Surface plasmon resonance imaging device and refractive index detection method thereof

Abstract

The application belongs to the field of biochemical substance detection, and particularly relates to a surface plasmon resonance imaging device and a refractive index detection method thereof. The surface plasmon resonance imaging device adopts a wavelength scanning device to realize switching of incident light wavelength, a voltage-adjusted transmittance device is used to compensate for transmittance deviation of a polarizer under different incident light wavelengths, an acquisition control system controls an applied voltage of the voltage-adjusted transmittance device and a wavelength of an output light beam of the wavelength scanning device, and simultaneously acquires data of an array detector under different wavelengths to perform differential operation and differential operation of average reflection light intensity of a sample point region and a sample point background region. The surface plasmon resonance imaging device and the refractive index detection method thereof reduce operation difficulty in the detection process, reduce operation time and equipment development cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical substance detection, and particularly relates to a surface plasmon resonance imaging device and a refractive index detection method thereof. BACKGROUND

[0002] Surface plasmon resonance imaging (SPRi) is a high-throughput, label-free technology that can measure the refractive index change of the metal surface of a microarray chip in real time. When a p-polarized light beam of a certain wavelength and incident angle is coupled through a certain medium coupler on the metal surface of a microarray chip-medium coupler interface, the incident light couples into the surface plasmon wave (SPW) existing on the interface in the form of evanescent wave, which decays exponentially along the vertical direction of the interface. The coupling ratio is maximum when the corresponding incident angle is the resonance angle. In the SPRi technology, when a p-polarized parallel light beam enters the coupler at an incident angle close to the resonance angle, the change in the intensity of the reflected light beam of the metal-medium interface can be measured in real time by an array detector to monitor the refractive index change at different positions of the interface. In the above-mentioned SPRi measurement, a microarray is prepared on the metal surface of the microarray chip, and by measuring the average reflected light intensity of the region corresponding to each sample point in the array, high-throughput real-time detection of the interaction between the sample to be measured and each sample point can be achieved.

[0003] When the sample to be measured interacts with each sample point, the refractive index change of the sample point region is composed of two parts, i.e., the bulk refractive index change of the sample to be measured and the refractive index change of the binding substance produced by the interaction. The former is the same as the sample background region of the sample point and is caused by the change of the sample to be measured, so in order to measure the refractive index change of the binding substance, the refractive index changes of the sample point and the sample background region need to be calculated, and the bulk refractive index change of the sample to be measured in the sample background region needs to be deducted from the refractive index change of the sample point region.

[0004] To achieve this detection goal, the existing SPRi device usually needs to convert the reflected light intensity into refractive index by a calibration step with the aid of a standard sample. The calibration conversion method based on the standard sample is to take the initial medium refractive index as the reference, to calculate the change of the reflected light intensity corresponding to the deviation by replacing the standard sample to produce a fixed refractive index deviation, to calculate the intensity-refractive index detection sensitivity of each pixel or each region of the array detector or the microarray, and to make the detector unit output signal be the result of the sample point refractive index measurement by adjusting the bias voltage and the sensitivity coefficient of the array detector or to convert the measured reflected light intensity value into the real refractive index change by dividing the measured reflected light intensity value by the detection sensitivity. After the calibration, the deduction operation of the refractive index change of the sample point background area of the sample to be measured is performed. The above calibration and deduction steps increase the operation time and the cost of device development, and the operations such as adjusting the bias voltage and the sensitivity coefficient are complex, which greatly limits the real-time performance and the ease of use of the SPRi device detection. SUMMARY

[0005] The embodiment of the present application provides a surface plasmon resonance imaging device and a refractive index detection method thereof, which aims to reduce the operation difficulty of the detection process and reduce the operation time and the cost of device development.

[0006] To this end, according to one aspect of the present application, a surface plasmon resonance imaging device is provided, comprising:

[0007] A detection system comprising a medium coupler, a microarray chip, and a sample cell in contact with the microarray chip, wherein the incident light is coupled to the metal surface of the microarray chip via the medium coupler;

[0008] An optical system comprising a light source, a beam shaper, a wavelength scanning device, a voltage-adjusted transmittance device, a polarizer, and an array detector, wherein the light source, the beam shaper, the wavelength scanning device, the voltage-adjusted transmittance device, and the polarizer are sequentially arranged along the optical path of the incident light, the wavelength scanning device is used for switching the wavelength of the incident light, the voltage-adjusted transmittance device is used for compensating the transmittance deviation of the polarizer under different incident light wavelengths, and the array detector is used for receiving the reflected light beam of the metal-medium interface of the microarray chip and measuring the intensity spatial distribution thereof; and

[0009] A collection control system, which collects the data of the array detector before the experiment, records the spatial positions of each sample point region on the microarray chip, controls the wavelength of the output light beam of the wavelength scanning device and the applied voltage of the voltage-adjusted transmittance device during the experiment, simultaneously collects the data of the array detector under different wavelengths and performs differential operation, and performs differential operation on the average reflected light intensity of the sample point region and the sample point background region.

[0010] Optionally, the wavelength scanning device comprises an acousto-optic tunable filter.

[0011] Optionally, the wavelength scanning device comprises a motor and a filter in transmission connection with the motor, the motor being configured to adjust the position of the filter so that the light beam emitted from the light beam shaper is projected onto different filtering areas of the filter.

[0012] Optionally, the voltage-adjusted transmittance device changes the wavelength transmittance by adjusting the voltage applied on the transmittance-adjustable medium to compensate for the transmittance deviation of the polarizer under different incident light wavelengths.

[0013] Optionally, the transmittance-adjustable medium comprises a nanowire assembly.

[0014] Optionally, the light source comprises a white light emitting diode or a red light emitting diode; and the array detector comprises a photodiode, a phototriode or a photomultiplier tube.

