract

This application belongs to the field of biochemical substance detection, and particularly relates to a measuring device and its detection method. The measuring device includes a detection system, an SPRi system, a terahertz imaging system, and a data acquisition and control system. The measuring device incorporates a terahertz imaging system and sets four channels on the sample cell at the bottom of the microarray chip. A standard sample is sequentially introduced into the first channel, the analyte into the second channel, the standard sample into the third channel, and the analyte into the fourth channel. The data acquisition and control system controls the injection of different samples into the four channels, while simultaneously controlling the movement of the light source assembly and the terahertz emitter, so that the p-polarized beam and the terahertz signal simultaneously illuminate the same channel. By calculating the signals between different channels, the terahertz imaging system can determine the sample type within the fluid by measuring the refractive index change of the analyte compared to the standard sample, thereby improving the accuracy of determining the start and end times of specific binding.

Description

Technical Field

[0001] This application relates to the field of biochemical substance detection technology, and in particular to a measuring device and its detection method. Background Technology

[0002] Biochemical molecular affinity analysis is a technique for measuring and calculating the strength of intermolecular binding, primarily used to determine whether a specific affinity exists between two molecules. This technique can be used to study the mechanisms of intermolecular interactions and to explore the interactions between drugs, proteins, and biomolecules. Currently, commonly used biochemical molecular affinity analysis mainly relies on intermolecular interaction kinetic detection methods. Affinity information is obtained by fitting and monitoring signals generated by intermolecular binding through kinetic models (such as optical and thermodynamic methods). Compared to single-channel analysis methods such as thermodynamics, which can only detect binding at a dynamic equilibrium state, surface plasmon resonance imaging (SPRi) has advantages such as real-time monitoring of signals at various stages of intermolecular binding and multi-channel expansion. Therefore, it has wide applications in drug development, biomolecule structure prediction, and disease diagnosis and treatment.

[0003] The basic principle of biochemical molecular affinity analysis based on SPRi technology is as follows: after chemically modifying a metal surface and immobilizing biochemical molecules (hereinafter referred to as "immobilized molecules"), a solution of molecules that bind to them (hereinafter referred to as "analyte molecules") is introduced to induce specific binding between the two on the modified surface. During this process, a p-polarized light beam with a specific wavelength and incident angle is excited at the metal-modified surface interface by a dielectric coupler (such as a prism or grating) and energy transfer occurs. The energy transfer ratio changes with the introduction and specific binding of the analyte molecules, thereby altering the intensity of reflected light at the interface continuously collected by the array detector. The affinity data of the specific binding can be obtained by fitting the reflected light intensity signal through a kinetic model. One limitation of using SPRi sensors for biochemical molecular affinity analysis is that the introduction and specific binding of the analyte molecules occur in the same space, making it difficult to effectively distinguish the introduction time point and the start and end times of specific binding. This makes it difficult to effectively subtract the volume refractive index change caused by the introduction of the analyte molecules, and also has a certain impact on the accuracy of affinity analysis. Summary of the Invention

[0004] This application provides a measuring device and a detection method thereof, which can effectively distinguish the refractive index change of the analyte molecule compared with the standard sample, and the start and end times of the binding between the analyte molecule and the fixed molecule, thereby improving the accuracy of affinity analysis.

[0005] Therefore, according to one aspect of this application, a measuring device is provided, comprising:

[0006] The detection system includes a microarray chip and a sample cell in contact with the microarray chip. The sample cell has a first channel, a second channel, a third channel, and a fourth channel. The microarray chip has fixed molecules corresponding to the third and fourth channels.

[0007] The SPRi system includes a light source assembly, a first linear module, and a first array detector. The light source assembly is disposed on the first linear module and can change its position under the drive of the first linear module to emit a p-polarized beam into the first channel, the second channel, the third channel, or the fourth channel. The first array detector is used to receive the reflected beam from the metal-dielectric interface of the microarray chip.

[0008] A terahertz imaging system includes a terahertz transmitter, a second linear module, and a second array detector. The terahertz transmitter is mounted on the second linear module and can change position under the drive of the second linear module to transmit terahertz signals to a first, second, third, or fourth channel. The second array detector is used to receive terahertz signals reflected from the surface of a microarray chip on the fluid side.

[0009] The data acquisition and control system is used to control the operation of the light source assembly, the first linear module, the terahertz transmitter, and the second linear module, as well as to acquire and calculate the data of the first array detector and the second array detector.

[0010] Optionally, the detection system also includes a multi-channel fluid valve, a standard sample injection pump, a analyte molecule injection pump, and a controller. The first, second, third, and fourth channels are respectively connected to the five outlets of the multi-channel fluid valve. The standard sample injection pump and the analyte molecule injection pump are respectively connected to the two inlets of the multi-channel fluid valve. The controller is electrically connected to the acquisition and control system and is used to control the operation of the multi-channel fluid valve, the standard sample injection pump, and the analyte molecule injection pump.

