CN104237170B - Surface plasma resonance imaging sensor detection system - Google Patents

Abstract

The invention discloses a surface plasmon resonance imaging sensing detection system, which includes a light source. The light emitted by the light source then passes through a Galileo beam expander to diffuse the light emitted by a point light source into a uniform beam, and then the light beam passes through a polarizer, The variable grating, and then the light is irradiated on the surface of the sensor chip on the flow cell, and finally the light is reflected by the prism and the chip, enters the CCD imaging system, and then outputs the corresponding sensing curve and real-time imaging results through the computer. According to the surface plasmon resonance imaging sensing detection system of the present invention, the surface plasmon resonance imaging technology (SPRi) is a new label-free, high-throughput optical sensing technology integrating SPR technology, CCD imaging and arrayed chips. Applied to the study of biomolecular interactions.

Description

A Surface Plasmon Resonance Imaging Sensing Detection System

technical field

The invention relates to a surface plasmon resonance imaging technology, in particular to a surface plasmon resonance imaging sensing and detection system.

Background technique

Surface plasmon resonance imaging (SPRi) technology is an optical imaging technology developed based on surface plasmon resonance (SPR) technology, combined with CCD imaging and arrayed chips. Surface plasmon resonance (SPR) is a charge-density vibrational resonance wave that exists at the interface of a metal and a dielectric and can be excited by incident electromagnetic waves. When the incident light propagates from the optically denser medium to the optically rarer medium and the incident angle is in an appropriate range, total internal reflection occurs at the interface of the two substances. SPR is extremely sensitive to small changes in the refractive index of the interface medium. When the sample is in contact with the interface, the refractive index of the medium at the interface will change due to adsorption or chemical reactions. Based on this principle, biomolecules and their interactions can be monitored in real time, quickly and in situ without damage, the kinetic process of binding and dissociation between biomolecules can be studied, and the physical and chemical adsorption process at the interface can be understood. As a powerful biomolecular analysis tool, SPR biosensor has the advantages of real-time, fast, label-free, good reproducibility, high sensitivity, and no need for sample purification. It has been widely used in the detection and analysis of proteins, nucleic acids, and small molecules. . The surface plasmon resonance imaging technology not only has the advantages of surface plasmon resonance technology, but also relies on the combination of SPRi technology and arrayed biochip technology to modify the surface of the biosensing chip with different probe arrays to realize the target substance in the sample to be tested. High-throughput detection and screening. In recent years, in order to adapt to the arrival of the "post-genome era", various molecular biology studies such as genomics, proteomics and metabolomics have been deepened, and surface plasmon resonance imaging technology has become more and more widely used by many scientific researchers. Attention and attention, it is considered to be a high-throughput biological analysis method with great potential.

Contents of the invention

In view of the requirements of the prior art, the object of the present invention is to provide a surface plasmon resonance imaging sensing detection system.

A surface plasmon resonance imaging sensing detection system, including a light source, the light emitted by the light source then passes through a Galilean beam expander to diffuse the light emitted by a point light source into a uniform beam, and then the light beam passes through a polarizer and a variable grating in sequence, Then the light is irradiated on the surface of the sensor chip on the flow cell, and finally the light is reflected by the prism and the chip, enters the CCD imaging system, and then outputs the corresponding sensing curve and real-time imaging results through the computer.

Preferably, the flow cell includes a base, the base is provided with a main part of the flow cell, and the main part of the flow cell is provided with a osmotic block laterally inside, and the osmotic block is cylindrical with one end closed, corresponding to the osmotic block. The main body of the flow cell at the open end of the block is sealed by an end cover, and at least two openings are provided on the end cover. Small holes are provided on the closed end face of the permeation block, and there are holes on the flow main part corresponding to the closed end of the permeation block. Gasket groove, a gasket is embedded in the gasket groove, the outer side of the gasket is bonded to the chip to form a working cavity, the chip is compressed by a prism, and the prism is compressed by a flow cell attachment, and the flow cell attachment corresponds to Set behind the main part of the flow cell, the accessory of the flow cell includes a fixed seat fastened on the base, a compression block is arranged between the fixed seat and the prism, and an adjustable screw is arranged laterally on the fixed seat Used to compress the compression block.

Preferably, the chip is made of a glass slide through a gold-plating process to form an arrayed dot matrix chip.

Preferably, the side of the pressing block is provided with grooves running through the upper and lower surfaces, and the grooves fix the edges of the triangular prism.

Preferably, a horizontal gradient adjustment table (mainly used to adjust the height and level of the flow cell) and a rotary table at the bottom (mainly used to adjust the overall angle of the flow cell) are linked under the flow cell.

Preferably, a sampling system is also connected to the flow cell, mainly including a six-way valve and a precision micro-injection pump.