[0015] Optionally, the method for preparing the microarray chip comprises:

[0016] cleaning the surface of the glass substrate by performing ultrasonic cleaning on the glass substrate with a first pre-set volume ratio of ethanol-ether mixture for a first pre-set time length;

[0017] placing the glass substrate into an electron beam evaporation instrument and performing vacuum pumping to reduce the air pressure value in the electron beam evaporation instrument to a pre-set air pressure value;

[0018] evaporating a first pre-set thickness of chromium as an adhesion layer and a second pre-set thickness of zinc sulfide as an adhesion enhancement layer on the surface of the glass substrate at a first pre-set rate;

[0019] evaporating a third pre-set thickness of gold or silver as a metal surface for exciting surface plasmon resonance on the surface of the glass substrate at a second pre-set rate;

[0020] immersing the glass substrate in a first pre-set concentration of mercapto acid solution for a second pre-set time length to form a monomolecular self-assembled layer;

[0021] forming a pre-set biomolecular microarray on the surface of the glass substrate by using a biomolecular spotting instrument to obtain a microarray chip, each spot of the microarray chip having a pre-set diameter and a pre-set shape.

[0022] Optionally, the pre-set volume ratio is in a range of 1:1-1:10.

[0023] The first pre-set time length is greater than or equal to 30 min.

[0024] The pre-set air pressure value is less than or equal to 10 -5 mTorr.

[0025] The first pre-set rate is less than or equal to 0.1 nm / s.

[0026] The first preset thickness is in a range of 0.5 nm to 2.5 nm;

[0027] The second preset thickness is in a range of 0.5 nm to 3 nm;

[0028] The second preset rate is greater than or equal to 0.01 nm / s;

[0029] The third preset thickness is in a range of 40 nm to 70 nm;

[0030] The first preset concentration is greater than or equal to 0.1 mM;

[0031] The carbon chain length of the thiol acid in the thiol acid solution is in a range of 3 to 15;

[0032] The second preset time length is greater than or equal to 30 min;

[0033] The preset diameter is greater than or equal to 10 um;

[0034] The preset shape can be a circle or a circle-like shape.

[0035] According to another aspect of the present application, a refractive index detection method is provided, based on the surface plasmon resonance imaging device described above, and the refractive index detection method comprises the following steps:

[0036] Under the condition that the sample cell is a standard sample, by taking a picture of the surface microarray of the microarray chip, marking each sample point area and the sample point background area;

[0037] After the sample cell is connected to the sample to be detected, by adjusting the wavelength scanning device and the voltage adjustment transmittance device, the wavelength of the incident light is cyclically changed under the condition that the intensity of the incident light is kept unchanged;

[0038] The data difference of the array detector corresponding to different wavelengths in the sample point background area is calculated in each cycle, the refractive index change between the sample to be detected and the standard sample is obtained, and the data difference of the array detector corresponding to different wavelengths in the sample point area is subtracted from the corresponding difference of the sample point background area to obtain the refractive index change between the binder and the standard sample.

[0039] Optionally, before the experiment, the sample pool is kept as a standard sample. The array detector collects the reflected light intensity on the surface of the microarray chip under the control of the collection control system. The spatial position of each sample point area is obtained by distinguishing different intensity values. The wavelength scanning device is adjusted to change the wavelength to λ1 and λ2, respectively. The average reflected light intensity of the sample point area collected by the array detector at the wavelengths λ1 and λ2 is recorded. The applied voltage of the transmittance device is adjusted to make the average reflected light intensity of the sample point area collected by the array detector at the wavelengths λ1 and λ2 consistent. The refractive index of the standard sample is denoted as n1. The reflected light intensity of the sample point area at the wavelength λ1 is denoted as I1(λ1, n1), and the reflected light intensity of the sample point background area is denoted as I2(λ1, n1). The reflected light intensity of the sample point area at the wavelength λ2 is denoted as I1(λ2, n1), and the reflected light intensity of the sample point background area is denoted as I2(λ2, n1). The incident light intensity-body refractive index sensitivity of the sample point area and the sample point background area at the wavelength λ1 is denoted as C2, and the incident light intensity-body refractive index of the sample point area and the sample point background area at the wavelength λ2 is denoted as C’2.

[0040] For the sample point background area, there is an expression shown in formula (1) at λ1 and λ2, where Δn represents the refractive index difference between n1 and n2:

[0041]

[0042] The relationship between C’2 and C2 can be linearly approximated by the expression shown in formula (2), where Δλ represents the wavelength difference between λ1 and λ2:

[0043]

[0044] Substitute formula (2) into formula (1), subtract the lower formula from the upper formula in formula (1), and the calculation result is shown in formula (3):

[0045]

[0046] In the experiment, the first time length of the standard sample is introduced into the sample cell, and then the second time length of the immunoglobulin G with a certain concentration is introduced, and the refractive index is denoted as n2, and finally the third time length of the standard sample is introduced. The wavelength and the applied voltage of the wavelength scanning device are controlled by the acquisition control system, and the reflected light intensity on the surface of the microarray chip is collected by the array detector. The acquisition control system controls the wavelength scanning device to realize the switching of the wavelengths λ1 and λ2, wherein the reflected light intensity of the sample point region at the wavelength λ1 is denoted as I1(λ1, n2), and the reflected light intensity of the sample point background region is denoted as I2(λ1, n2); the reflected light intensity of the sample point region at the wavelength λ2 is denoted as I1(λ2, n2), and the reflected light intensity of the sample point background region is denoted as I2(λ2, n2); the incident light intensity-body refractive index of the sample point region and the sample point background region at the wavelength λ1 remains unchanged, the incident light intensity-binder refractive index of the sample point region is set as C1, the incident light intensity-body refractive index corresponding to the wavelength λ2 remains unchanged, and the incident light intensity-binder refractive index of the sample point region is set as C’1;

[0047] The sample point region has an expression shown in formula (4) when the wavelengths are λ1 and λ2, wherein Δn represents the refractive index difference between n1 and n2, which can be calculated by formula (3), and Δb represents the refractive index change caused by the binder:

