CN112505003A - Porous microstructure optical fiber array type single virus sensing system based on microscopic imaging - Google Patents
Porous microstructure optical fiber array type single virus sensing system based on microscopic imaging Download PDFInfo
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- CN112505003A CN112505003A CN202011133902.4A CN202011133902A CN112505003A CN 112505003 A CN112505003 A CN 112505003A CN 202011133902 A CN202011133902 A CN 202011133902A CN 112505003 A CN112505003 A CN 112505003A
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- 238000003384 imaging method Methods 0.000 title claims abstract description 40
- 239000013307 optical fiber Substances 0.000 title claims abstract description 36
- 241000700605 Viruses Species 0.000 title claims abstract description 27
- 239000000835 fiber Substances 0.000 claims abstract description 26
- 238000012545 processing Methods 0.000 claims abstract description 4
- 238000001228 spectrum Methods 0.000 claims abstract description 4
- 239000011521 glass Substances 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 7
- 239000004038 photonic crystal Substances 0.000 claims description 5
- 238000001179 sorption measurement Methods 0.000 claims description 5
- 108091023037 Aptamer Proteins 0.000 claims description 4
- 238000000799 fluorescence microscopy Methods 0.000 claims description 4
- 239000010453 quartz Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 230000003595 spectral effect Effects 0.000 claims description 2
- 239000012491 analyte Substances 0.000 claims 1
- 239000002094 self assembled monolayer Substances 0.000 claims 1
- 239000013545 self-assembled monolayer Substances 0.000 claims 1
- 108020004707 nucleic acids Proteins 0.000 abstract description 9
- 102000039446 nucleic acids Human genes 0.000 abstract description 9
- 150000007523 nucleic acids Chemical class 0.000 abstract description 9
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 13
- 239000011148 porous material Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 230000004907 flux Effects 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000013076 target substance Substances 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000000701 chemical imaging Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000010460 detection of virus Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
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- 239000007788 liquid Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/144—Imaging characterised by its optical setup
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1486—Counting the particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6484—Optical fibres
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention discloses a porous microstructure optical fiber array type single virus sensing system based on microscopic imaging, which comprises: the device comprises a porous microstructure optical fiber, a sample area, a microscopic imaging unit and a computer. The porous microstructure fiber is used as an array for virus sample circulation to circulate a virus sample to the sample area, the microscopic imaging unit is used for carrying out microscopic imaging on each air hole on the end face of the porous microstructure fiber to obtain a spectrogram, and the computer is connected with the microscopic imaging unit and used for processing and analyzing the spectrogram obtained by the microscopic imaging unit and recording the quantity of the air holes with characteristic spectrums. The invention can accurately and quantitatively measure the trace virus nucleic acid with ultralow abundance in the sample without nucleic acid amplification, and simultaneously realizes ultrahigh sensitivity, strong specificity and high-flux measurement.
Description
Technical Field
The invention relates to the field of medical sensing, in particular to a porous microstructure optical fiber array type single virus sensing system based on microscopic imaging.
Background
Viral infectious diseases pose a threat to the health and even life of people worldwide. To date, researchers have conducted a great deal of research on how to screen viruses in time, and have developed numerous products. Timely screening of viruses is an important prevention and control means for reducing the transmission coefficient, which is not only beneficial to reducing the death risk of infected people, but also beneficial to timely controlling the infection sources and reducing the infection risk of closely contacted people.
The methods currently used for virus detection are mainly nucleic acid detection and immunological detection. The nucleic acid detection method mainly comprises various semi-quantitative or quantitative detection methods based on PCR amplification.
The nucleic acid detection method is an important basis for patients to determine virus infection, but the method is not only easily influenced by sample quality, virus infection parts, detection environment and the like, but also has the problem of insufficient detection flux. Optical fiber-based biosensors have been developed rapidly in the future, and optical fibers have many advantages such as anti-electromagnetic interference, large bandwidth, low cost, and good biocompatibility.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a micro-imaging-based porous microstructure optical fiber array type single virus sensing system, which can realize the accurate measurement of a trace target in a complex environment, can be applied to multiple fields of nucleic acid detection, protein detection and the like, and is suitable for the detection of viruses such as coronavirus detection, HIV (human immunodeficiency Virus) (P24 protein antigen-antibody) and the like.
