CN107764776B - Multi-wavelength adjustable surface plasma resonance imaging device and application thereof - Google Patents
Multi-wavelength adjustable surface plasma resonance imaging device and application thereof Download PDFInfo
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Abstract
The invention designs a multiband adjustable surface plasma resonance imaging device and application thereof, wherein light beams emitted by an LED light source with broadband spectrum are filtered into monochromatic light beams by a linear optical filter, then form a p-polarized light source by a collimating mirror and a polarizing film, and are projected onto an SPR generating device, and emergent light beams of the device are imaged by a CCD camera; the moving platform can also be used for controlling the linear optical filter to linearly adjust the wavelength of the light source, multiband incident light can be obtained for SPR imaging, all areas on the surface of the chip can be scanned in a wave band mode simultaneously to obtain a resonance spectrogram of each area, and the resonance spectrum offset generated by the environmental change of each area on the surface of the sensing chip can be accurately measured under the condition that the SPRI imaging is not distorted. By using the method, the data of all areas on the surface of the chip can reach the optimal sensitivity condition and the dynamic range and sensitivity of the detection of an instrument can be improved.
Description
Technical Field
The invention belongs to the field of instruments and equipment, and particularly relates to a Kretschmann SPR imaging device and method.
Background
The Surface Plasmon Resonance (SPR) phenomenon is a physical phenomenon, which refers to a physical optical process in which free vibrating electrons existing on a metal Surface are excited by light under a certain condition and the ability of absorbing light resonates.
When light is projected from an optically dense medium to an optically thinner medium, a total reflection phenomenon occurs when an incident angle is larger than a certain angle (critical angle). Incident light and the normal line of the surface of the medium form an incident plane, the electric field of incident light waves can be decomposed into two mutually orthogonal polarized light components, one is transverse magnetic waves in the incident plane and is called TM waves or p-polarized waves; the other is a transverse electric wave perpendicular to the incident surface and parallel to the interface, and is called a TE wave or an s-polarized wave. One part of the incident light is reflected to form reflected light, and the other part of the incident light penetrates through the metal surface to form refracted waves, and the refracted waves are attenuated exponentially along the direction perpendicular to the interface, and the reflected light is also called Evanescent waves. The effective penetration depth of the evanescent wave in the optically thinner medium is generally 200-300 nm. The physical reason for the attenuation is that free electrons exist in the metal, and under the action of electromagnetic waves, induced current appears in the metal, joule heat is generated, the energy of the electromagnetic waves is consumed, and therefore the amplitude of the waves is weakened. The evanescent wave is always attenuated perpendicular to the interface regardless of the direction of the incident light, but its penetration depth is related to the direction of the incident light.
If a metal thin film (generally, a gold/silver film of about 50 nm) of several tens of nanometers exists between two media, a p-polarized Wave of an evanescent Wave generated by total reflection enters the metal thin film, couples with free electrons in the metal thin film, and excites a Surface Plasmon Wave (SPW) propagating along the Surface of the metal thin film. And the s-polarized light is parallel to the interface, so that the surface plasma cannot be generated by excitation. When the angle or wavelength of the incident light reaches a certain value, most of the incident light is converted into the energy of the surface plasmon wave, so that the reflected light energy is greatly reduced, and a resonance absorption peak appears on the reflection spectrum, at which time the angle or wavelength of the incident light becomes the resonance angle or the resonance wavelength.
The Surface Plasmon Resonance (SPR) sensing method has the characteristics of no need of marking, high sensitivity, real-time monitoring and the like, and is widely applied to the fields of biology and chemistry. However, with the wide application of gene arrays, protein arrays and other biological arrays (i.e., biochips), the sensors for measuring the refractive index of a single point are not able to meet the research requirements, and a high-throughput detection method, i.e., imaging detection, capable of detecting a two-dimensional plane is required. The traditional imaging method using SPR, namely spm (surface plasma microscope), directly detects the reflected light intensity of the whole chip by fixing the wavelength and incident angle of the incident parallel light beam, and the correspondence between the gray value of the image and the refractive index is not a linear relationship, so that the change of the refractive index can only be qualitatively explained; moreover, the method can only measure the refractive index range with little change, and the two same refractive indexes can correspond to different gray values when the refractive index range is exceeded; such devices, which in addition use laser illumination, seriously affect the imaging effect due to the strong coherence of the laser.