[0048]

[0049] According to formula (3) and (4), the expression of Δb is as formula (5):

[0050]

[0051] The surface plasmon resonance imaging device and the refractive index detection method provided by the application have the advantages that, compared with the prior art, the surface plasmon resonance imaging device of the application uses a wavelength scanning device to switch the wavelength of incident light, a beam shaper and a polarizer are used to generate a p-polarized parallel light beam, a voltage-adjusted transmittance device is used to compensate for the transmittance deviation of the polarizer under different incident light wavelengths, the acquisition control system controls the applied voltage of the voltage-adjusted transmittance device and the wavelength of the light beam output by the wavelength scanning device, and simultaneously acquires the data of the array detector under different wavelengths and performs differential operation on the average reflected light intensity of the sample point region and the sample point background region. In the detection method based on the device, the sample point region and the sample point background region are marked by taking a picture of the microarray on the surface of the microarray chip under standard sample conditions, and after the sample to be detected is introduced, the wavelength scanning device and the voltage-adjusted transmittance device are adjusted to cyclically change the wavelength of the incident light beam under the condition that the intensity of the incident light is kept unchanged, the difference between the array detector data corresponding to different wavelengths of the sample point background region is calculated in each cycle to obtain the refractive index change between the sample body of the sample to be detected and the standard sample, and the difference between the array detector data corresponding to different wavelengths of the sample point region and the corresponding difference of the sample point background region is subtracted to obtain the refractive index change between the conjugate and the standard sample.

[0052] The surface plasmon resonance imaging device and the refractive index detection method provided by the application have the advantages that, compared with the prior art, the surface plasmon resonance imaging device of the application uses a wavelength scanning device to switch the wavelength of incident light, a beam shaper and a polarizer are used to generate a p-polarized parallel light beam, a voltage-adjusted transmittance device is used to compensate for the transmittance deviation of the polarizer under different incident light wavelengths, the acquisition control system controls the applied voltage of the voltage-adjusted transmittance device and the wavelength of the light beam output by the wavelength scanning device, and simultaneously acquires the data of the array detector under different wavelengths and performs differential operation on the average reflected light intensity of the sample point region and the sample point background region. In the detection method based on the device, the sample point region and the sample point background region are marked by taking a picture of the microarray on the surface of the microarray chip under standard sample conditions, and after the sample to be detected is introduced, the wavelength scanning device and the voltage-adjusted transmittance device are adjusted to cyclically change the wavelength of the incident light beam under the condition that the intensity of the incident light is kept unchanged, the difference between the array detector data corresponding to different wavelengths of the sample point background region is calculated in each cycle to obtain the refractive index change between the sample body of the sample to be detected and the standard sample, and the difference between the array detector data corresponding to different wavelengths of the sample point region and the corresponding difference of the sample point background region is subtracted to obtain the refractive index change between the conjugate and the standard sample. BRIEF DESCRIPTION OF DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0054] Among them:

[0055] Figure 1 is a structural schematic diagram of the surface plasmon resonance imaging device shown in the application;

[0056] Figure 2 is a schematic diagram of the spatial position of each sample point region of a surface microarray of a microarray chip before the experiment shown in the application;

[0057] Figure 3is a reflection light intensity distribution graph of the microarray chip at different wavelengths when the incident angle is 53.29° in Embodiment I of the present application;

[0058] Figure 4 is a reflection light intensity-refractive index detection sensitivity graph of the microarray chip at different wavelengths when the incident angle is 53.29° in Embodiment I of the present application;

[0059] Figure 5 is a sample body refractive index measurement result graph of a sample point background area of a sample to be detected obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment I of the present application;

[0060] Figure 6a is a band body refractive index measurement affinity curve graph obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment I of the present application;

[0061] Figure 6b is a body refractive index change graph obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment I of the present application;

[0062] Figure 6c is a conjugate refractive index change graph obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment I of the present application;

[0063] Figure 7 is a reflection light intensity distribution graph of the microarray chip at different wavelengths when the incident angle is 55.25° in Embodiment II of the present application;

[0064] Figure 8 is a reflection light intensity-refractive index detection sensitivity graph of the microarray chip at different wavelengths when the incident angle is 55.25° in Embodiment II of the present application;

[0065] Figure 9 is a sample body refractive index measurement result graph of a sample point background area of a sample to be detected obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment II of the present application;

[0066] Figure 10a is a band body refractive index measurement affinity curve graph obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment II of the present application;

[0067] Figure 10b is a body refractive index change graph obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment II of the present application;

[0068] Figure 10c is a conjugate refractive index change graph obtained by a detection method based on the surface plasmon resonance imaging device in Embodiment II of the present application.

[0069] Explanation of main element symbols:

[0070] 10. Detection system;

[0071] 11. Medium coupler;

[0072] 12. Microarray chip;

[0073] 13. Sample cell;

[0074] 20. Optical system;

[0075] 21. Light source;

[0076] 22. Beam shaper;

[0077] 23. Wavelength scanning device;

[0078] 24. Voltage-adjusted transmittance device;

[0079] 25. Polarizer;

[0080] 26. Array detector;

[0081] 30. Acquisition control system. DETAILED DESCRIPTION

[0082] For the purpose of promoting an understanding of the present application, the present application will now be described in greater detail with reference to the relevant drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0083] It should be noted that when an element is referred to as being "fixed" or "set" on another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or indirectly connected to the other element.

[0084] It should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, which indicate the orientation or positional relationship based on the orientation or position relationship shown in the drawings, are only for the purpose of facilitating the description of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0085] In addition, the terms "first", "second", etc. are used only for descriptive purposes and do not connote or imply relative importance. Thus, a feature described as "first" or "second" can implicitly or explicitly include one or more of the features.