A porous microstructure optical fiber array type single virus sensing system based on microscopic imaging comprises a porous microstructure optical fiber, a sample area, a microscopic imaging unit and a computer; the porous microstructure fiber is used as an array for virus sample circulation to circulate a virus sample to the sample area, the microscopic imaging unit is used for carrying out microscopic imaging on each air hole on the end face of the porous microstructure fiber to obtain a spectrogram, and the computer is connected with the microscopic imaging unit and is used for processing and analyzing spectral information obtained by the microscopic imaging unit and analyzing the quantity of the air holes with characteristic spectra; the size of the air holes of the porous microstructure optical fiber is adapted to the object to be measured in the sample, and each air hole can contain at most one particle of the object to be measured.
The porous microstructure fiber comprises a photonic crystal fiber and a photonic band gap fiber.
The surface of the porous microstructure optical fiber adopts a self-assembled monomolecular film structure to inhibit nonspecific adsorption.
The surface of the porous microstructure optical fiber is functionally modified and is connected with an aptamer or an antibody of an object to be detected.
The sample area is a quartz plate or a glass slide.
The microscopic imaging unit comprises a high-resolution fluorescence imaging system and a hyperspectral meter.
Compared with the prior art, the porous microstructure optical fiber array type single virus sensing system based on microscopic imaging can perform advanced optical microscopic imaging such as hyperspectral imaging and counting on each air hole on the end face of the optical fiber by using the inherent air hole of the porous microstructure optical fiber as an array for sample circulation under the condition of not performing nucleic acid amplification, accurately measure trace viruses with ultralow abundance in a sample, and inhibit nonspecific adsorption by using a self-assembled monomolecular membrane structure, so that the device can work in a complex body fluid environment, and ultrahigh sensitivity and strong specificity are realized at the same time. By using a single molecule counting method, the target substance is directly detected on the premise of not amplifying a sample or accumulating signals. The flux of detection can be further improved by adjusting the size and the period of the air holes of the porous microstructure optical fiber.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
Fig. 2 is a schematic structural diagram of an embodiment of the present invention.
FIG. 3 is an end-view SEM of a porous microstructured optical fiber used in an embodiment of the present invention.
FIG. 4 is a schematic representation of a functional biological surface of an aptamer or antibody protein with target capture capability.
The device comprises a porous microstructure optical fiber 1, a sample area 2, a liquid drop 3, a high-magnification high-NA oil lens 4, a light source 5, a lens 6, a dichroic mirror 7, a filter 8, a reflector 9, a receiver 10, a microscopic imaging unit 11 and a computer 12.
Detailed Description
The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings, and it is apparent that the embodiments are only a part of the embodiments of the present invention, and not all embodiments.
It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or intervening elements may also be present.
Unless defined otherwise, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Please refer to fig. 1, which is a schematic structural diagram of the present invention. The invention provides a porous microstructure optical fiber array type single virus sensing system based on microscopic imaging, which comprises: porous microstructure fiber 1, sample area 2, micro-imaging unit 11, computer 12.
The porous microstructure optical fiber 1 is used as an array for virus sample circulation to circulate a virus sample to the sample area, the microscopic imaging unit 11 performs microscopic imaging on each air hole on the end face of the porous microstructure optical fiber to obtain a spectrogram, and the computer 12 is connected with the microscopic imaging unit 11 and is used for processing and analyzing the spectrogram obtained by the microscopic imaging unit and recording the quantity of the air holes with characteristic spectrums.
In an alternative embodiment, the porous microstructured optical fiber is a porous optical fiber, including photonic crystal fibers and photonic band gap fibers. Such as photonic crystal fibers, may be used.
In an alternative embodiment, the sample region connecting the micro-imaging unit and the porous micro-structured fiber may be quartz, a quartz plate made of glass, or a glass slide.
In an alternative embodiment, the microscopic imaging unit comprises an optical microscopic imaging unit such as a high resolution fluorescence imaging system, a high spectrometer, or the like. Such as high resolution fluorescence imaging systems.
Examples
As shown in FIG. 2, the high density uniform air holes inherent to the porous microstructured optical fiber 1 are used as an array for sample flow, the porous microstructured optical fiber 1 contacts the sample (solution containing virus), and the sample can reach the sample region 2 (in this case, a glass slide) along the air holes by capillary action. And the microscopic imaging unit 11 is used for carrying out single-particle optical microscopic imaging on each air hole on the end face of the porous microstructure optical fiber through a glass slide. In this example, microscopic imaging unit 11 is based on the oil mirror, including high NA oil mirror 4 of high magnification, light source 5, lens 6, dichroic mirror 7, filter 8, speculum 9, receiver 10, the light that light source 5 jetted out passes through lens 6 in proper order, dichroic mirror 7, the high NA oil mirror 4 of high magnification, reach the terminal surface of porous microstructure optic fibre 1 behind the sample district 2, reach receiver 10 behind sample district 2, the high NA oil mirror 4 of high magnification, filter 8, speculum 9 in proper order after the light is reflected by the sample on the terminal surface. The light information received by the receiver 10 may be transmitted to a computer 12 for further analysis.