[0011] Optionally, the light source assembly includes a visible light source, a collimating lens, and a polarizer arranged sequentially along the optical path.

[0012] Optionally, both the first linear module and the second linear module are powered by stepper motors.

[0013] Optionally, the stepper motor has an accuracy of 0.1mm.

[0014] Optionally, the sample cell is made of polydimethylsiloxane.

[0015] Optionally, the fabrication method of the microarray chip is as follows:

[0016] The glass substrate is ultrasonically cleaned for a first preset time using a mixture of ethanol and ether with a preset volume ratio to clean the surface of the glass substrate.

[0017] The cleaned glass substrate is placed in an electron beam evaporation apparatus, and a vacuum is drawn to reduce the pressure to a preset value. Chromium of a first preset thickness is deposited at a first preset rate as an adhesion layer. Gold or silver of a second preset thickness is deposited on the adhesion layer at a second preset rate as an upper metal surface. Polycarbonate of a preset mass fraction is spin-coated on the upper metal surface at a third preset rate as a waveguide layer. Gold or silver of a fourth preset thickness is deposited on the waveguide layer at a fourth preset rate as a lower metal surface. Cadmium telluride of a fifth preset thickness is deposited on the metal surface at a fifth preset rate as a terahertz signal enhancement layer.

[0018] The evaporated glass substrate is immersed in a mercaptoalkanoic acid solution of a first preset concentration for a second preset time to form a monomolecular self-assembled layer.

[0019] A hydrogel surface is formed by immersing the sample in a polymer solution for a third preset time.

[0020] Immobilized molecules are coated on the hydrogel surface in regions corresponding to the third and fourth channels.

[0021] Optionally, the preset volume ratio ranges from 1:1 to 1:10; the first preset duration is greater than 30 minutes.

[0022] The preset air pressure value is less than 10⁻⁵ mTorr;

[0023] The first preset speed is less than 0.1 nm / s; the first preset thickness ranges from 0.5 nm to 2.5 nm.

[0024] The second preset rate is greater than 0.01 nm / s; the second preset thickness ranges from 25 nm to 35 nm.

[0025] The third preset rate is 2000 r / s; the preset mass fraction is 1%.

[0026] The fourth preset rate is greater than 0.01 nm / s; the fourth preset thickness ranges from 25 nm to 35 nm.

[0027] The fifth preset speed is 0.01 nm / s; the fifth preset thickness ranges from 10 nm to 20 nm.

[0028] The first preset concentration is greater than 0.1 mM, and the second preset duration is greater than 30 min;

[0029] The third preset duration is greater than 30 minutes.

[0030] According to another aspect of this application, a detection method is provided, based on the above-described measuring device, the detection method comprising the following steps:

[0031] S1: Before the experiment, measure the terahertz absorption coefficient of the SPRi signal of the standard sample, the analyte molecule, and the fixed molecule;

[0032] S11: Move the light source assembly and terahertz emitter to allow the p-polarized beam and terahertz signal to enter the first channel, introduce a standard sample into the first channel, and measure the SPRi signal and terahertz signal by the first array detector and the second array detector respectively. The acquisition and control system calculates the terahertz absorption coefficient at the preset frequency.

[0033] S12: Move the light source assembly and terahertz emitter to allow the p-polarized beam and terahertz signal to enter the second channel. Introduce the target molecule, immunoglobulin, at a preset concentration into the second channel. The SPRi signal and terahertz signal are measured by the first array detector and the second array detector, respectively. The acquisition and control system calculates the terahertz absorption coefficient at the preset frequency and calculates the SPRi signal difference between the standard sample and the target molecule.

[0034] S13: Move the light source assembly and terahertz emitter to allow the p-polarized beam and terahertz signal to enter the third channel, and introduce a standard sample into the third channel. The SPRi signal and terahertz signal are measured by the first array detector and the second array detector, respectively. The acquisition and control system calculates the terahertz absorption coefficient at the preset frequency.

[0035] S2: In the experiment, the light source assembly and terahertz transmitter were moved to allow the p-polarized beam and terahertz signal to enter the fourth channel.

[0036] S21: Pass a standard sample of the fourth preset duration into the fourth channel, measure its SPRi signal and terahertz signal, and calculate the terahertz absorption coefficient at the preset frequency;

[0037] S22: Introduce immunoglobulin G at a preset concentration of the target molecule into the fourth channel for a certain duration, measure its SPRi signal and terahertz signal, and calculate the terahertz absorption coefficient at the preset frequency. Determine the diffusion time of immunoglobulin G in the hydrogel of the microarray chip from the terahertz absorption measurement results at the preset frequency when the target molecule generates specific binding.

