CN109507162B - A laser detection system and method based on resonant cavity and FRET effect - Google Patents

Abstract

A laser detection system and method based on resonator and FRET effect, comprising a laser source module, a resonator module, an optical collimation module and a signal receiving module, the laser source module is used to provide a laser source, and the optical collimation module is used to The source is coupled to the resonator module and the signal light generated by the resonator module is collected into the spectrometer; the resonator module is used to generate optical resonance and FRET effect, including a microcavity capable of generating optical resonance, and fluorescence around the microcavity capable of generating FRET effect Receptor substance; the signal receiving module is used to receive, analyze and store the signal light. The laser source module generates laser light, and the laser source is coupled to the resonator cavity module through the optical collimation module to excite the microcavity to generate resonance, and then excite the fluorescent receptor substances around the microcavity to generate the FRET effect and generate signal light; if the obtained signal light and There is a specific difference in the standard signal light, which indicates the existence of the molecule to be detected, and the detection of trace substances outside the resonant cavity is realized.

Description

A laser detection system and method based on resonant cavity and FRET effect

technical field

The present invention is mainly based on the optical resonant cavity of the whispering gallery mode and the effect of fluorescence resonance energy transfer (FRET). Using a specific laser source to pump and excite the fluorescent beads, the beads generate a cluster of laser light and excite the fluorescent medium outside the beads through the FRET effect, and the system outputs another cluster of lasers in adjacent wavelength bands. to achieve ultra-high-sensitivity sensing of biomolecules.

Background technique

The Whispering Gallery mode (WGM) phenomenon exists in the Temple of Heaven in Beijing and St. Paul's Church in London, mainly because the sound waves are constantly reflected on the curved and smooth walls and the loss is extremely low, so when you speak at a certain point on the wall, the wall is far away. Another point can still be heard. The whispering gallery principle of the optical mode is similar to that. The glass beads made of quartz material fused and sintered are the most primitive whispering gallery microcavities. Due to the small roughness of the inner boundary of the small ball, the difference in the refractive index between the inside and outside is large, and the small ball is scored. The beam is easily totally reflected in the cavity and is not easily absorbed by scattering. Because the size of the sphere is similar to the wavelength of light, the photons resonate in the cavity, which can form a simple microlaser with low threshold. The composition of the resonant cavity extends from various silicon-based materials or crystals to semiconductors and polymers, and can have a variety of cavity modes, such as spherical, microdisk, double-column ring, and so on. The advantages of small size, diverse structures, and simple fabrication make whispering gallery-mode microcavities attract more and more researchers.

Compared with the traditional Fabry-Perot cavity, the size of the whispering gallery cavity is smaller and easier to integrate. Due to its extremely high quality factor, and the size can range from hundreds of nanometers to 500μm, different refractive indices The microcavity has different characteristics from the absorption rate, and has very good application prospects in the fields of optical sensing, filter delay, nonlinear optics and biological imaging. For example, when the size of the optical cavity is in the micrometer level, the output change will be observed by a small perturbation, and the whispering gallery sphere can be used as an extremely sensitive optical micro-motion sensing element. The coupling system based on the waveguide and the microcavity can couple the signal consistent with the resonance wavelength of the cavity, so the whispering gallery ball can act as a filter. In recent years, the whispering gallery mode microcavity has emerged in the field of biophotonics. In 2002, F. Vollmer et al. realized the detection of proteins by using the microsphere resonant cavity. detection. In 2011, Seok HyunYun of Harvard University took the lead in realizing the single-cell biolaser, and then some researchers combined the concept of echoing gallery with cells, and found that cells can engulf echo-gallery spheres of the same size as themselves, and can survive and metabolize normally for about a week. until programmed apoptosis. Through the feedback light of the cell-whispering gallery, various optical data can be obtained and analyzed. It can be seen that the whispering gallery cavity has great potential in the field of optical biophotonics, and future research work on this will focus more on the design of integrated microplatforms.