Preferably, the LED with a light source wavelength of 650nm is connected to a small DC power supply, and the voltage of the light source is stabilized through a low-ripple and low-noise linear voltage stabilization method to ensure stable light intensity.

Preferably, the light source is fixed by a threaded base, and the threaded base is fixed on the adjustable pinhole base, so that the XY direction of the light source can be precisely adjusted to ensure the accuracy of the optical path.

Preferably, the polarizer is fixed on a polarizer rotating table, and the direction of the polarizer can be adjusted to achieve the function of controlling polarized light in different directions to pass through the polarizer.

Preferably, the base, main parts and accessories of the flow cell are made of hard aluminum alloy.

Preferably, the side of the pressing block is provided with grooves running through the upper and lower surfaces, and the grooves fix the edges of the triangular prism.

Preferably, the osmotic block is processed from polymethyl methacrylate.

Preferably, the chip is made of a glass slide through a gold-plating process to form an arrayed dot matrix chip.

Preferably, the gasket is processed from polytetrafluoroethylene.

Preferably, the triangular prism is made of flint glass.

As mentioned above, according to the surface plasmon resonance imaging sensing detection system of the present invention, it has the following beneficial effects:

Surface plasmon resonance imaging (SPRi) is a new label-free, high-throughput optical sensing technology that integrates SPR technology, CCD imaging and arrayed chips, and is widely used in the study of biomolecular interactions. SPRi has the advantages of real-time, fast, label-free, good reproducibility and high sensitivity. It can imitate the biological environment and perform high-throughput biochemical analysis, especially in monitoring molecular interactions and dynamic analysis.

Description of drawings

FIG. 1 shows a schematic structural diagram of a detection system implemented in the present invention.

FIG. 2 is a schematic structural diagram I of a flow cell according to an embodiment of the present invention.

FIG. 3 is a schematic structural diagram II of a flow cell according to an embodiment of the present invention.

Fig. 4 is a side cross-sectional view of a flow cell according to an embodiment of the present invention.

Fig. 5 shows the spectrogram obtained by using the detection system of this embodiment.

Part number description

1 base,

2 circulation main parts,

3 infiltration blocks,

4 round end caps,

5 washers,

6 chips,

7 prisms,

8 compression block,

9 Flow cell accessories,

10 Adjustable screw.

detailed description

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

This embodiment provides a surface plasmon resonance imaging sensing detection system. First, an LED with a wavelength of 650nm is selected as a light source, connected to a small DC power supply, and the voltage of the light source is stabilized through a linear voltage stabilization method with low ripple and low noise. Ensure stable light intensity; then fix the light source through the threaded base, and fix the threaded base on the adjustable pinhole base, so the XY direction of the light source can be precisely adjusted to ensure the accuracy of the optical path; the light emitted by the light source then passes through The Galileo beam expander diffuses the light emitted by the point light source into a uniform beam, all parallel and the energy of the beam is the same, without deviation, and then passes through the polarizer, which is fixed on the polarizer rotating table, which can be adjusted The direction of the polarizer is adjusted to achieve the function of controlling polarized light in different directions to pass through the polarizer; then the light will pass through a variable grating, and the amount of incident light can be controlled by the adjusting rod; then the light is irradiated on the surface of the sensor chip on the flow cell , there is a horizontal slope adjustment platform for adjusting the height and level of the flow cell and a rotary table at the bottom for adjusting the overall angle of the flow cell; finally, the light is reflected by the prism and the chip and enters the CCD for imaging The system then outputs the corresponding sensing curve and real-time imaging results on the computer through the supporting software.

The "small DC power supply, low ripple and low noise linear voltage regulation method" used in this embodiment has the disadvantages of low conversion efficiency of the transformer, large voltage difference of the voltage regulation chip, and imperfect filter circuit in the traditional linear voltage regulation power supply. There are problems such as large output ripple, high heat generation, low efficiency, and indirectly adding thermal noise to the system. On this small DC power supply, the step-down module, rectifier and filter module, linear voltage regulator module and low-pass filter module Therefore, the power supply not only has the advantages of high stability and low ripple, but also has small output voltage fluctuations, adjustable voltage, strong load capacity, and small size.

The threaded base used in this embodiment is mainly based on the adjustable pinhole base, and is mainly used for fixing and adjusting the position of the light source.

The 'variable grating used in this embodiment can control the amount of incident light through an adjustment rod', on the variable grating, there is an adjustment rod, the size of the central aperture of the variable grating can be adjusted by adjusting the adjustment rod, so it can be adjusted The amount of light passing through the variable grating is controlled and regulated.