The measurement method of Surface Plasmon Resonance Imaging (Surface Plasmon Resonance Imaging) usually adopts a reflectivity modulation mode (RIM) to achieve the optimal sensitivity of the instrument operation by fixing the optimal incident light wavelength and incident angle, so as to detect the molecular interaction of each region on the Surface of the sensor chip. However, the optimum working point (lambda) on the surface of the gold film0，θ0) Depending on the refractive index of the corresponding region on the surface of the gold film, the difference in the content or species of the immobilized biomolecules in each region also results in the difference in the refractive index of each region. When the reflectivity modulation mode is applied to an SPR imaging experiment, the data acquisition of different probe point areas prepared on the surface of a chip has great data difference. More importantly, when the biological sensing detection is carried out by adopting an RIM detection mode, along with the change of the refractive index of the buffer system, the change of the refractive index of the surface of the sensing chip and the response of an instrument can easily exceed a linear range, so that the dynamic range under the mode is narrower.
The problem of narrow dynamic range in the reflectivity modulation mode can be solved by adopting a wavelength modulation mode or an angle modulation mode, and the offset of the formant of each sample point can be accurately measured. For the SPRI detection of wavelength modulation type and angle modulation type, the sensitivity of each sample point on the surface of the sensing chip is measured independently. In order to accurately measure the shift amount of the resonance peak of each sample point on the chip surface individually, each sample point must be scanned and measured by angle or wavelength scanning. In the angle modulation mode, the SPR image on the chip is subjected to distortion along with the change of the incident angle; in the wavelength modulation mode, the resonant spectral distribution is obtained by scanning the wavelength of the incident light or using a spectrometer as a detector; the former measurement mode can perform synchronous scanning detection on a 2-dimensional array, has 2-dimensional imaging capability, and the latter provides instantaneous spectral information, but can only be limited to scanning of a 1-dimensional array. Therefore, the SPRI technique combining wavelength modulation with high-throughput data acquisition remains a challenge.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a multi-wavelength adjustable SPRI imaging method, which effectively improves the dynamic range of instrument detection and can accurately determine the resonance angle (resonance wavelength) offset of each sample area on a 2-dimensional array.
The invention provides a multi-wavelength adjustable SPRI imaging device, wherein a light source, a collimating mirror, an objective lens and a CCD camera form an SPR imaging light path, and the device is characterized in that an optical filter is arranged between the light source and the collimating mirror, the light source is filtered by the optical filter to form a beam of monochromatic light and is projected onto an SPR sensing chip, when a sample point of a chip array is detected, the incident light wavelength is adjusted by accurately controlling the position of the optical filter by using a moving platform, the reflected light intensity under different incident light wavelengths is detected by the CCD, and a computer is used for recording and processing data. The method can be used for accurately measuring the displacement of the resonance peak of each sample point, and can improve the dynamic range of the instrument detection.
Preferably, the moving platform is a piezoelectric ceramic translation platform or an electric moving platform. More preferably, the mobile platform is an electric mobile platform. The electric moving platform is a displacement translation platform capable of accurately and stably controlling movement, and has the advantages of accurately controlling the movement distance, high reliability and the like.
The invention further provides a multi-wavelength adjustable SPRI imaging method, which comprises the following steps:
(1) the optical filter is arranged between a light source and a collimating mirror of the conventional SPRI imaging device, the position of the optical filter is adjusted by using an electric moving platform, so that incident light beams are projected to different filter areas of the optical filter, then the change of the wavelength of the incident light along with the movement of a displacement platform is recorded, the coupling degree of each area of a chip is changed, the change of the reflected light intensity is caused, and the formant offset of each sample point is processed and fitted by a computer.
