CN116297377B - Single-molecule fluorescence detection chip, preparation method thereof and single-molecule fluorescence detector - Google Patents
Single-molecule fluorescence detection chip, preparation method thereof and single-molecule fluorescence detector Download PDFInfo
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
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- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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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
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a single-molecule fluorescence detection chip and a preparation method thereof, and a single-molecule fluorescence detector, wherein the single-molecule fluorescence detection chip comprises a chip substrate and a cover plate, and the chip substrate comprises a substrate, and a reflecting layer, a first low refractive index layer, a high refractive index layer and a second low refractive index layer which are sequentially laminated on the substrate; the upper surface of the chip substrate, which is close to the cover plate, is provided with two first grooves which are parallel to each other, two second grooves which extend forwards and outwards along each first groove respectively, a third groove which is connected with the end parts of the two second grooves, which are far away from the first grooves, and micro-channel grooves which are arranged at intervals with the third grooves and extend in the same direction; the cover plate seals the first groove, the second groove, the third groove and the micro flow channel groove. The invention realizes the segmentation of the high-flux sample volume by utilizing the micro-channel technology, and simultaneously amplifies the excitation light intensity and focuses the excitation light beam by utilizing the photon nano jet technology, thereby enhancing the signal-to-noise ratio of fluorescent signals.
Description
Technical Field
The invention relates to the technical field of single-molecule fluorescence detection, in particular to a single-molecule fluorescence detection chip, a preparation method thereof and a single-molecule fluorescence detector.
Background
The immunological detection utilizes the specific combination between antibody antigens to detect the concentration of specific proteins, is widely applied to diagnosis of various diseases, and is one of the most commonly used in vitro detection means at present. The most commonly used immunodetection techniques at present include chemiluminescence, enzyme-linked immunosorbent assay, co-immunoprecipitation, colloidal gold chromogenic methods and the like. Among them, the chemiluminescent method has the highest sensitivity and occupies the main share of the market. With the development of medicine and biology, the sensitivity of the chemiluminescence method cannot completely meet the requirements of some researches and applications, including rapid detection of acute cardiovascular disease markers, accurate rapid detection of major transfected disease antigens, early screening of cancers, early blood detection of neurodegenerative diseases and the like.
The detection limit of the single-molecule fluorescence detection technology is far beyond the commercial chemiluminescence immune detection technology, can be used for detecting and analyzing femtomolar biological molecules, and has wide application in the fields of basic life science research, novel disease marker development, accurate and rapid diagnosis, ultra-low abundance protein marker detection and the like. Single molecule fluorescent immunodetection is used as a next generation immunodetection technique that can achieve 100-to 1000-fold higher sensitivity than chemiluminescence using cleavage of analyte volumes and analysis of each cleavage unit.
The basic technical requirements of single-molecule fluorescence detection include: first, cleaving the sample volume using spatial confinement or selective acceptance such that there is substantially only one or less analyte molecules per small cell; secondly, analyzing whether each small unit contains the analyte molecules by using a hypersensitive optical detection means, and obtaining the total concentration of the analyte by using a statistical method.
Currently, there are two main single-molecule fluorescence detection technologies, one of which is developed by Quanterix corporation, and its implementation mode is: a chip with a micropore array is manufactured by utilizing a semiconductor process, a sample marked by an antigen is divided into micropores, and the luminous condition of each micropore is observed by utilizing an imaging system, so that the concentration of an analyte is obtained. Another developed by Singulex corporation, implemented as: the confocal microscopy principle is utilized to excite and observe the fluorescence intensity in the optical focal range, so that the aim of cutting the sample volume is fulfilled.