The 'horizontal gradient adjustment table for adjusting the height and horizontal state of the flow cell and a rotary table for the overall angle adjustment of the flow cell at the bottom' used in this embodiment, above the horizontal slope adjustment table are fixed flow cells, For the prism and chip, through the adjustment of the adjustment platform, the center point of the flow cell, prism and chip can be located at the center point of the entire platform, and it can be kept at a level with the incident light, which is beneficial to subsequent experiments. The rotary table at the bottom mainly rotates and adjusts the upper prism through the post connection system, so as to adjust the angle of incident light to adjust the optimal angle of different refractive index solutions.

The 'supporting software' software used in this embodiment is based on the latest Labview. According to the required functions of the experimental platform, the corresponding operating functions are designed and programmed to realize the control software with easy operation and complete functions.

The flow cell used in this embodiment includes a base 1, the base 1 is a square three-dimensional block, the side above the base 1 is provided with a flow cell main part 2, and the flow main part 2 is an upwardly extending three-dimensional block integrated with the base 1 manufacture. A circular through hole is arranged laterally in the middle of the main body 2 of the flow cell, and a permeation block 3 is arranged in the through hole. The permeation block 3 is a cylinder with one end closed, forming a circulation cavity, and the liquid circulation volume in the circulation cavity is 30-50 ul. A circular end cap 4 is fixedly installed by threads on the flow cell corresponding to the opening end of the permeation block 3. There are two openings distributed on the circular end cap 4. In this embodiment, the lower opening is the sample inlet, and the upper opening is the sample outlet. mouth. All openings are connected to external sample delivery lines. A small hole is provided on the closed end surface of the permeation block 3, and the small hole is used for the liquid to be measured in the circulation cavity to pass through. The flow body 2 corresponding to the closed end of the permeable block 3 is provided with a gasket groove, and a gasket 5 is embedded in the gasket groove, and the embedded gasket 5 is slightly higher than the back plane of the flow cell body 2 . The flow cell of this embodiment also includes a chip 6, and the chip 6 presses the gasket 5 so that the chip 6 and the gasket 5 form a reaction chamber. The chip 6 is pressed by an equilateral triangular prism 7 with a side length of 25 mm. One vertical surface of the triangular prism 7 is in close contact with the chip, and the opposite edge of the adhering surface is compressed by a pressing block 8 . The pressing block 8 is a three-dimensional block, and a groove is vertically arranged, and the edges cooperate with the groove.

A flow cell accessory 9 is arranged on the back of the flow cell main part 2 . The flow cell accessory 9 is a fixing seat fixed on the base, steps are provided on both sides of the fixing seat, mounting holes are provided on the steps, and the mounting screws are connected to the base 1 through the mounting holes. Two installation holes are horizontally arranged on the fixing seat, and an adjustable screw rod 10 equal to the aperture is penetrated in the installation hole, and the end of the adjustable screw rod 10 is pressed against the compression block 8 to compress the triangular prism 7 and the chip 6 .

The base 1, main part 2 and accessories 9 of the flow cell in this embodiment are made of hard aluminum alloy.

The permeable block 3 of this embodiment is processed from polymethyl methacrylate. The osmotic block 3 using this material is not easy to break, has high transparency, and is easy to observe the internal liquid state and whether there is interference of air bubbles; and has chemical reagent and solvent resistance.

The chip 6 of this embodiment is made of a glass slide through a gold-plating process to form an arrayed dot matrix chip. Can be used for high-throughput analytical applications. The gold-plating process is to wash the glass slides with piranha solution and acid solution, rinse them with deionized water, dry them with nitrogen gas, heat them at 70°C for one hour, and finally carry out electron beam evaporation (electron beam evaporation). Evaporation) treatment, plate a layer of 2nm thick Cr on the processed glass slide, and then plate a layer of 51nm thick gold. The dot matrix chip of this embodiment is an arrayed chip, that is, when making the chip, according to the needs of the experiment, when coating with electron beam evaporation technology, selectively carry out 4×4, 8×8, 10×10 Each point is a basic unit of reaction monitoring, so high-throughput detection can be performed.

The gasket 5 of this embodiment is processed from polytetrafluoroethylene. It has excellent heat resistance, moisture resistance, wear resistance and corrosion resistance, and is suitable for various conventional buffer solutions in different detection systems. Its outer diameter is 19.05mm and inner diameter is 15.88mm.

The triangular prism 7 of the present embodiment is an equilateral triangular prism with a side length of 25 mm, and is made of F2 glass material. F2 is a kind of flint glass, which has good performance in the visible light and near-infrared spectral ranges, and has high reflectivity and low Abbe number, and compared with N-SF11, it has higher chemical resistance and slightly higher transmittance.