(2) And (3) carrying out accurate filtering processing by utilizing a moving platform, and determining the resonance peak offset of each sample point, wherein:
the optical filter is loaded on the electric platform capable of accurately controlling the displacement and assembled between the light source and the collimating mirror interface, incident light is firstly projected onto the optical filter, and monochromatic light beams formed after the optical filter is used for filtering the light source are projected onto the gold film coupled with the prism. At the moment, the moving platform is controlled by the computer to perform spiral movement from inside to outside, the optical filter shifts along with the movement of the moving platform, and the position of the light source is always kept fixed, so that different filtering areas of the optical filter continuously pass through light beams of the light source, and the light source is filtered to obtain a series of light beams with different wavelengths. When the incident angle is fixed and light beams with different wavelengths are projected on the surface of the SPRI sensing chip, coupling degrees generated by evanescent waves and electrons in the metal are different, so that the light intensity weakening degree is different, and finally the light intensity of reflected light is different. Thus different wavelengths correspond to different reflected light intensities. And recording the wavelength of incident light and the intensity signal of reflected light, processing to obtain the reflectivity curve of each area on the surface of the sensing chip, and obtaining a good imaging graph with the shape of the SPRI image unchanged.
Because the traditional multi-wavelength adjustment surface plasma resonance imaging device adopts a plurality of monochromatic light sources to form a multi-wavelength incident light source emitter after being connected in parallel, or adopts a halogen lamp with multi-band as a light source, and uses a wavelength scanning device to scan the wavelength of polychromatic light, the multi-wavelength adjustable SPRI imaging structure is completed. Both of these approaches result in complex wavelength modulation device construction and halogen lamps experience temperature drift during use, which can cause high baseline noise. Therefore, the surface plasma resonance imaging device with the adjustable wavelength can be greatly and simply realized by adopting the mode, and good instrument detection performance is obtained.
Has the advantages that: compared with the prior art, the multiband adjustable SPR imaging device can obtain the optimal resonance wavelength offset of each area on the surface of the sensing chip under the condition of not losing the image distortion degree, and can improve the dynamic range of SPR sensing to a greater extent, so that the detection performance of an instrument is improved.
Drawings
FIG. 1 is a schematic diagram of the principle of the SPRI of the Kretschmann type.
Fig. 2 is a schematic diagram of a conventional SPRI imaging apparatus.
Fig. 3 is a schematic diagram of the multi-wavelength tunable SPRI imaging apparatus according to the present invention.
Fig. 4 is a schematic diagram of filtering by a linear bandpass filter, wherein the origin and the straight line after filtering are monochromatic light beams with different wavelengths.
Detailed Description
The invention designs a system formed by combining a multi-band broadband LED Light source (Light source), a Linear Variable band pass filter (Linear Variable Bandpass Filters), a collimating mirror (prism lens), an electric displacement stage (Motorized Stages), an Objective lens (Objective) and a CCD camera on the basis of the traditional single-wavelength filtering SPRI imaging Light path, and mainly uses the Linear Variable band pass filter and the electric displacement stage in the traditional SPRI imaging device. The structure is shown in fig. 3.
In fig. 1 to 4, 1 is a light source, 2 is a collimator lens, 3 is a diaphragm, 4 is a polarizing plate, 5 is a filter, 6 is a prism, 7 is a lens, 8 is a camera, 9 is a longitudinal adjusting stage, 10 is an angle adjusting stage, 11 is a linear band-pass filter, 12 is an electric shift stage, and 13 is a connecting rod.
A linear variable band-pass filter 11 of a filter device is added between a light source 1 and a collimating mirror 2, the wavelength of the light source is filtered, and the electric displacement table 12 is used for controlling the filtering processing of different areas of the filter to the light source, so that multiband incident light is achieved. The SPRI experiment is carried out by using the incident light with different wavelengths, so that the reflection spectrum of each region on the surface of the sensing chip, which changes along with the wavelength, can be obtained, and the offset of the optimal formant of each corresponding region after the environment on the surface of the sensing chip is changed can be accurately obtained. And the condition of generating the optimal linear response range for each area can be achieved by adjusting the optimal incident wavelength, and the dynamic range of the detection of the instrument is improved.
The schematic diagram of the linear variable bandpass filter is shown in fig. 4. After the light filter filters the light source, the collimating mirror collimates the projected monochromatic light beam, and the collimated monochromatic light beam is projected onto the surface of the SPRI sensing chip and then reflected onto the CCD camera for imaging.