[0038] S23: Introduce a standard sample with a preset duration into the fourth channel and measure its SPRi signal and terahertz absorption coefficient. Subtract the diffusion time in S22 and the SPRi signal difference between the standard sample and the analyte in S12 from the SPRi signal result graph of the analyte generating specific binding when the analyte is introduced into the fourth channel to obtain the corrected SPRi measurement result of the analyte's specific binding.

[0039] Optionally, the preset frequency is 1THz or 2THz;

[0040] The preset concentration is 10 nM or 100 nM;

[0041] The fourth preset duration is 200 seconds;

[0042] The duration is 200 seconds.

[0043] The fifth preset duration is 600 seconds.

[0044] The beneficial effects of the measuring device and detection method provided in this application are as follows: Compared with the prior art, the measuring device of this application introduces a terahertz imaging system and sets four channels on the sample cell at the bottom of the microarray chip. Standard samples are introduced into the first channel, analytes into the second channel, standard samples into the third channel, and analytes into the fourth channel in sequence. The acquisition and control system controls the injection of different samples into the four channels. At the same time, the first linear module carrying the light source assembly and the second linear module carrying the terahertz transmitter are controlled to move, so that the p-polarized beam and the terahertz signal simultaneously irradiate the same channel. By calculating the signals between different channels, the terahertz imaging system can determine the type of sample in the fluid by realizing the refractive index change of the analyte compared with the standard sample, and improve the accuracy of determining the start and end times of specific binding. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] in:

[0047] Figure 1 This is a schematic diagram of the overall structure of the measuring device shown in one embodiment of this application;

[0048] Figure 2 This is a schematic diagram showing the connection of the sample cell, multi-channel fluid valve, standard sample injection pump, analyte molecule injection pump and controller in a measuring device according to an embodiment of this application.

[0049] Figure 3 This application illustrates the variation of reflected light intensity of a microarray chip with incident angle under different samples.

[0050] Figure 4This is a schematic diagram of the spatial electric field intensity distribution of a microarray chip on the metal surface toward the chemically modified structure at the resonance angle of a standard sample, as shown in an embodiment of this application.

[0051] Figure 5 This is a schematic diagram of the absorption rates of different samples in the terahertz band;

[0052] Figure 6 This is a graph showing the SPRi signal results of the specific binding of the analyte molecule when the incident angle is 55.6 degrees in Embodiment 1 of this application.

[0053] Figure 7 This is a terahertz absorption measurement result at a frequency of 1THz when the analyte molecule is introduced to generate specific binding in Embodiment 1 of this application;

[0054] Figure 8 This is a graph showing the results of SPRi measurement of the specific binding of the analyte molecule, obtained by time node correction based on the terahertz absorption measurement results in Embodiment 1 of this application.

[0055] Figure 9 This is a graph showing the SPRi signal results of specific binding of the analyte molecule when the incident angle is 55.7 degrees in Embodiment 2 of this application.

[0056] Figure 10 This is a terahertz absorption measurement result at a frequency of 2THz when the analyte molecule is introduced and specific binding occurs in Embodiment 2 of this application.

[0057] Figure 11 This is a graph showing the results of SPRi measurement of the specific binding of the analyte molecule, obtained by time node correction based on the terahertz absorption measurement results in Embodiment 2 of this application.

[0058] Explanation of key component symbols:

[0059] 100. Detection system;

[0060] 110. Microarray chip;

[0061] 120. Sample cell; 121. First channel; 122. Second channel; 123. Third channel; 124. Fourth channel;

[0062] 130. Dielectric coupler;

[0063] 140. Multi-way fluid valve;

[0064] 150. Standard sample injection pump;

[0065] 160. Sample pump for the analyte molecule;

[0066] 170. Controller;

[0067] 200, SPRi system;

[0068] 210. Light source assembly; 211. Light source; 212. Collimating lens; 213. Polarizer;

[0069] 220. First linear module;

[0070] 230. First array detector;

[0071] 300. Terahertz Imaging System;

[0072] 310. Terahertz transmitter;

[0073] 320. Second linear module;

[0074] 330. Second array detector;

[0075] 400. Data Acquisition and Control System. Detailed Implementation

[0076] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many other different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0077] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0078] Furthermore, the terms "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first," "second," "third," or "fourth" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0079] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0080] Embodiments of this application provide a measuring device, such as... Figures 1-2As shown, the measuring device includes a detection system 100, an SPRi system 200, a terahertz imaging system 300, and an acquisition and control system 400.

[0081] The detection system 100 includes a microarray chip 110 and a sample cell 120 in contact with the microarray chip 110. The sample cell 120 has a first channel 121, a second channel 122, a third channel 123 and a fourth channel 124. The microarray chip 110 has fixed molecules corresponding to the third channel 123 and the fourth channel 124.

[0082] The SPRi system 200 includes a light source assembly 210, a first linear module 220, and a first array detector 230. The light source assembly 210 is disposed on the first linear module 220 and can change its position under the drive of the first linear module 220 to emit a p-polarized beam into the first channel 121, the second channel 122, the third channel 123, or the fourth channel 124. The first array detector 230 is used to receive the reflected beam from the metal-dielectric interface of the microarray chip 110.