However, both of the above approaches require a long waiting time to collect enough optical signals to achieve an acceptable signal-to-noise ratio, making the measurement time much longer than that of chemiluminescence.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a single-molecule fluorescence detection chip, a preparation method thereof and a single-molecule fluorescence detector, which comprise a photon nano jet technology and a micro-channel technology, wherein the micro-channel technology is utilized to realize the segmentation of high flux and detection volume of a sample, and the photon nano jet technology is utilized to amplify the excitation light intensity and focus the excitation light beam, so that the signal-to-noise ratio of a single-molecule fluorescence detection signal is enhanced.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the single-molecule fluorescence detection chip comprises a chip substrate and a cover plate arranged on the chip substrate, wherein the chip substrate comprises a substrate, and a reflecting layer, a first low refractive index layer, a high refractive index layer and a second low refractive index layer which are sequentially laminated on the substrate, and the refractive indexes of the first low refractive index layer and the second low refractive index layer are respectively lower than that of the high refractive index layer;
the upper surface of the chip substrate, which is close to the cover plate, is provided with two first grooves which are parallel to each other, two second grooves which extend forwards and outwards along each first groove, a third groove which is connected with the end parts of the two second grooves, which are far away from the first grooves, and a micro-channel groove which is arranged at intervals with the third groove and extends in the same direction, wherein the micro-channel groove is used for allowing single molecules of a sample to flow through, and the depths of the first grooves, the second grooves, the third grooves and the micro-channel groove are respectively equal to the depth of the reflecting layer;
the structure between the two first grooves forms a straight waveguide core, the structure between the two second grooves and the third grooves forms a gradual change waveguide core, and the structure between the third grooves and the micro-channel grooves forms a light-gathering waveguide core;
the interface between the graded waveguide core and the third groove is a first arc-shaped interface protruding towards the direction of the third groove, and the interface between the concentrating waveguide core and the third groove is a second arc-shaped interface protruding towards the direction of the third groove;
the cover plate is connected with the second low-refractive-index layer, and seals the first groove, the second groove, the third groove and the micro-channel groove;
the irradiation source beam is transmitted to the gradual change waveguide core through the straight waveguide core to be diverged, then is collimated and emitted to the condensing waveguide core through the first arc-shaped interface, and is converged to the micro-channel groove through the second arc-shaped interface to form an excitation beam, and the excitation beam is used for carrying out fluorescence excitation on sample single molecules flowing in the micro-channel groove to generate fluorescent photons.
The invention also provides a preparation method of the single-molecule fluorescence detection chip, which comprises the following steps:
providing a substrate, and sequentially forming the reflecting layer, the first low refractive index layer, the high refractive index layer and the second low refractive index layer on the substrate to obtain an initial chip substrate;
etching the initial chip substrate to form the first groove, the second groove, the third groove and the micro-channel groove, so as to obtain the chip substrate;
and providing a cover plate, and sealing the first groove, the second groove, the third groove and the micro-channel groove by using the cover plate.
The invention also provides a single-molecule fluorescence detector comprising the single-molecule fluorescence detection chip.
The implementation of the embodiment of the invention has the following beneficial effects:
according to the embodiment of the invention, the low-high-low refractive index medium layers of the first low refractive index layer, the high refractive index layer and the second low refractive index layer are arranged, so that the propagation of the irradiation source beam in the high refractive index layer is limited by utilizing the principle that light rays are totally reflected at the interface of the high-low refractive index medium layers; the first grooves and the second grooves are arranged, the medium in the grooves is gas, the refractive index of the gas is lowest and is lower than that of the high refractive index layer, so that the straight waveguide core and the first grooves on two sides of the straight waveguide core and the second grooves on two sides of the straight waveguide core respectively form low-high-low refractive index medium layers, and the irradiation source light beam is limited to be transmitted forwards in the straight waveguide core and the gradual waveguide core; the width of the irradiation source beam is enlarged by arranging the gradual change waveguide core for collimation and focusing; through setting up the third slot, make the gas in gradual change waveguide core and the third slot and make spotlight waveguide core and the gas in the third slot constitute high, low refractive index dielectric layer respectively, through setting up first arc interface, make divergent light turn into collimation light, and through setting up the second arc interface, make collimation light assemble, thereby obtain comparing the extremely reduced and extremely enlarged excitation light beam of shining to the microchannel slot of light beam irradiation area of irradiation source, this excitation light beam can carry out fluorescence excitation to the sample single molecule that flows through the microchannel slot, thereby can obtain the fluorescence signal of the single molecule that the light intensity is stronger and more accurate.
The straight waveguide core, the gradual change waveguide core, the third groove and the light-gathering waveguide core form a photon nanometer jet structure, a photon nanometer jet effect is formed in the micro-channel groove by utilizing the high effective refractive index of the waveguide core, optical focusing smaller than 1 micron is obtained, the energy density of an excitation light beam is improved to 5-10 times of the energy density of an irradiation source light beam, the excitation light intensity is amplified, and the signal-to-noise ratio of a single-molecule detection fluorescent signal is improved.
According to the invention, the photon nano jet technology and the micro-channel technology are combined, the photon nano jet effect is utilized to focus the excitation light beam, the excited area of the sample is reduced, stray light caused by scattering is reduced, the energy density of the excitation light beam is improved, the excitation light intensity is amplified, and the signal-to-noise ratio of single molecule detection fluorescence signals is improved; the invention can realize high-flux and high-accuracy single-molecule fluorescence detection on the sample by utilizing the micro-channel technology to carry out high-flux sample volume segmentation.