The working process of the invention is as follows:
fixing the linear band-pass filter 11 on a filter fixing frame, then assembling the filter fixing frame and the electric displacement table 12 together by using a connecting rod 13, firstly adjusting the electric displacement table 12, so that the edge of the filter 11 filters the light source 1, and recording the coordinates of the electric displacement table 12. The moving speed of the electric displacement platform 12 is set to be 0.5mm/s through a computer program, the wavelength of the light source can be adjusted to be 10nm changing speed per second, the acquisition speed of the CCD camera is set to be 1 second, one CCD camera is accessed, data is processed through a computer, the wavelength-reflection curve of each area on the surface of the sensing chip is obtained, and in the process of scanning incident light, the imaging spectrogram on the surface of the SPR sensing chip does not generate distortion. The multiband adjustable SPRI function is achieved by using the accurate adjustment displacement platform to change the moving distance.
By using the multiband adjustable SPRI imaging device, the following effects can be achieved:
the formant offset is obtained without the cost of image distortion: when the traditional Kretschmann surface plasma resonance imaging method is used for measuring a resonance peak spectrogram of a sample on the surface of a sensing chip, the method is obtained by adjusting or scanning the incident angle of incident light, detecting and recording the change of the reflected light intensity along with the incident angle and processing data. When the wavelength of incident light is fixed and the incident angle is changed, the image of SPR imaging can be distorted along with the change of the incident angle, and an instrument needs to adopt an accurate angle adjusting device in an angle scanning mode. And the adoption of the multiband adjustable SPRI imaging device does not need to use an additional angle regulator, and can conveniently determine the formant spectrogram without losing the truth of the imaging image.
Claims (4)
1. A multiband adjustable surface plasmon resonance imaging method is characterized by comprising the following steps:
(1) an optical filter is arranged between a light source of surface plasma resonance imaging and a collimating mirror, an electric moving platform is used for adjusting the position of the optical filter, so that incident light beams are projected to different filtering areas of the optical filter, then the change of the wavelength of the incident light along with the movement of a displacement platform is recorded, the coupling degree of each area of a chip is changed, the change of the reflected light intensity is caused, and the resonance peak offset of each sample point is processed and fitted through a computer;
(2) and (3) carrying out accurate filtering processing by utilizing a moving platform, and determining the resonance peak offset of each sample point, wherein:
the optical filter is loaded on an electric platform capable of accurately controlling displacement and assembled between a light source and a collimating mirror interface, incident light is firstly projected onto the optical filter, and monochromatic light beams formed after the optical filter is used for filtering the light source are projected onto a gold film coupled with a prism; at the moment, the computer controls the mobile platform to perform spiral movement from inside to outside, the optical filter shifts along with the movement of the mobile platform, and the position of the light source is always fixed, so that different filtering areas of the optical filter continuously pass through light beams of the light source, and the light source is filtered to obtain a series of light beams with different wavelengths;
when the incident angle is fixed and light beams with different wavelengths are projected on the surface of the SPRI sensing chip, coupling degrees generated by evanescent waves and electrons in the metal are different, so that the light intensity weakening degree is different, and finally the light intensity of reflected light is different, so that different wavelengths correspond to different reflected light intensities, the reflectivity-wavelength curves of all areas on the surface of the sensing chip are obtained by recording incident light wavelength and reflected light intensity signals and processing, and an imaging graph with the shape of the SPRI image unchanged is obtained.
2. The method of claim 1 wherein the filter is a linear variable bandpass filter or a linear variable edge filter.
3. The method of claim 1 wherein the moving platform is a piezo-ceramic translation stage or an electro-dynamic moving platform.
4. The method of claim 1 wherein the filters are assembled with the motorized stage via a connecting rod.
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| CN109444078A (en) * | 2019-01-14 | 2019-03-08 | 兰州理工大学 | Length scanning index sensor based on unsymmetrical metal cladding Medium Wave Guide |
| CN112098371A (en) * | 2020-09-15 | 2020-12-18 | 深圳大学 | Strength type SPRi sensing system and method based on dual-wavelength difference |
| CN113049544A (en) * | 2021-03-11 | 2021-06-29 | 中国科学院上海光学精密机械研究所 | Computing optical imaging device and method based on white light illumination |
| CN113324954A (en) * | 2021-05-29 | 2021-08-31 | 东北师范大学 | Prism coupling surface plasmon resonance test system based on spectral imaging |
| CN114895722B (en) * | 2022-05-17 | 2024-05-03 | 南京理工大学 | Humidity adjustment control and detection device and humidity detection method |
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