[0083] The terahertz imaging system 300 includes a terahertz transmitter 310, a second linear module 320, and a second array detector 330. The terahertz transmitter 310 is mounted on the second linear module 320 and can change position under the drive of the second linear module 320 to transmit terahertz signals to the first channel 121, the second channel 122, the third channel 123, or the fourth channel 124. The second array detector 330 is used to receive the terahertz signals reflected by the surface of the microarray chip 110 on the fluid side; and

[0084] The data acquisition and control system 400 is used to control the operation of the light source assembly 210, the first linear module 220, the terahertz transmitter 310 and the second linear module 320, and to acquire and calculate the data of the first array detector 230 and the second array detector 330.

[0085] In this embodiment, the measuring device incorporates a terahertz imaging system 300 and has four channels on the sample cell 120 at the bottom of the microarray chip 110. Standard samples are introduced into the first channel 121, the analyte is introduced into the second channel 122, the standard sample is introduced into the third channel 123, and the analyte is introduced into the fourth channel 124. The acquisition and control system 400 controls the injection of different samples into the four channels. At the same time, the first linear module 220 carrying the light source assembly 210 and the second linear module 320 carrying the terahertz transmitter 310 are controlled to move, so that the p-polarized beam and the terahertz signal simultaneously illuminate the same channel. By calculating the signals between different channels, the terahertz imaging system 300 can determine the type of sample in the fluid by the refractive index change of the analyte compared to the standard sample, and improve the accuracy of determining the start and end times of specific binding.

[0086] In one embodiment, such as Figure 2 As shown, the detection system also includes a multi-channel fluid valve 140, a standard sample injection pump 150, a analyte molecule injection pump 160, and a controller 170. The first channel 121, the second channel 122, the third channel 123, and the fourth channel 124 are respectively connected to the five outlets of the multi-channel fluid valve 140. The standard sample injection pump 150 and the analyte molecule injection pump 160 are respectively connected to the two inlets of the multi-channel fluid valve 140. The standard sample injection pump 150 is used to pump in standard samples, and the analyte molecule injection pump 160 is used to pump in analytes. The controller 170 is electrically connected to the acquisition and control system 400. The controller 170 is used to control the operation of the multi-channel fluid valve 140, the standard sample injection pump 150, and the analyte molecule injection pump 160.

[0087] By setting up a multi-channel fluid valve 140, a standard sample injection pump 150, a molecule injection pump 160, and a controller 170, the data acquisition and control system 400 can accurately control the introduction of standard samples or molecules to be tested into different channels.

[0088] In one specific embodiment, such as Figure 1 As shown, the light source assembly 210 includes a visible light source 211, a collimating lens 212, and a polarizer 213 arranged sequentially along the optical path. The collimating lens 212 and the polarizer 213 are used to generate a p-polarized parallel beam.

[0089] In the application, the p-polarized parallel beam emitted by the light source component 210 is coupled to the metal surface of the microarray chip 110 via the dielectric coupler 130. The dielectric coupler 130 can be implemented by a glass prism or waveguide element. The glass prism can be made of optical glass with high light transmittance according to actual needs, such as ZF3 glass, BK7 glass, etc. Figure 1The example shows a dielectric coupler 130 as a triangular prism-shaped glass prism, with a microarray chip 110 disposed on one side surface of the dielectric coupler 130.

[0090] The first array detector 230 and the second array detector 330 can be implemented by photoelectric conversion devices such as photodiodes, phototransistors, and photomultiplier tubes, for example, charge-coupled device (CCD) sensors or complementary metal-oxide-semiconductor (CMOS) sensors.

[0091] The acquisition and control system 400 can be a Central Processing Unit (CPU), or other general-purpose processors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. The general-purpose processor can be a microprocessor or any conventional processor, such as a control card with an STM32F407 or STM32F103 chip. The control system can communicate with other components via any wired communication method.

[0092] Example 1

[0093] Reference Figure 1 This embodiment provides a multi-parameter measurement device based on terahertz imaging and surface plasmon resonance imaging. The device includes a detection system 100, an SPRi system 200, a terahertz imaging system 300, and a data acquisition and control system 400. Additionally, the measurement device may include an optional temperature control system. The SPRi system 200 uses a 632.8nm laser as a light source 211, a 0.1mm precision stepper motor as the power source for the first linear module 220, a visible light-band charge-coupled device (CCD) as the first array detector 230, and ZF3 glass as the glass prism material.