[0086] 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 in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0087] As described in the background, the existing SPRi device usually needs to convert the reflected light intensity into the refractive index by a calibration step with a standard sample, and then subtract the refractive index change of the sample from the background area of the sample point after calibration. The above calibration and subtraction steps increase the operation time and the cost of device development, and the operations of adjusting the bias voltage and the sensitivity coefficient are complex, which greatly limits the real-time performance and ease of use of the SPRi device.

[0088] To solve the above problems, according to one aspect of the present application, as shown in Figure 1 An embodiment of the present application provides a surface plasmon resonance imaging device, which comprises a detection system 10, an optical system 20 and a collection control system 30.

[0089] The detection system 10 comprises a medium coupler 11, a microarray chip 12 and a sample cell 13 in contact with the microarray chip 12, and the incident light is coupled to the metal surface of the microarray chip 12 via the medium coupler 11.

[0090] The optical system 20 comprises a light source 21, a beam shaper 22, a wavelength scanning device 23, a voltage-adjusted transmittance device 24, a polarizer 25 and an array detector 26, the light source 21, the beam shaper 22, the wavelength scanning device 23, the voltage-adjusted transmittance device 24 and the polarizer 25 are sequentially arranged on the light path of the incident light, the wavelength scanning device 23 is used for switching the wavelength of the incident light, the voltage-adjusted transmittance device 24 is used for compensating the transmittance deviation of the polarizer 25 under different incident light wavelengths, and the array detector 26 is used for receiving the reflected light beam of the metal-medium interface of the microarray chip 12 and measuring the intensity spatial distribution thereof.

[0091] The acquisition control system 30 acquires data of the array detector 26 before the experiment, records the spatial positions of each sample point area on the microarray chip 12, controls the wavelength scanning device 23 to output the wavelength of the light beam and the voltage adjustment transmittance device 24 to output the applied voltage during the experiment, and simultaneously acquires data of the array detector 26 under different wavelengths and performs differential operation and differential operation of the average reflection intensity of the sample point area and the sample point background area.

[0092] In the embodiment of the present application, the surface plasmon resonance imaging device adopts the wavelength scanning device 23 to switch the wavelength of the incident light, the light beam shaper 22 and the polarizer 25 are used to generate a p-polarized parallel light beam, the voltage adjustment transmittance device 24 is used to compensate for the transmittance deviation of the polarizer 25 under different incident light wavelengths, and the acquisition control system 30 controls the applied voltage of the voltage adjustment transmittance device 24 and the wavelength of the light beam output by the wavelength scanning device 23, simultaneously acquires data of the array detector 26 under different wavelengths and performs differential operation and differential operation of the average reflection intensity of the sample point area and the sample point background area. Based on the surface plasmon resonance imaging device adopting the wavelength scanning device 23, during the detection process, the sample point area and the sample point background area are marked by taking a picture of the microarray on the surface of the microarray chip 12 under standard sample conditions, and after the sample to be detected is introduced, the wavelength scanning device 23 and the voltage adjustment transmittance device 24 are adjusted to cyclically change the wavelength of the incident light beam under the condition that the intensity of the incident light is kept unchanged. The difference between the refractive index changes of the sample body and the standard sample is calculated by calculating the difference between the data of the array detector 26 corresponding to different wavelengths of the sample point background area in each cycle. The difference between the refractive index changes of the binding substance and the standard sample is obtained by subtracting the difference between the data of the array detector 26 corresponding to different wavelengths of the sample point area from the difference between the data of the array detector 26 corresponding to different wavelengths of the sample point background area. As can be seen from the above, the surface plasmon resonance imaging device proposed by the present application can directly obtain the sample body refractive index change of the sample point background area and the binding substance refractive index change of the sample point area at the same time without adjusting the bias voltage and sensitivity coefficient of the array detector 26, and without the calibration conversion method based on the standard sample. The operation difficulty of the detection process is reduced, the calculation time is reduced, and the equipment development cost is reduced.

[0093] In application, the positions between the optical system 20 and the detection system 10 are relatively fixed, so that the incident light can be incident to the microarray chip 12, and the incident angle can be any acute angle.

[0094] The medium coupler 11 can be realized by a glass prism or a waveguide element or a grating, etc. The glass prism can be prepared by using optical glass with high light transmittance according to actual needs, for example, ZF3 glass, BK7 glass, etc. Figure 1 The medium coupler 11 is an exemplary triangular glass prism, and the microarray chip 12 is arranged on one surface of the medium coupler 11.

[0095] The light beam shaper 22 can be one of an expander lens group and a collimator lens. The array detector 26 can be implemented by a photoelectric conversion device such as a photodiode, a phototriode, a photomultiplier, a charge-coupled device (CCD) sensor, or a complementary metal oxide semiconductor (CMOS) sensor. The size of each pixel in the array detector 26 can be selected according to actual needs.

[0096] The acquisition control system 30 can be a central processing unit (CPU), and can also be another general-purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or another programmable logic device, a discrete gate or transistor logic device, a discrete hardware component, or the like. The general-purpose processor can be a microprocessor or any conventional processor, such as a control card with a chip model of STM32F407 or STM32F103. The control system can be in communication connection with other components through any wired communication mode.

[0097] In an embodiment, the wavelength scanning device 23 includes an acousto-optic tunable filter. Alternatively, the wavelength scanning device 23 includes a motor and a filter plate in transmission connection with the motor, and the motor is used to adjust the position of the filter plate so that the light beam emitted from the light beam shaper 22 is projected onto different filter regions of the filter plate.

[0098] In an embodiment, the preparation method of the microarray chip 12 is as follows:

[0099] The glass substrate is cleaned by ultrasonic cleaning with a first preset volume ratio of ethanol-ether mixture for a first preset time period.

[0100] The glass substrate is placed in an electron beam evaporation instrument and vacuumized so that the air pressure value in the electron beam evaporation instrument is reduced to a preset air pressure value.