与偶极子之间的距离

满足公式

,其中系数

由偶极子本身的光谱特 性决定，

指FRET效率达到50%时偶极子之间的距离。现在，FRET已经是为数不多的能够实 现纳米级距离检测的几个技术手段之一，当然这也有赖于不断发展的新型荧光探针染料和 日益优化的算法。The FRET effect refers to the phenomenon that excitation energy is conducted from the donor molecule (D) to the acceptor molecule (A) when the distance is 1–10 nm. For a given donor-acceptor pair, the energy lost by the donor is the energy gained by the acceptor. FRET efficiency

distance from the dipole

satisfy the formula

, where the coefficient

Determined by the spectral properties of the dipole itself,

Refers to the distance between dipoles when the FRET efficiency reaches 50%. Now, FRET is one of the few technical means that can realize nanoscale distance detection, which of course also depends on the continuous development of new fluorescent probe dyes and increasingly optimized algorithms.

SUMMARY OF THE INVENTION

The invention utilizes the fluorescent-doped micro resonator cavity and realizes the detection of trace substances outside the resonator cavity through the FRET principle. In the presence of the substance to be detected, the laser generated by pumping the fluorescence-doped micro-resonator with a light source will excite another fluorescent substance (fluorescence acceptor) outside the microsphere through the FRET effect, causing it to generate laser output. By adjusting the concentration of the fluorescent dopant, two laser beams can be radiated simultaneously. The spectrum of the two beams of light will shift with the change of the concentration of the object to be tested, so as to realize the sensing detection of the object to be tested.

The 473nm laser light source was used to excite the dye Dragon Green (DG) doped whispering gallery balls, and the balls were excited to emit fluorescence peaks at 500-600nm wavelength. A cluster of laser peaks. We can use the FRET effect to transfer the energy in the sphere to the outside of the sphere, continue to excite the liquid with the fluorescent dye dissolved outside the sphere, and generate a new fluorescence peak in the adjacent long wavelength band, or after reaching the threshold value. A cluster of laser peaks between 540 and 570 nm, experiments have verified the above invention. Theory can predict similar "spectral shift" effects in other bands.

The main purpose of the present invention is to construct a microcavity-based optical system and method for precise biological sensing. The system includes a laser source module (1), a resonant cavity module (2), an optical collimation module (3) and a signal receiving module (4), wherein the laser source module (1) is used to provide a laser source, including a laser source ( 11), a continuous attenuation sheet (12); the optical collimation module (3) is used to couple the laser source to the resonator module (2), and collect the signal light generated by the resonator module (2) into the spectrometer, including two A first microscope objective (31), a second microscope objective (32), a dichroic mirror (33), a convex lens (34) and a camera (35) with overlapping focal lengths; the resonator module (2) is used to generate light Resonance and FRET effects, including a microcavity capable of generating optical resonance, a fluorescent acceptor substance capable of generating FRET effects around the microcavity, and a three-dimensional adjustment frame; the signal receiving module (4) is used for receiving, analyzing, and storing signal light, including filtering plate (41), convex lens (42), spectrometer (43); the output laser light of the laser source (11) sequentially passes through the continuous attenuation plate (12), the dichroic mirror (33), the first microscope objective lens (31), the resonance The cavity module (2) generates signal light, the signal light is divided into two parts, and a part of the signal light sequentially passes through the second microscope objective lens (32), the filter (41), the convex lens (42), and finally enters the spectrometer (43) for reception , analysis, and storage, and another part of the signal light passes through the first microscope objective lens (31), dichroic mirror (33), convex lens (34) and camera (35) in sequence to observe the coupling effect between the laser and the resonator in real time. Based on the images observed in real time, adjust the position of the microcavity with a three-dimensional adjustment frame to achieve the best coupling.

The two first microscope objective lenses ( 31 ) and the second microscope objective lenses ( 32 ) with overlapping focal lengths can also be replaced by two convex lenses, or one microscope objective lens and one convex lens.

The camera (35) can be a CCD or CMOS image sensor, or a whiteboard.

The material of the microcavity can be quartz, doped quartz, polymer or a material capable of forming optical resonance.

The microcavity can be in the form of microspheres, microdisks, microrings, microtubes, and micropolygons, and its geometric radius should be larger than the single wavelength size of the excitation light source (1) and smaller than 1 mm.

Fluorescent substances can be added to the microcavity, or not added.

The extra-cavity fluorescent acceptor material can be in a gaseous state, a liquid state or a solid state, and its refractive index is smaller than that in the resonant cavity; the extra-cavity fluorescent acceptor material can contain a variety of fluorescent materials for multiple FRET effect.