[0094] The fabrication process and key parameter range of the microarray chip 110 are as follows: The glass substrate is ultrasonically cleaned for more than 30 minutes with a mixture of ethanol and diethyl ether at a volume ratio of 1:1 to 1:10 to clean its surface; it is then placed in an electron beam evaporation instrument and the vacuum is reduced to 10. -5Below millitor level; a 2.5 nm chromium layer is deposited at a rate of less than 0.1 nm per second as an adhesion layer; a 30 nm gold layer is deposited on the adhesion layer at a rate of greater than 0.01 nm per second as the upper metal surface of WCSPR (Waveguide-Coupled Surface Plasmon Resonance); a 1% polycarbonate layer is spin-coated on the upper metal surface at a rate of 2000 rpm as the waveguide layer of WCSPR; a 30 nm gold layer is deposited on the waveguide layer at a rate of greater than 0.01 nm per second as the lower metal surface of WCSPR structure-excited SPR (Surface Plasmon Resonance); and a 15 nm cadmium telluride layer is deposited at a rate of 0.01 nm per second as the terahertz signal enhancement layer. After soaking in a solution of mercaptoalkanoic acid (with a carbon chain length between 3 and 15) at a concentration of 0.1 mM or higher for 30 minutes to form a monomolecular self-assembled layer, the surface of the hydrogel is formed by soaking in a dextran solution with a molecular weight of 5000 Daltons for 1 hour. Protein A at a fixed concentration of 20 nM is then coated on the corresponding regions of the third channel 123 and the fourth channel 124 on the hydrogel surface.

[0095] The four channels of the sample pool (i.e., the first channel 121, the second channel 122, the third channel 123 and the fourth channel 124) and the pipeline for conveying fluid are all made of polydimethylsiloxane, with each channel having dimensions of length × width × height (mm): 10 × 3 × 0.1.

[0096] The variation of reflected light intensity of microarray chip 110 with incident angle under different samples is as follows: Figure 3 As shown, the spatial electric field intensity distribution on the metal surface towards the chemically modified structure at the resonance angle of the standard sample is as follows: Figure 4 As shown. The terahertz imaging system 300 uses a fiber-coupled terahertz photoconductive antenna excited by a femtosecond laser with a center wavelength of approximately 1560nm as the terahertz transmitter 310, a stepper motor with an accuracy of 0.1mm as the power source for the second linear module 320, and a terahertz band charge-coupled device (CCD) as the second array detector 330.

[0097] Based on the multi-parameter measurement device based on terahertz imaging and surface plasmon resonance imaging provided in this embodiment, this embodiment provides a detection method for simultaneously detecting the introduction time of the analyte molecule, the binding start and end times, and the introduction time of the standard sample. This method can achieve time calibration of the real-time binding signals of the analyte molecule and the fixed molecule measured by surface plasmon resonance imaging. For the SPR signal: the first channel 121 and the second channel 122 are used to calculate the volume refractive index change and are used for subtraction in the fourth channel 124. For the terahertz signal: the first channel 121 is used to measure the absorption of the standard sample, the second channel 122 is used to measure the absorption of the analyte molecule, the third channel 123 subtracts the first channel 121 to calculate the fixed molecule absorption, the appearance of the second channel 122 signal after subtracting the third channel 123 in the fourth channel 124 indicates the presence of the analyte molecule, and the subsequent shift after subtracting the second channel 122 indicates the start of binding absorption. If the absorption of the analyte molecule and the binding absorption appear simultaneously, it indicates that both occur at the same time.

[0098] Before the experiment, it is necessary to measure the terahertz absorption coefficients of the SPRi signals of the standard sample, the analyte molecule, and the fixed molecule.

[0099] The first step involves moving the light source assembly 210 and the terahertz transmitter 310 to allow the p-polarized beam and terahertz signal to enter the first channel 121. The multi-channel fluid valve 140 is adjusted to introduce a standard sample into the first channel 121. The first array detector 230 and the second array detector 330 measure the SPRi signal and the terahertz signal respectively. The acquisition and control system 400 calculates the terahertz absorption coefficient at a frequency of 1 THz.

[0100] The second step involves moving the light source assembly 210 and the terahertz emitter 310 to allow the p-polarized beam and terahertz signal to enter the second channel 122. The multi-channel fluid valve 140 is adjusted to introduce immunoglobulin with a concentration of 10 nM into the second channel 122. The SPRi signal and terahertz signal are measured by the first array detector 230 and the second array detector 330, respectively. The acquisition and control system 400 calculates the terahertz absorption coefficient at a frequency of 1 THz and calculates the SPRi signal difference between the standard sample and the analyte.

[0101] The third step involves moving the light source assembly 210 and the terahertz emitter 310 to direct the p-polarized beam and terahertz signal into the third channel 123. The multi-channel fluid valve 140 is adjusted to introduce a standard sample into the third channel 123. The first array detector 230 and the second array detector 330 measure the SPRi signal and the terahertz signal respectively. The acquisition and control system 400 calculates the terahertz absorption coefficient at a frequency of 1 THz. The absorption rates of different samples in the terahertz band are shown below. Figure 5 As shown.