[0101] A first preset thickness of chromium is evaporated on the surface of the glass substrate as an adhesion layer, and a second preset thickness of zinc sulfide is evaporated as an adhesion enhancement layer, at a first preset rate.

[0102] evaporating gold or silver with a third preset thickness on the surface of the glass substrate at a second preset rate as a metal surface for exciting surface plasmon resonance;

[0103] immersing the glass substrate in a solution of a mercapto acid with a first preset concentration for a second preset time duration to form a monomolecular self-assembled layer;

[0104] spotting a preset biomolecule microarray on the surface of the glass substrate by using a biomolecule spotter to obtain a microarray chip 12, each spot of the microarray chip 12 having a preset diameter and a preset shape.

[0105] In applications, each preset parameter in the preparation method of the microarray chip 12 can be set according to actual needs, for example:

[0106] The preset volume ratio has a value range of 1:1-1:10, and can be specifically 1:2, 1:4 or 1:10;

[0107] The first preset time duration is greater than or equal to 30 min (minutes), and can be specifically 30 min, 2 h (hours) or 4 h;

[0108] The preset air pressure value is less than or equal to 10 -5 mTorr (millitorr), and can be specifically 10 -7 mTorr, 10 -6 mTorr or 10 - 5 mTorr;

[0109] The first preset rate is less than or equal to 0.1 nm / s (nanometers per second), and can be specifically 0.01 nm / s or 0.03 nm / s;

[0110] The first preset thickness has a value range of 0.5 nm (nanometers)-2.5 nm, and can be specifically 1.5 nm or 2.5 nm;

[0111] The second preset thickness has a value range of 0.5 nm-3 nm, and can be specifically 1 nm, 2 nm or 3 nm;

[0112] The second preset rate is greater than or equal to 0.01 nm / s, and can be specifically 0.01 nm / s, 0.03 nm / s or 0.08 nm / s;

[0113] The third preset thickness has a value range of 40 nm-70 nm, and can be specifically 45 nm or 50 nm;

[0114] The first preset concentration is greater than or equal to 0.1 mM (mmol / L, millimoles per liter), and can be specifically 1 mM or 1.5 mM;

[0115] The carbon chain length of the mercapto acid in the mercapto acid solution ranges from 3 to 15, and can be 8, 11 or 13;

[0116] The second preset time length is greater than or equal to 30 minutes, and can be 24 hours.

[0117] The preset biomolecule can be a protein, a nucleic acid fragment, a polypeptide molecule, a tissue slice, a cell, etc., and can be bovine serum albumin.

[0118] The preset diameter is greater than or equal to 10 microns, and can be 50 microns or 100 microns.

[0119] The preset shape can be circular or quasi-circular, for example, elliptical.

[0120] In application, the sample pool is arranged on the side where the metal surface of the microarray chip 12 is located, so that when the sample is introduced into the sample pool, the incident light is internally reflected at the interface between the sample and the metal surface, and at the same time, the evanescent wave is excited at the interface and the transfer of light energy is realized, so that the intensity of the reflected light obtained by reflection is sharply attenuated relative to the incident light. The standard sample can be deionized water.

[0121] Embodiment I

[0122] Reference Figure 1 The embodiment provides a surface plasmon resonance imaging device, which mainly comprises a detection system 10, an optical system 20 and an acquisition control system 30. In addition, the device can also include an unnecessary temperature control system (not shown in the figure). The light source 21 of the optical system 20 adopts a white light emitting diode, a wavelength 930nm-980nm continuous adjustable acousto-optic modulator is used as a wavelength scanning device 23, and a vanadium oxide / tungsten oxide composite is used as a material of a transmittance adjustable medium of a voltage adjustable transmittance device 24.

[0123] The preparation process and key parameter range of the microarray chip 12 are as follows: the glass substrate is ultrasonically cleaned in an ethanol-ethyl ether mixed solution with a volume ratio of 1:1-1:10 for more than 30 minutes to clean the surface, and then is placed in an electron beam evaporation instrument to be vacuumized to reduce the air pressure value to 10 -5Below millitor level; 0.5nm-2.5nm chromium as an adhesion layer and 0.5nm-2.5nm zinc sulfide as an adhesion enhancement layer are deposited at a rate less than 0.1nm / s; 50nm gold or silver is deposited at a rate greater than 0.01nm / s as the metal surface for excitation SPR; after immersion in a solution of mercaptoalkanoic acid (with carbon chain length between 3-15) at a concentration of 0.1mM or higher for more than 30 minutes to form a monomolecular self-assembled layer, a 10nM protein A microarray is formed using a biomolecular spotting instrument, with each spot being a circle with a diameter of 10μm or more. A fluid cell is in contact with the surface of the microarray chip 12 to hold phosphate buffer standard samples and test samples using phosphate buffer as a solvent. Under standard conditions, the intensity distribution of reflected light at different wavelengths of the microarray chip 12 is measured by a spectrometer as shown below. Figure 3 As shown. After changing the refractive index of the sample in the fluid cell, the intensity of reflected light at different wavelengths was remeasured using a spectrometer. The sensitivity of the reflected light intensity versus sample refractive index at different wavelengths was calculated as follows. Figure 4 As shown.

[0124] Based on the surface plasmon resonance imaging device provided in this embodiment, this embodiment also provides a method for simultaneously measuring the volume refractive index of the sample background region and the refractive index of the sample aggregate without calibration. This method includes three steps: recording the spatial position of the microarray sample points before the experiment, scanning the incident beam wavelength and compensating for the incident beam transmittance during the experiment, calculating the difference between the sample region data and the sample background region data at different wavelengths, and calculating the difference between the two difference calculation results.