The resonant cavity module (2) can be transformed into a microfluidic structure.

The resonance frequency of the excitation light source module (1) is the same as the intrinsic resonance frequency of the resonant cavity module (2).

A laser detection method based on a resonant cavity and FRET effect, wherein a laser source module (1) generates laser light, couples the laser source to the resonator cavity module (2) through an optical collimation module (3), excites a microcavity to generate resonance, and then excites The fluorescent acceptor material around the microcavity produces the FRET effect and generates signal light;

If there is no specific difference between the obtained signal light and the standard signal light, it means that the molecule to be tested does not exist;

If there is a specific difference between the obtained signal light and the standard signal light, it indicates that the molecule to be tested exists;

Wherein, the standard signal light is the signal light generated by the FRET effect when the medium around the microcavity contains only the fluorescent acceptor substance preset by the system.

beneficial effect

Compared with the prior art, the present invention has the following advantages:

1. Traditional methods for detecting biological enzymes generally need to destroy the normal physiological environment of cells. For example, for the observation of a certain enzyme activity in cells, it is usually necessary to first disrupt the cells and then add the reaction substrate. By observing the concentration of the substrate change to reflect the catalytic effect of the enzyme. If the invention is adopted, the optical mode of the whispering gallery is combined with the FRET effect, and the protein molecule activity can be monitored under the condition of normal cell growth and metabolism, and the physiological characteristics of the enzyme that are closer to the natural state of the cells can be obtained by analyzing the spectral characteristics.

2. The core structure of the present invention is in micron size, and the material used is plastic polymer or quartz glass with stable performance, which can be used repeatedly. Combined with the current mature light source and micro-nano lens design technology, the system can be more easily miniaturized or even made into a system-on-chip.

3. Single-stranded DNA molecules and immune protein molecules generally do not exceed the nanometer size, which is beyond the scope that can be observed by optical microscopes. Most of the previous methods for DNA hybridization and immunoassay are isotope labeling or molecular imprinting and re-imaging. Our microcavity sensing system can detect molecular binding or conformational changes in the range of several micrometers. and immunoassays provide new approaches.

4. Combining the whispering gallery mode with FRET has unique advantages. Although there have been many reports on detection using the whispering gallery mode, the applicable detection systems are often relatively simple. The combination of the whispering gallery mode and FRET, and the use of appropriate fluorescent dyes to label the detected molecules, can realize the specific detection of target molecules in complex systems.

In general, laser sensing detection systems based on the whispering gallery mode and FRET principle have great potential and applications in biomolecular dynamics, immunoassays, nucleic acid detection, and protein-protein interactions. The research on the metabolic process of cell life has an extremely important position in biology, and the present invention provides a new way to observe this process in real time.

Description of drawings:

Figure 1 is a schematic diagram of the laser sensing detection system based on the whispering gallery mode and the FRET principle in Example 1

Figure 2 is a schematic diagram of the laser sensing detection system based on the whispering gallery mode and the FRET principle in Example 2

Figure 3 is a schematic diagram of the laser sensing detection system based on the whispering gallery mode and the FRET principle in Example 3

Figure 4 is a schematic diagram of the laser sensing detection system based on the whispering gallery mode and the FRET principle in Example 4

Figure 5 is a schematic diagram of the laser sensing detection system based on the whispering gallery mode and the FRET principle in Example 5

Figure 6 is a schematic diagram of the appearance of the resonator sample in the laser sensing detection system based on the whispering gallery mode and FRET principle

Figure 7 is a detailed schematic diagram of the FRET effect of the laser sensing detection system based on the whispering gallery mode and the FRET principle

Figure 8 is a microscopic schematic diagram of the FRET effect of the laser sensing detection system based on the whispering gallery mode and the FRET principle

Figures 9a-d are schematic diagrams of the signal spectrum of the laser sensing detection system based on the whispering gallery mode and the FRET principle

Among them, laser source (11), continuous attenuation plate (12), silver mirror (13), cavity sample (21), dichroic mirror (33), convex lens (34), filter (41), convex lens (42) ), spectrometer (43)

Detailed ways

The present invention will be further described below in conjunction with the examples, but the present invention is not limited to the following examples.