[0102] In the experiment, the light source assembly 210 and the terahertz transmitter 310 were moved so that the p-polarized beam and the terahertz signal were injected into the fourth channel 124.

[0103] First, adjust the multi-channel fluid valve 140, introduce a standard sample into the fourth channel 124 for 200 seconds, measure its SPRi signal and terahertz signal, and calculate the terahertz absorption coefficient at a frequency of 1THz.

[0104] The second step involves adjusting the multi-channel fluid valve 140, introducing 10 nM immunoglobulin G (the analyte) into the fourth channel 124 for 200 seconds, measuring its SPRi and terahertz signals, and calculating the terahertz absorption coefficient at a frequency of 1 THz. Figure 6 It is known that immunoglobulin G binds to protein A within channel 4, causing an increase in SPRi signal. However, the timing of when immunoglobulin G ceases diffusion in the hydrogel and begins to react with protein A is unknown. Figure 7 The change in the terahertz absorption coefficient revealed that the diffusion time was 10 seconds.

[0105] The third step involves adjusting the multi-channel fluid valve 140, introducing a standard sample into the fourth channel 124 for 600 seconds, and measuring its SPRi signal and terahertz absorption coefficient. Figure 6 It is known that immunoglobulin G and protein A undergo a dissociation reaction, but the timing of when the standard sample finishes diffusion in the hydrogel and triggers the dissociation reaction between immunoglobulin G and protein A is unknown. Figure 7 The change in terahertz absorption coefficient revealed that the diffusion time was 10 seconds. The difference between the above diffusion time and the SPRi signal of the standard sample and the analyte was calculated from... Figure 6 After deduction, we get Figure 8 The results of SPRi measurements on the specific binding of the analyte are shown below after correction.

[0106] Example 2

[0107] In this embodiment, the SPRi system 200 uses a light-emitting diode with a center wavelength of 630nm as the light source 211, a stepper motor with an accuracy of 0.1mm as the power source of the first linear module 220, a visible light band charge-coupled device (CCD) as the first array detector 230, and the glass prism material is ZF3 glass.

[0108] The fabrication process and key parameter range of the microarray chip 110 are as follows: The glass substrate is ultrasonically cleaned for more than 30 minutes with a mixture of ethanol and diethyl ether at a volume ratio of 1:1 to 1:10 to clean its surface; it is then placed in an electron beam evaporation instrument and the vacuum is reduced to 10. -5Below millitor level; 1.5 nm chromium is deposited as an adhesion layer at a rate of less than 0.1 nm / s; 30 nm gold is deposited on the adhesion layer at a rate of greater than 0.01 nm / s as the upper metal surface of the WCSPR; 1% polycarbonate is spin-coated on the upper metal surface at a rate of 2000 rpm as the waveguide layer of the WCSPR; 30 nm gold is deposited on the waveguide layer at a rate of greater than 0.01 nm / s as the lower metal surface of the WCSPR structure-excited SPR; 15 nm cadmium telluride is deposited on the lower metal surface at a rate of 0.01 nm / s as the terahertz signal enhancement layer. After immersing in a solution of mercaptoalkanoic acid (with carbon chain length between 3 and 15) at a concentration of 0.1 mM or higher for 30 minutes to form a monomolecular self-assembled layer, it is immersed in a dextran solution with a molecular weight of 10000 Daltons for 1 hour to form a hydrogel surface. Protein A at a fixed concentration of 200 nM is coated on the corresponding regions of the third channel 123 and the fourth channel 124 on the hydrogel surface.

[0109] The four channels of the sample pool (i.e., the first channel 121, the second channel 122, the third channel 123 and the fourth channel 124) and the pipeline for conveying fluid are all made of polydimethylsiloxane, with each channel having dimensions of length × width × height (mm): 20 × 5 × 0.2.

[0110] Based on the multi-parameter measurement device based on terahertz imaging and surface plasmon resonance imaging provided in this embodiment, this embodiment provides a detection method that simultaneously detects the molecule introduction time, binding start and end times, and standard sample introduction time, which can realize time calibration of the real-time binding signal of the molecule and the fixed molecule measured by surface plasmon resonance imaging.

[0111] Before the experiment, it is necessary to measure the terahertz absorption coefficients of the SPRi signals of the standard sample, the analyte molecule, and the fixed molecule.

[0112] The first step involves moving the light source assembly 210 and the terahertz transmitter 310 to allow the p-polarized beam and terahertz signal to enter the first channel 121. The multi-channel fluid valve 140 is adjusted to introduce a standard sample into the first channel 121. The first array detector 230 and the second array detector 330 measure the SPRi signal and the terahertz signal respectively. The acquisition and control system 400 calculates the terahertz absorption coefficient at a frequency of 2THz.