[0125] Specifically, before the experiment, the sample cell 13 was kept unchanged with standard samples. The acquisition and control system 30 used a microcontroller control card to control the CCD to collect the intensity of reflected light from the surface of the microarray chip 12. By distinguishing different intensity values, the spatial position of each sample point area in the microarray was obtained, such as... Figure 2 As shown. By adjusting the frequency of the external signal of the acousto-optic modulator, the center wavelength of the filter was changed to 945 nm and 955 nm, respectively. The average reflected light intensity of the CCD sample collection area was recorded at both wavelengths. The applied voltage of the transmittance device 24 was adjusted to make the average reflected light intensity of the CCD sample collection area consistent at both wavelengths. The refractive index of the standard sample is denoted as n1, the wavelength of 945 nm is denoted as λ1, and the wavelength of 955 nm is denoted as λ2. At a wavelength of 945 nm, the reflected light intensity of the sample area is denoted as I1(λ1, n1), and the reflected light intensity of the sample background area is denoted as I2(λ1, n1); at a wavelength of 955 nm, the reflected light intensity of the sample area is denoted as I1(λ2, n1), and the reflected light intensity of the sample background area is denoted as I2(λ2, n1), as shown. Figure 4As shown, the incident light intensity- bulk refractive index sensitivity of the sample point region and the sample point background region at wavelength 945 nm is set as C2, and the incident light intensity- bulk refractive index of the sample point region and the sample point background region at wavelength 955 nm is set as C'2.

[0126] For the sample point background region, there is an expression shown in formula (1) at wavelengths 945 nm and 955 nm, wherein Δn represents the refractive index difference between n1 and n2:

[0127]

[0128] According to Figure 4 As shown, the relationship between C'2 and C2 can be linearly approximated by the expression shown in formula (2), wherein Δλ represents the wavelength difference between λ1 and λ2:

[0129]

[0130] Substituting formula (2) into formula (1), subtracting the lower expression from the upper expression in formula (1) gives the calculation result shown in formula (3), and the refractive index change measurement result of the sample to be measured with a known refractive index is shown in formula (4): Figure 5 As shown:

[0131]

[0132] In the experiment, the standard sample was first introduced into the sample cell 13 for 500 seconds, then the 500 nM concentration immunoglobulin G was introduced for 500 seconds, the refractive index was recorded as n2, and finally the standard sample was introduced for 1000 seconds. The single-chip microcomputer control card synchronously controls the filter center wavelength of the acousto-optic modulator, the voltage of the voltage-adjusted transmittance controllable device, and the CCD to collect the reflected light intensity on the surface of the microarray chip 12 at a frequency signal of 32 Hz. The filter center wavelength of the acousto-optic modulator is changed to switch the wavelength of the light beam incident on the microarray chip 12 between 945 nm and 955 nm. The reflected light intensity of the sample point region at wavelength 945 nm is recorded as I1(λ1, n2), and the reflected light intensity of the sample point background region at wavelength 945 nm is recorded as I2(λ1, n2). The reflected light intensity of the sample point region at wavelength 955 nm is recorded as I1(λ2, n2), and the reflected light intensity of the sample point background region at wavelength 955 nm is recorded as I2(λ2, n2). The incident light intensity- bulk refractive index of the sample point region and the sample point background region at wavelength 945 nm remains unchanged at C2, the incident light intensity- analyte refractive index of the sample point region is set as C1, the incident light intensity- bulk refractive index at wavelength 955 nm remains unchanged at C'2, the incident light intensity- analyte refractive index of the sample point region is set as C'1, and the reflected light intensity of the sample point region changes with time at wavelength 945 nm is shown in Figure 6a As shown.

[0133] When the wavelength of the sample area is 945 nm and 955 nm, the expression shown in equation (4) is given, where Δn represents the difference in refractive index between n1 and n2, which can be calculated using equation (3). The result is as follows: Figure 6b As shown, Δb represents the refractive index change caused by the composite:

[0134]

[0135] Based on equations (3) and (4), the expression for Δb is given in equation (5), and the result is as follows: Figure 6c As shown:

[0136]

[0137] Example 2

[0138] In this embodiment, the optical system 20 uses a red light-emitting diode with a wavelength coverage range of 630nm-660nm as the light source 21, a motor rotating filter device as the wavelength scanning device 23, and liquid crystal as the material of the voltage-adjustable transmittance device 24.

[0139] The fabrication process and key parameter range of the microarray chip 12 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. Then, it is placed in an electron beam evaporation instrument and the vacuum is evacuated to reduce the pressure to 10. -5 Below millitor level; 0.5nm-2.5nm chromium as an adhesion layer and 0.5nm-2.5nm zinc sulfide as an adhesion enhancement layer are deposited at a rate less than 0.1nm / s; 50nm gold or silver is deposited at a rate greater than 0.01nm / s as the metal surface for SPR excitation; after soaking in a solution of mercaptoalkanoic acid (with carbon chain length between 3-15) at a concentration of 0.1mM or higher for more than 30 minutes to form a monomolecular self-assembled layer, a 20nM protein A microarray is formed using a biomolecular spotting instrument, with each sample spot being a circle with a diameter of 10μm or more. The fluid cell is in contact with the surface of the microarray chip 12 and is used to hold phosphate buffer standard samples and test samples using phosphate buffer as a solvent. Under standard conditions, the spatial position diagram of each sample spot area on the surface of the microarray chip 12 received by the CCD is shown in the figure. Figure 2 As shown, the intensity distribution of reflected light at different wavelengths of the microarray chip 12 was obtained by spectrometer measurement. Figure 7 As shown. After changing the refractive index of the sample in the fluid cell, the intensity of reflected light at different wavelengths was remeasured using a spectrometer. The sensitivity of the reflected light intensity versus sample refractive index at different wavelengths was calculated as follows. Figure 8 As shown.