Implementation Case 1

As shown in Figure 1, the resonant cavity sample (21) uses polymer beads (Bangs Laboratories, FS07F) doped with dragon green dye with an average diameter of about 15 μm. ) solution, mix well, drop a drop of reagent on the cover glass, cover it with another cover glass, and apply nail polish around it to seal it (Figure 6). After the sealed part is dried, the above-mentioned sample is fixed on the glass slide, so that the sample to be tested can be made into a precision three-dimensional adjustment frame.

A continuous attenuator (12) is installed behind the laser source (11), and after being reflected by a silver mirror (13) at 90°, the beam is refracted by a 500nm low-pass high-reflection dichroic mirror (33) by 90°, so that the The light beam is focused and collimated by a 40× first microscope objective lens (31), and is coupled to a resonant cavity sample (21) mounted on a precision three-dimensional adjustment frame. The other side of the dichroic mirror (33) is equipped with a camera (35). The rear end of the resonator sample (21) is equipped with a 60× microscope objective (32), a 500nm long-pass filter (41) and a convex lens (42) with a focal length of f=18.40mm, which enters the probe of the spectrometer (43). The experimental device adopts the method of forward detection, and installs a convex lens (34) with a focal length of f=100mm and a camera (35) on the other side of the dichroic mirror (33) to observe the position of the ball in real time (Fig. 7).

The sample is placed vertically in the optical path between the two microscope objectives during detection. Adjust the continuous attenuator (12) to adjust the light intensity of the light source to a lower intensity, adjust the position of the sample continuously, and observe whether the laser source (11) with a wavelength of 473 nm is coupled to the small ball through the CCD camera (35) at the other end, Until the light output from the pellet is the strongest on the spectrometer (Figure 8). Then, the same ball is injected with increasing laser coupling. Because the ball has a strong whispering gallery effect, when the 473nm laser is coupled into the ball, the fluorescent substance dragon green in the ball will be excited to emit fluorescence in the 500-600nm band. When the intensity of the 473 nm laser source reaches the threshold value of the sphere, a cluster of laser peaks will be emitted (Fig. 9a). In a suitable concentration of fluorescent substance solution RhB, the FRET effect will be generated, and this cluster of laser peaks will be transferred to another wavelength band f ₁ (transfer of energy to longer wavelength bands) (Fig. 9b). On the above basis, the molecules to be tested are added around the spheres. When the distance between the molecules to be tested and the spheres is less than 10 nm, the original FRET effect will be disturbed and a new FRET spectrum f ₂ will be formed (Fig. 9c). Due to the different perturbations caused by different molecules to the sensing system, the generated spectral characteristics are also different. Comparing the spectral changes before and after the perturbation can specifically detect the presence or absence and movement of the molecule.

Implementation case 2

As shown in Figure 2, the silver mirror (13) in Example 1 is removed, and the light exit angle of the laser source (11) is adjusted to a direction perpendicular to the optical collimation system. Other execution methods are the same as those in Embodiment 1, and the same detection effect can be achieved.

Implementation Case 3

As shown in Fig. 3, the silver mirror (13) in Example 1 is removed, and the light exit angle of the laser source (11) is adjusted to a direction perpendicular to the optical collimation system. The 40× first microscope objective lens ( 31 ) and the 60× second microscope objective lens ( 32 ) are replaced with convex lenses, and other implementation methods are the same as those in Embodiment 1, and the same detection effect can be achieved.

Implementation Case 4

As shown in Fig. 3, the silver mirror (13) in Example 1 is removed, and the light exit angle of the laser source (11) is adjusted to a direction perpendicular to the optical collimation system. The 40x first microscope objective lens (31) and the 60x second microscope objective lens (32) are replaced by convex lenses, and the camera (35) is replaced by a whiteboard. Other implementation methods are the same as those in Example 1, and the same detection effect can be achieved.

Implementation Case 5

As shown in Figure 5, the microfluidic channel (21) can precisely control the flow of the solution. This sensing system uses polymer beads (Bangs Laboratories, FS07F) doped with dragon green dye with an average diameter of about 15 μm. First, take an appropriate amount of the original solution of the beads and add it to the prepared Rhodamine (RhB) solution and mix well. The mixed solution is then injected into the microfluidic channel (21).