[0113] In the second step, the light source assembly 210 and the terahertz emitter 310 are moved so that the p-polarized beam and the terahertz signal are injected into the second channel 122. The multi-channel fluid valve 140 is adjusted so that the immunoglobulin with a concentration of 100 nM of the analyte molecule is introduced into the second channel 122. The SPRi signal and the terahertz signal are measured by the first array detector 230 and the second array detector 330, respectively. The acquisition and control system 400 calculates the terahertz absorption coefficient at a frequency of 2 THz and calculates the SPRi signal difference between the standard sample and the analyte molecule.

[0114] The third step involves moving the light source assembly 210 and the terahertz transmitter 310 to allow the p-polarized beam and terahertz signal to enter the third channel 123. The multi-channel fluid valve 140 is adjusted to introduce a standard sample into the third channel 123. The first array detector 230 and the second array detector 330 measure the SPRi signal and the terahertz signal respectively. The acquisition and control system 400 calculates the terahertz absorption coefficient at a frequency of 2THz.

[0115] In the experiment, the light source assembly 210 and the terahertz transmitter 310 were moved so that the p-polarized beam and the terahertz signal were injected into the fourth channel 124.

[0116] First, adjust the multi-channel fluid valve 140, introduce a standard sample into the fourth channel 124 for 200 seconds, measure its SPRi signal and terahertz signal, and calculate the terahertz absorption coefficient at a frequency of 2THz.

[0117] The second step involves adjusting the multi-channel fluid valve 140 to introduce 100 nM immunoglobulin G (the analyte) into the fourth channel 124 for 200 seconds. The SPRi and terahertz signals are then measured, and the terahertz absorption coefficient at a frequency of 2 THz is calculated. Figure 9 It is known that immunoglobulin G binds to protein A in channel 124, causing an increase in SPRi signal. However, the timing of when immunoglobulin G ends its diffusion in the hydrogel and begins to react with protein A is unknown. Figure 10 The change in the terahertz absorption coefficient revealed that the diffusion time was 10 seconds.

[0118] The third step involves adjusting the multi-channel fluid valve 140, introducing a standard sample into the fourth channel 124 for 600 seconds, and measuring its SPRi signal and terahertz absorption coefficient. Figure 9 It is known that immunoglobulin G and protein A undergo a dissociation reaction, but the timing of when the standard sample finishes diffusion in the hydrogel and triggers the dissociation reaction between immunoglobulin G and protein A is unknown. Figure 10 The change in terahertz absorption coefficient revealed that the diffusion time was 10 seconds. The difference between the above diffusion time and the SPRi signal of the standard sample and the analyte was calculated from... Figure 9 After deduction, we get Figure 11The results of SPRi measurements on the specific binding of the analyte are shown below after correction.

[0119] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A measuring device, characterized in that The detection system comprises a microarray chip and a sample cell in contact with the microarray chip, the sample cell having a first channel, a second channel, a third channel and a fourth channel, and the microarray chip having immobilized molecules corresponding to the third channel and the fourth channel; The SPRi system comprises a light source assembly, a first linear module and a first array detector, the light source assembly being arranged on the first linear module and capable of changing position under the drive of the first linear module to emit a p-polarized light beam to the first channel, the second channel, the third channel or the fourth channel, and the first array detector being used to receive the reflected light beam of the metal-dielectric interface of the microarray chip; The terahertz imaging system comprises a terahertz emitter, a second linear module and a second array detector, the terahertz emitter being arranged on the second linear module and capable of changing position under the drive of the second linear module to emit a terahertz signal to the first channel, the second channel, the third channel or the fourth channel, and the second array detector being used to receive the terahertz signal reflected on the fluid side of the surface of the microarray chip; The acquisition control system is used to control the actions of the light source assembly, the first linear module, the terahertz emitter and the second linear module, and to acquire and calculate the data of the first array detector and the second array detector. The detection system further comprises a multi-way fluid valve, a standard sample injection pump, a sample injection pump and a controller, the first channel, the second channel, the third channel and the fourth channel being respectively communicated with five outlets of the multi-way fluid valve, the standard sample injection pump and the sample injection pump being respectively communicated with two inlets of the multi-way fluid valve, and the controller being electrically connected to the acquisition control system, and the controller being used to control the working of the multi-way fluid valve, the standard sample injection pump and the sample injection pump. The light source assembly comprises a visible light source, a collimating lens and a polarizer arranged in sequence along the light path.

2. The measuring device of claim 1, wherein, The power sources of the first linear module and the second linear module are both step motors.