[0140] Based on the surface plasmon resonance imaging device provided in this embodiment, this embodiment also provides a method for simultaneously measuring the volume refractive index of the sample background region and the refractive index of the sample aggregate without calibration. The method is as follows: Before the experiment, the sample cell 13 is kept unchanged with a standard sample. The microcontroller control card controls the CCD to collect the intensity of reflected light from the surface of the microarray chip 12. By distinguishing different intensity values, the spatial position of each sample area in the microarray is obtained, such as... Figure 2 As shown. The wavelengths were changed to 635 nm and 645 nm by adjusting the rotating filter device of the motor. The average reflected light intensity of the CCD sample collection area at both wavelengths was recorded. The applied voltage of the transmittance controllable device was adjusted to make the average reflected light intensity of the CCD sample collection area consistent at both wavelengths. The refractive index of the standard sample is denoted as n1, the wavelength of 535 nm as λ1, and the wavelength of 645 nm as λ2. The reflected light intensity of the sample area at wavelength 635 nm is denoted as I1(λ1, n1), and the reflected light intensity of the sample background area is denoted as I2(λ1, n1); the reflected light intensity of the sample area at wavelength 645 nm is denoted as I1(λ2, n1), and the reflected light intensity of the sample background area is denoted as I2(λ2, n1). Figure 8 As shown, the incident light intensity-volume refractive index sensitivity of the sample area and the sample background area at a wavelength of 635 nm is set to C2, and the incident light intensity-volume refractive index of the sample area and the sample background area at a wavelength of 645 nm is set to C'2.

[0141] For the sample background region, at wavelengths of 635 nm and 645 nm, the expression shown in equation (6) applies, where Δn represents the difference in refractive index between n1 and n2:

[0142]

[0143] according to Figure 4 As shown, the relationship between C'2 and C2 can be linearly approximated by the expression shown in equation (7), where Δλ represents the wavelength difference between λ1 and λ2:

[0144]

[0145] Substituting equation (2) into equation (1), and subtracting the lower equation from the upper equation in equation (1), the calculation result is shown in equation (8). The measurement result of the refractive index change of the sample to be tested with a known refractive index is as follows: Figure 9 As shown:

[0146]

[0147] In the experiment, 20 nM immunoglobulin G was introduced into sample cell 13, with a refractive index denoted as n2. A microcontroller control card synchronously controlled the center wavelength of the rotating filter device, the applied voltage of the voltage-adjustable transmittance controllable device, and the intensity of reflected light from the surface of the CCD collecting microarray chip 12 using a 16 Hz frequency signal. The center wavelength of the rotating filter device allowed switching between 635 nm and 645 nm for the incident light beam on the microarray chip 12. The reflected light intensity of the sample area at wavelength 635 nm was denoted as I1(λ1, n2), and the reflected light intensity of the background area was denoted as I2(λ1, n2); the reflected light intensity of the sample area at wavelength 645 nm was denoted as I1(λ2, n2), and the reflected light intensity of the background area was denoted as I2(λ2, n2). At a wavelength of 635 nm, the incident light intensity-volume refractive index of both the sample area and the sample background area remained constant at C2, while the incident light intensity-boundary refractive index of the sample area was set to C1. Similarly, at a wavelength of 645 nm, the incident light intensity-volume refractive index remained constant at C'2, and the incident light intensity-boundary refractive index of the sample area was set to C'1. The measured change in reflected light intensity over time at a wavelength of 635 nm is shown below. Figure 10a As shown.

[0148] When the wavelength of the sample area is 945 nm and 955 nm, the expression shown in equation (9) applies, where Δn represents the difference in refractive index between n1 and n2, which can be calculated using equation (8). The result is as follows: Figure 10b As shown, Δb represents the refractive index change caused by the composite:

[0149]

[0150] Based on equations (8) and (9), the expression for Δb is given in equation (10), and the result is as follows: Figure 10c As shown:

[0151]

[0152] The technical features of the example can be combined arbitrarily. 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.

[0153] 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 surface plasmon resonance imaging device, characterized by, The application relates to a detection system, an optical system and a collection control system. The detection system comprises a medium coupler, a microarray chip and a sample pool in contact with the microarray chip, and incident light is coupled to a metal surface of the microarray chip via the medium coupler. The optical system comprises a light source, a beam shaper, a wavelength scanning device, a voltage-adjusted transmittance device, a polarizer and an array detector, the light source, the beam shaper, the wavelength scanning device, the voltage-adjusted transmittance device and the polarizer are sequentially arranged on an optical path of the incident light, the wavelength scanning device is used for switching of the wavelength of the incident light, the voltage-adjusted transmittance device is used for compensation of transmittance deviation of the polarizer under different wavelengths of the incident light, and the array detector is used for receiving a reflected light beam of a metal-medium interface of the microarray chip and measuring an intensity spatial distribution of the reflected light beam. The collection control system collects data of the array detector before the experiment, records spatial positions of each sample point area on the microarray chip, controls the wavelength scanning device to output a wavelength of a light beam and the voltage-adjusted transmittance device to output an applied voltage during the experiment, simultaneously collects data of the array detector under different wavelengths and performs differential operation and differential operation of average reflected light intensity of the sample point area and a sample point background area.

2. The surface plasmon resonance imaging device of claim 1, wherein, The wavelength scanning device comprises an acousto-optic tunable filter.

3. The surface plasmon resonance imaging apparatus according to claim 1, wherein The wavelength scanning device comprises a motor and a filter sheet in transmission connection with the motor, the motor is used for adjusting a position of the filter sheet, so that a light beam emitted from the beam shaper is projected to different filter areas of the filter sheet.

4. The surface plasmon resonance imaging apparatus according to claim 1, wherein The voltage-adjusted transmittance device changes wavelength transmittance by adjusting an applied voltage on a transmittance-adjustable medium, so as to compensate for transmittance deviation of the polarizer under different wavelengths of the incident light.

5. The surface plasmon resonance imaging apparatus according to claim 1, wherein The light source comprises a white light emitting diode or a red light emitting diode; and the array detector comprises a photodiode, a phototriode or a photomultiplier.