The flow cell of this embodiment is connected to the six-way valve through the sample inlet on the end cover, and then powered by a precision micro-syringe pump for sample injection and detection, and then connected to the outside through the sample outlet on the end cover to remove waste. The liquid to be tested passes through the injection port, flows through the main channel of the permeation block in the flow chamber, and then passes through the holes on the permeation block to reach the closed space formed by the prism, chip and gasket at the other end of the flow cell. The liquid flows across the surface of the chip in this space, and the substance to be tested in the liquid fully contacts and reacts with the substance that has been modified on the chip surface to realize the process of interaction. Then, driven by the buffer, this part of the liquid to be tested is then From the main channel to the sample outlet, and then to the waste tank, the whole process is completed once. The flow cell uses a base and accessories made of hard aluminum alloy, and a liquid permeation block with a flow cavity processed by polymethyl methacrylate, forming a precise and micro flow system, which can treat a small amount of samples to be tested. It can be detected and combined with arrayed chips to achieve high-throughput analysis of multiple substances.

This embodiment is suitable for specific experiments:

First, the chip was cleaned with piranha solution (concentrated H ₂ SO ₄ :H ₂ O ₂ =7:3), then the piranha solution on the surface of the chip was rinsed with double distilled water, and the chip was blown dry with nitrogen; Drop one or two drops of matching fluid on the surface of the dried chip, and then assemble the chip, prism and flow cell; set the flow rate of the precision micro-injection pump to 1ml/min, and install it in the precision syringe in advance. Ionized water was used as the running buffer for the entire experiment; open the software and set the exposure time to 4ms, select the area to be detected, and then turn on the syringe pump. After the baseline is stable, adjust the six-way valve to the Load state and inject different Glucose solution with a high concentration, then adjust the six-way valve to the Inject state. While the liquid to be tested flows through the chip surface, observe the change of the dynamic curve in the control software interface. After the experiment is stopped, the saved data can be opened to draw the curve. As shown in Figure 5, each peak corresponds to different concentrations of glucose solutions. Since the refractive index of different concentrations of glucose solutions is different, it will lead to differences in reflected light intensity, which is the difference in height of the peaks shown in the figure.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (8)

1. a kind of surface plasma resonance image-forming sensing and detecting system is it is characterised in that include light source, the light that light source sends Subsequently pass through Galileo beam-expander, the light diffusion uniformly light beam that point source is sent, then light beam passes sequentially through Polariser, iris shutter, then it is irradiated to the censorchip surface on flow cell, last light passes through the reflection of prism and chip, Enter ccd imaging system and then sense curve and realtime imaging result by computer output is corresponding；Described flow cell, the bottom of including Seat, described base is provided with flow cell main part, and described flow cell main part inner transverse is provided with infiltration block, and described infiltration block is one Hold airtight tubular, airtight by end cap on the circulation tank main body of corresponding described infiltration block opening end, end cap is provided with least two Individual opening, the sealed end end face of described infiltration block is provided with aperture, and the circulation main component of corresponding described infiltration block sealed end sets There is gasket recess, described gasket recess is embedded in packing ring, formation working cavity of fitting with chip outside described packing ring, described chip Compressed by prism, described prism is compressed by flow cell adnexa, described flow cell adnexa is correspondingly arranged at flow cell master The rear of part, described flow cell adnexa includes the fixed seat being fastened on base, sets between described fixed seat and described prism There is compact heap, in described fixed seat, horizontally set adjusting screw is used for compressing compact heap.

2. detecting system according to claim 1 it is characterised in that described chip by microscope slide through gold-plated science and engineering Skill, forms the dot matrix chip of array.

3. detecting system according to claim 1 is it is characterised in that described compact heap side is provided with and runs through upper and lower surface Groove, the seamed edge of the fixing prism of described groove.

4. detecting system according to claim 1 is it is characterised in that link for adjusting flow cell below described flow cell Height and horizontality horizontal slope regulating platform, the bottom of described horizontal slope regulating platform is provided with for adjusting flow cell angle The turntable of degree.

5. detecting system according to claim 1 is it is characterised in that being connected with sampling system on described flow cell, described Sampling system includes six-way valve and precise micro syringe pump, and described six-way valve connects the opening on flow cell end cap.

6. detecting system according to claim 1 is it is characterised in that described light source is the LED lamp of wavelength 650nm, described LED lamp is connected on small-sized DC power supply, by the linear voltage stabilization mode of low ripple low noise, make light source voltage stable it is ensured that Stable light intensity.

7. detecting system according to claim 1 is it is characterised in that described light source is fixed by threaded base, by this spiral shell Stricture of vagina base is fixed on scalable pin hole base, and the regulation that therefore the xy direction of light source can be carried out with precision is it is ensured that the essence of light path Accurate.

8. detecting system according to claim 1 is it is characterised in that described polariser is fixed on polariser turntable, Polariser direction can be adjusted, can reach the function by polariser for the polarized light controlling different directions.