A continuous attenuator (12) is installed after the laser source (11), and the light beam is refracted by a 500nm low-pass high-reverse dichroic mirror (33) by 90°, so that the light beam passes through the 40× first microscope objective lens (31). Focused and collimated, coupled to a microfluidic channel (21) mounted on a precision 3D mount. The other side of the dichroic mirror (33) is equipped with a camera (35). The rear end of the resonator sample (21) is equipped with a 60× microscope objective (32), a 500nm long-pass filter (41) and a convex lens (42) with a focal length of f=18.40mm, which enters the probe (43) of the spectrometer. The experimental device adopts the method of forward detection, and installs a convex lens (34) with a focal length of f=100mm (34) and a camera (35) for imaging on the other side of the dichroic mirror (33) to observe the spherical coupling effect in real time.

The FRET effect spectrum f1 produced by the system is first recorded. On the basis of the above, the molecules to be tested are added to the microfluidic channel. When the distance between the molecules to be tested is less than 10 nm from the sphere, the original FRET effect will be disturbed and a new FRET spectrum f2 will be formed. Comparing the difference between f1 and f2 enables specific molecular detection.

Implementation Case 6

As shown in FIG. 1 , the resonant cavity module (2) in Example 1 is slightly modified, and other equipment is the same as that in Example 1, which can monitor the physiological activities of cells. The specific transformation is as follows: if the cells are co-cultured with the pellets for a period of time, the pellets will be phagocytosed by the cells, and the cells will survive and metabolize normally. Take an appropriate amount of cells that phagocytose the beads and add it to the prepared rhodamine (RhB) solution, mix well, drop a drop of reagent on the cover glass, cover it with another cover glass, and apply nail polish around it to seal deal with. After the sealed part is dried, the above-mentioned sample is fixed on the glass slide, so that a resonant cavity sample (21) that can be placed on a precise three-dimensional adjustment frame is produced.

During detection, the resonator sample (21) is placed vertically on the optical path between the two microscope objective lenses. Adjust the continuous attenuator (12) to adjust the light intensity of the light source to a lower intensity, constantly adjust the position of the sample, and observe through the CCD camera (35) at the other end whether the laser source (11) with a wavelength of 473 nm is coupled to the small cell in the cell. on the sphere until the light output from the sphere appears strongest on the spectrometer. Then, the same ball is injected with increasing laser coupling. Because the ball has a strong whispering gallery effect, when the 473nm laser is coupled into the ball, the fluorescent substance dragon green in the ball will be excited to emit fluorescence in the 500-600nm band. When the intensity of the 473 nm laser source reaches the threshold of the sphere, a cluster of laser peaks will be emitted. The fluorescent receptor RhB also exists in the cytoplasm due to absorption, so the intracellular globules will produce the FRET effect, which will transfer this cluster of laser peaks to another waveband f1 (Fig. 9b). On the basis of the above, when the cell is metabolized, the molecules in the cytoplasm move continuously around the globule. When the distance between the cytoplasm and the globule is less than 10 nm, it will disturb the original FRET effect and form a new FRET spectrum f2 (Fig. 9c). ). Due to the different perturbations caused by different molecules to the sensing system, the generated spectral characteristics are also different. Comparing the spectral changes before and after perturbation can specifically detect the physiological activities of cells.

Implementation Case 7

As shown in FIG. 5 , the resonant cavity module (2) in Example 5 is slightly modified, and other equipment is the same as that in Example 5, which can monitor the DNA single-strand hybridization activity. The specific modification is as follows: the single-stranded DNA is fixed to the surface of the whistle-blower ball by the method of thiolation and covalent bonding on the surface of the whispering gallery ball. With the help of imaging tools such as super-resolution confocal microscopy, the amount of modified DNA can be determined, and conditions such as reaction concentration and time can be controlled to realize the modification of one or several chains on the beads. After immobilizing DNA single strands on the outside of the whispering-gallery globules, they were placed in a microfluidic channel. The laser light source and the coupling position pair are adjusted for excitation, and the fluid solution contains a certain concentration of substances such as quantum dots that can produce the FRET effect. After the system is stable, measure and record the spectral information. When the solution containing the DNA single strand to be tested is slowly passed in, the anchor strand will react with the anchor strand on the bead. Since double-stranded DNA is more compact and firmer in structure than single-stranded DNA, the hybridized DNA will change in shape and direction outside the sphere, which will interfere with the microenvironment outside the sphere. Since the conditions for generating FRET between the sphere and the liquid environment outside the sphere are very sensitive, the interference caused by the change of the external environment can be directly displayed on the change of the spectrum. Using this detection method, we can realize the detection of nucleic acid molecules with specific sequences.