6. The surface plasmon resonance imaging device according to any one of claims 1 to 5, wherein The preparation method of the microarray chip comprises the following steps: a first preset volume ratio of ethanol-ether mixed solution is used to ultrasonically clean a glass substrate for a first preset time length, so as to clean a surface of the glass substrate; the glass substrate is placed into an electron beam evaporation instrument and vacuumized, so that an air pressure value in the electron beam evaporation instrument is reduced to a preset air pressure value; a first preset thickness of chromium is evaporated on the surface of the glass substrate as an adhesion layer and a second preset thickness of zinc sulfide is evaporated on the surface of the glass substrate as an adhesion enhancement layer at a first preset rate; a third preset thickness of gold or silver is evaporated on the surface of the glass substrate as a metal surface for exciting surface plasmon resonance at a second preset rate; the glass substrate is soaked in a first preset concentration of mercapto acid solution for a second preset time length, so as to form a monomolecular self-assembled layer; a preset biomolecular microarray is spotted on the surface of the glass substrate by using a biomolecular spotting instrument, so as to obtain a microarray chip, each sample point of the microarray chip has a preset diameter and a preset shape.

7. The surface plasmon resonance imaging apparatus according to claim 6, wherein The preset volume ratio ranges from 1:1 to 1:10; The first preset time length is greater than or equal to 30 min; The preset air pressure value is less than or equal to 10 -5 mTorr; The first preset rate is less than or equal to 0.1 nm / s. The first preset thickness is 0.5nm-2.5nm; The second preset thickness is 0.5nm-3nm; The second preset rate is greater than or equal to 0.01nm / s; The third preset thickness is 40nm-70nm; The first preset concentration is greater than or equal to 0.1mM; The carbon chain length of the thiol acid in the thiol acid solution is 3-15; The second preset time length is greater than or equal to 30min; The preset diameter is greater than or equal to 10um; The preset shape is circular or quasi-circular.

8. A refractive index detection method characterized by, The surface plasmon resonance imaging device according to any one of claims 1-7, wherein the refractive index detection method comprises the following steps: Under the condition that the sample cell contains a standard sample, mark each sample point area and sample point background area by taking a picture of the surface microarray of the microarray chip; After the sample cell is filled with the sample to be detected, the wavelength scanning device and the voltage adjustment transmittance device are adjusted to cyclically change the wavelength of the incident light while keeping the intensity of the incident light unchanged; In each cycle, the data difference of the array detector corresponding to different wavelengths in the sample point background area is calculated to obtain the refractive index change between the sample to be detected and the standard sample, and the data difference of the array detector corresponding to different wavelengths in the sample point area is subtracted from the corresponding difference of the sample point background area to obtain the refractive index change between the binder and the standard sample.

9. The refractive index detecting method according to claim 8, wherein Before the experiment, the sample cell is kept with the standard sample, the array detector is controlled by the acquisition control system to collect the intensity of the reflected light on the surface of the microarray chip, the spatial positions of each sample point area are obtained by distinguishing different intensity values, the wavelength scanning device is adjusted to change the wavelengths to λ1 and λ2, the average intensity of the reflected light collected by the array detector in the sample point area at the wavelengths λ1 and λ2 is recorded, and the average intensity of the reflected light collected by the array detector in the sample point area at the wavelengths λ1 and λ2 is made consistent by adjusting the applied voltage of the voltage adjustment transmittance device; the refractive index of the standard sample is denoted as n1, the intensity of the reflected light in the sample point area at the wavelength λ1 is denoted as I1(λ1, n1), and the intensity of the reflected light in the sample point background area is denoted as I2(λ1, n1); the intensity of the reflected light in the sample point area at the wavelength λ2 is denoted as I1(λ2, n1), and the intensity of the reflected light in the sample point background area is denoted as I2(λ2, n1); the incident light intensity-body refractive index sensitivity of the sample point area and the sample point background area at the wavelength λ1 is denoted as C2, and the incident light intensity-body refractive index of the sample point area and the sample point background area at the wavelength λ2 is denoted as C’2; For the sample point background area, there is an expression shown in formula (1) at the wavelengths λ1 and λ2, wherein Δn represents the refractive index difference between n1 and n2: （1） The relationship between C’2 and C2 can be linearly approximated by the expression shown in formula (2), wherein Δλ represents the wavelength difference between λ1 and λ2: （2） Formula (2) is substituted into formula (1), and the calculation result is shown in formula (3) by subtracting the lower formula from the upper formula in formula (1): （3） In the experiment, first, the standard sample is introduced into the sample cell for a first time duration, then the immunoglobulin G of a certain concentration is introduced for a second time duration, and the refractive index is denoted as n2, and finally the standard sample is introduced for a third time duration. The acquisition control system controls the wavelength of the output light beam of the wavelength scanning device and the applied voltage of the voltage-adjusted transmittance device, and controls the array detector to collect the reflected light intensity on the surface of the microarray chip. The acquisition control system controls the wavelength scanning device to realize the switching of the wavelengths λ1 and λ2. When the wavelength is λ1, the reflected light intensity of the sample point region is denoted as I1(λ1, n2), and the reflected light intensity of the sample point background region is denoted as I2(λ1, n2). When the wavelength is λ2, the reflected light intensity of the sample point region is denoted as I1(λ2, n2), and the reflected light intensity of the sample point background region is denoted as I2(λ2, n2). When the wavelength is λ1, the incident light intensity-body refractive index of the sample point region and the sample point background region remains unchanged, the incident light intensity-binder refractive index of the sample point region is set as C1, the incident light intensity-body refractive index corresponding to the wavelength λ2 remains unchanged, and the incident light intensity-binder refractive index of the sample point region is set as C’1. The sample point region has an expression shown in formula (4) when the wavelengths are λ1 and λ2, wherein Δn represents the refractive index difference between n1 and n2, which can be calculated by formula (3), and Δb represents the refractive index change caused by the binder: （4） According to formula (3) and (4), the expression of Δb is as shown in formula (5): （5）。

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