A solution containing aminosilane absolute ethyl alcohol solution (1-2%) is absorbed into the interior of the porous microstructure optical fiber by using a siphon or capillary effect. The solvent covers the glass portion to form an aminated glass surface on the glass surface. Then adding a polyethylene glycol heterobifunctional coupling agent (polyethylene glycol crosslinker) PBS solution containing succinimide (NHS) end groups. The addition of the coupling agent forms a functional biological surface with the capability of targeted capture on the inner surface of the porous microstructure optical fiber, which has the capability of nonspecific adsorption resistance and can be used for further functional modification, such as connection of an aptamer or an antibody of a virus, as shown in fig. 4. Each porous microstructure fiber 1 has many pores, each of which is a micro reaction chamber, a sample is distributed into each pore when contacting the porous microstructure fiber 1, because the porous microstructure fiber has several or tens of extending empty pores along the length direction of the fiber, the size and the interval of the pores filled in the fiber can be adjusted when the fiber is manufactured, single particles with the sample in each pore are controlled by selecting the porous microstructure fiber with proper pore size, and when the single particles are an object to be detected (such as a virus), the single particles are captured by an antibody or a nucleic acid aptamer on the surface. The single particle is other substances in serum, such as serum protein, and will not be adsorbed on the surface of the optical fiber due to the presence of the non-specific adsorption layer. On this basis, the secondary antibody labeled with a single-molecule dye (such as alexa647 or TAMRA) is continuously introduced, so that the target substance can be accurately labeled. That is, the stomata containing the virus-specific sample will emit a fluorescence signal and show a positive result, while none will emit no fluorescence and show a negative result. When the concentration of the object to be measured is extremely low (for example, the number of the object to be measured is less than that of the holes of the photonic crystal optical fiber), each air hole can only pass through one object to be measured at most. Since the target is labeled with a secondary antibody that is attached by a fluorescent molecule that can be used for single molecule imaging, direct observation can be performed using a system such as that of fig. 2. And analyzing the trace target substances in the sample by utilizing a molecular direct counting method and a computer.
The invention can accurately and quantitatively measure the trace virus nucleic acid with ultralow abundance in the sample without nucleic acid amplification, simultaneously realizes three characteristics of ultrahigh sensitivity, strong specificity and high flux, and realizes the hypersensitive detection of the sample with ultralow abundance (10 ^ -18 mol). The dynamic range of detection can be further improved by adjusting the size and the period of the air holes of the porous microstructure optical fiber.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Claims (6)
1. A porous microstructure optical fiber array type single virus sensing system based on microscopic imaging is characterized by comprising a porous microstructure optical fiber, a sample area, a microscopic imaging unit and a computer; the porous microstructure fiber is used as an array for virus sample circulation to circulate a virus sample to the sample area, the microscopic imaging unit is used for carrying out microscopic imaging on each air hole on the end face of the porous microstructure fiber to obtain a spectrogram, and the computer is connected with the microscopic imaging unit and is used for processing and analyzing spectral information obtained by the microscopic imaging unit and analyzing the quantity of the air holes with characteristic spectra; the size of the air holes of the porous microstructure optical fiber is adapted to the object to be measured in the sample, and each air hole can contain at most one particle of the object to be measured.
2. The system of claim 1, wherein the porous microstructured fiber comprises a photonic crystal fiber and a photonic band gap fiber.
3. The system of claim 1, wherein the surface of the porous microstructured optical fiber is configured as a self-assembled monolayer to inhibit non-specific adsorption.
4. The system of claim 1, wherein the surface of the porous microstructure fiber is functionally modified to connect an aptamer or an antibody of an analyte.
5. The system of claim 1, wherein the sample area is a quartz or glass slide.
6. The system of claim 1, wherein the microscopic imaging unit comprises a high resolution fluorescence imaging system, a hyperspectral meter.
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| CN202011133902.4A CN112505003B (en) | 2020-10-21 | Porous microstructure optical fiber array type single virus sensing system based on microscopic imaging |
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| CN202011133902.4A CN112505003B (en) | 2020-10-21 | Porous microstructure optical fiber array type single virus sensing system based on microscopic imaging |
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| CN112505003A true CN112505003A (en) | 2021-03-16 |
| CN112505003B CN112505003B (en) | 2024-07-05 |
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