Claims (10)

1. A laser detection system based on resonant cavity and FRET effect is characterized in that: the system comprises a laser source module (1), a resonant cavity module (2), an optical collimation module (3) and a signal receiving module (4), wherein the laser source module (1) is used for providing a laser source and comprises a laser source (11) and a continuous attenuation sheet (12); the optical collimation module (3) is used for coupling the laser source to the resonant cavity module (2) and collecting signal light generated by the resonant cavity module (2) into the spectrometer, and comprises a first micro objective (31), a second micro objective (32), a dichroic mirror (33), a convex lens (34) and a camera (35), wherein the two focal lengths of the first micro objective and the second micro objective are coincident; the resonant cavity module (2) is used for generating optical resonance and FRET effect, and comprises a microcavity capable of generating optical resonance, a fluorescent acceptor substance capable of generating FRET effect around the microcavity, and a three-dimensional adjusting frame;

the microcavity is formed by polymer microspheres doped with fluorescent substances and acceptor fluorescent substances capable of generating FRET effect, a proper amount of fluorescent microspheres are added into another prepared fluorescent substance solution, a drop of reagent is dripped on a cover glass after the fluorescent microspheres are fully and uniformly mixed, the other cover glass is covered on the cover glass, the periphery of the cover glass is coated with nail polish for sealing treatment, and a sample is fixed on a glass slide after a sealed part is dried, so that the microcavity is manufactured;

The signal receiving module (4) is used for receiving, analyzing and storing signal light and comprises a filter plate (41), an aspheric lens (42) and a spectrometer (43); the output laser of laser source (11) has produced signal light through continuous attenuation piece (12) in proper order, dichroic mirror (33), first micro objective (31), resonant cavity module (2), signal light divide into two parts, partly signal light passes through second micro objective (32) in proper order, filter (41), aspheric lens (42), it receives to get into spectrum appearance (43) at last, analysis, storage, another part signal light passes through first micro objective (31) in proper order, dichroic mirror (33), convex lens (34) and camera (35), a coupling effect for real-time observation laser and resonant cavity, according to the image of real-time observation, realize best coupling with three-dimensional adjustment frame adjustment microcavity position.

2. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the two first micro objective lenses (31) and the second micro objective lenses (32) with coincident focal lengths are replaced by two convex lenses, or one micro objective lens and one convex lens.

3. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the camera (35) is a CCD or CMOS image sensor or a white board.

4. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the microcavity material is a material capable of forming optical resonance.

5. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the micro-cavity is in the form of a microsphere, a micro-disk, a micro-ring, a micro-tube and a micro-polygon, and the geometric radius of the micro-cavity is larger than the single wavelength of the laser source (11) and smaller than 1 mm.

6. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the micro-cavity is added with or without fluorescent substances.

7. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the fluorescent acceptor substance outside the cavity is in a gas state, a liquid state or a solid state, and the refractive index of the fluorescent acceptor substance is smaller than that in the resonant cavity; the fluorescent acceptor substance outside the cavity contains a plurality of fluorescent substances for the multiple FRET effect to occur.

8. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the resonant cavity module (2) is transformed into a microfluidic structure.

9. A laser detection system based on resonator and FRET effects as claimed in claim 1, wherein: the resonant frequency of the laser source module (1) is the same as the intrinsic resonant frequency of the resonant cavity module (2).

10. A method of detection using a laser system based on the resonator and FRET effects as claimed in any of claims 1-9, wherein: the laser source module (1) generates laser, the laser is coupled to the resonant cavity module (2) through the optical collimation module (3), the microcavity is excited to generate resonance, and then fluorescent acceptor substances around the microcavity are excited to generate FRET effect to generate signal light;

if the obtained signal light has no specificity difference with the standard signal light, the molecule to be detected does not exist;

if the obtained signal light has specificity difference with the standard signal light, the existence of the molecule to be detected is indicated;

wherein, the standard signal light is the signal light generated by FRET effect when the medium around the microcavity only contains the fluorescence acceptor substance preset by the system.

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