3. The measuring device of claim 1, wherein, The precision of the step motor is 0.1mm.

4. The measuring device of claim 1, wherein, The material of the sample cell is polydimethylsiloxane.

5. The measuring device of claim 4, wherein, The preparation method of the microarray chip is as follows:

6. The measuring device of claim 1, wherein, cleaning the surface of the glass substrate by ultrasonic cleaning with a pre-set volume ratio of ethanol-ether mixed solution for a first pre-set time; 7. The measuring device according to any of claims 1-6, characterized in that putting the cleaned glass substrate into an electron beam evaporation instrument and vacuumizing to reduce the air pressure value to a pre-set air pressure value; evaporating chromium as an adhesion layer at a first pre-set rate and a first pre-set thickness; evaporating gold or silver as an upper metal surface on the adhesion layer at a second pre-set rate and a second pre-set thickness; rotary coating polycarbonate as a waveguide layer on the upper metal surface at a third pre-set rate and a pre-set mass fraction; evaporating gold or silver as a lower metal surface on the waveguide layer at a fourth pre-set rate and a fourth pre-set thickness, and evaporating cadmium telluride as a terahertz signal enhancement layer on the metal surface at a fifth pre-set rate and a fifth pre-set thickness; ​ ​ immersing the glass substrate after evaporation into a first pre-set concentration of mercapto alkanoic acid solution for a second pre-set time period to form a monomolecular self-assembled layer; immersing the glass substrate into a third pre-set time period of a high polymer solution to form a hydrogel surface; coating the immobilized molecules on the hydrogel surface in the regions corresponding to the third and fourth channels.

8. The measuring device of claim 7, wherein, the pre-set volume ratio ranges from 1:1 to 1:10, the first pre-set time period is greater than 30 min; The preset air pressure value is less than 10 -5 mTorr; the first pre-set rate is less than 0.1 nm / s, and the first pre-set thickness ranges from 0.5 nm to 2.5 nm; the second pre-set rate is greater than 0.01 nm / s, and the second pre-set thickness ranges from 25 nm to 35 nm; the third pre-set rate is 2000 r / s, and the pre-set mass fraction is 1%; the fourth pre-set rate is greater than 0.01 nm / s, and the fourth pre-set thickness ranges from 25 nm to 35 nm; the fifth pre-set rate is 0.01 nm / s, and the fifth pre-set thickness ranges from 10 nm to 20 nm; the first pre-set concentration is greater than 0.1 mM, and the second pre-set time period is greater than 30 min; the third pre-set time period is greater than 30 min.

9. A method of detection, characterized in that The measuring device of claim 1, wherein the detection method comprises the following steps: S1: Before the experiment, measuring the terahertz absorption coefficient of the SPRi signals of the standard sample, the molecule to be measured, and the immobilized molecules; S11: Moving the light source assembly and the terahertz emitter to make the p-polarized light beam and the terahertz signal enter the first channel, introducing the standard sample into the first channel, measuring the SPRi signal and the terahertz signal by the first array detector and the second array detector respectively, and calculating the terahertz absorption coefficient at the pre-set frequency by the acquisition control system; S12: Moving the light source assembly and the terahertz emitter to make the p-polarized light beam and the terahertz signal enter the second channel, introducing the immunoglobulin of the molecule to be measured into the second channel at a pre-set concentration, measuring the SPRi signal and the terahertz signal by the first array detector and the second array detector respectively, calculating the terahertz absorption coefficient at the pre-set frequency by the acquisition control system, and calculating the SPRi signal difference between the standard sample and the molecule to be measured; S13: Moving the light source assembly and the terahertz emitter to make the p-polarized light beam and the terahertz signal enter the third channel, introducing the standard sample into the third channel, measuring the SPRi signal and the terahertz signal by the first array detector and the second array detector respectively, and calculating the terahertz absorption coefficient at the pre-set frequency by the acquisition control system; S2: During the experiment, moving the light source assembly and the terahertz emitter to make the p-polarized light beam and the terahertz signal enter the fourth channel; S21: Introducing the standard sample into the fourth channel for a fourth pre-set time period, measuring the SPRi signal and the terahertz signal, and calculating the terahertz absorption coefficient at the pre-set frequency; S22: introducing the immunoglobulin G with a preset concentration of the to-be-tested molecule into the fourth channel for a certain time length, measuring the SPRi signal and the terahertz signal, and calculating the terahertz absorption coefficient at the preset frequency, determining the diffusion time of the immunoglobulin G in the hydrogel of the microarray chip from the terahertz absorption measurement result graph at the preset frequency when specific binding occurs due to the introduction of the to-be-tested molecule; S23: introducing the standard sample into the fourth channel for a fifth preset time length, measuring the SPRi signal and the terahertz absorption coefficient, and deducting the diffusion time in S22 and the SPRi signal difference between the standard sample and the to-be-tested molecule in S12 from the SPRi signal result graph when specific binding occurs due to the introduction of the to-be-tested molecule into the fourth channel, to obtain the corrected SPRi measurement result of specific binding of the to-be-tested molecule.

10. The detection method according to claim 9, characterized in that, The preset frequency is 1 THz or 2 THz; The preset concentration is 10 nM or 100 nM; The fourth preset time length is 200 s; The certain time length is 200 s; The fifth preset time length is 600 s.

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