The photonic nanometer jet flow structure and the micro-channel are integrated on one chip smaller than 1 square centimeter, so that the function integration is realized. Meanwhile, the small volume and the integrated chip can bring system stability, and can be used for miniaturized portable instruments.
In addition, the chip of the invention is simple to prepare, can be reused, and can obviously reduce the cost of the chip.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Wherein:
FIG. 1 is a schematic diagram showing an exploded structure of a single-molecule fluorescence detection chip according to an embodiment of the present invention.
Fig. 2 is a schematic overall structure of the structure shown in fig. 1.
Fig. 3 is a schematic top view of the chip substrate of fig. 1.
Fig. 4 is an enlarged schematic view of the area indicated by the broken line frame a in fig. 3.
Fig. 5 is a schematic cross-sectional structure along a line B in fig. 4.
Fig. 6 is a schematic cross-sectional structure along a line C in fig. 4.
Fig. 7 is a schematic cross-sectional structure along the line D in fig. 4.
Fig. 8 is a light field distribution of the graded region of fig. 4.
Fig. 9 is a light field distribution of the collimation areas of fig. 4.
Fig. 10 is a light field distribution of the nano-jet focusing region of fig. 4.
FIG. 11 is a schematic diagram of a single-molecule fluorescence detector according to an embodiment of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1 to 7, the invention discloses a single-molecule fluorescence detection chip 100, which comprises a chip substrate 10 and a cover plate 20 arranged on the chip substrate 10, wherein the chip substrate 10 comprises a substrate 11, and a reflecting layer 12, a first low refractive index layer 13, a high refractive index layer 14 and a second low refractive index layer 15 which are sequentially laminated on the substrate 11, and the refractive indexes of the first low refractive index layer 13 and the second low refractive index layer 15 are respectively lower than the refractive index of the high refractive index layer 14. By providing the low-high-low refractive index dielectric layers of the first low refractive index layer 13, the high refractive index layer 14 and the second low refractive index layer 15, the propagation of the irradiation source beam at the high refractive index layer 14 is restricted by utilizing the principle that light rays are totally reflected at the interface of the high-low refractive index dielectric layers. In the present invention, the low in both the first low refractive index layer 13 and the second low refractive index layer 15 is relative to the high in the high refractive index layer 14.
Referring to fig. 3 to 7, the upper surface of the chip substrate 10, which is close to the cover plate 20, is provided with two first grooves 16 parallel to each other, two second grooves 17 extending forward and outward along each first groove 16, a third groove 18 connecting the ends of the two second grooves 17, which are away from the first grooves 16, and a micro-channel groove 19 arranged at intervals from the third groove 18 and extending in the same direction, wherein the micro-channel groove 19 is used for allowing single molecules of a sample to flow therethrough, and the depths of the first groove 16, the second groove 17, the third groove 18 and the micro-channel groove 19 are respectively equal to the depth of the reflecting layer 12; the structure between the two first grooves 16 forms a straight waveguide core 161, the structure between the two second grooves 17 and the third groove 18 forms a gradual change waveguide core 171, and the structure between the third groove 18 and the micro-channel groove 19 forms a light-gathering waveguide core 181; the interface between the graded waveguide core 171 and the third groove 18 is a first arc-shaped interface 182 protruding toward the third groove 18, and the interface between the concentrating waveguide core 181 and the third groove 18 is a second arc-shaped interface 183 protruding toward the third groove 18; by providing the first grooves 16 and the second grooves 17, the medium in the grooves is gas, the refractive index of the gas is lowest and lower than that of the high refractive index layer 14, so that the straight waveguide core 161 and the first grooves 16 at two sides of the straight waveguide core and the graded waveguide core 171 and the second grooves 17 at two sides of the graded waveguide core respectively form low-high-low refractive index medium layers, and the irradiation source beam is limited to be transmitted forwards in the straight waveguide core 161 and the graded waveguide core 171; by providing a graded waveguide core 171, the width of the illumination source beam is enlarged for subsequent collimation and focusing; by providing the third groove 18, the gas in the graded waveguide core 171 and the third groove 18 and the gas in the condensed waveguide core 181 and the third groove 18 respectively constitute high and low refractive index medium layers, by providing the first arc interface 182, divergent light is converted into collimated light, and by providing the second arc interface 183, the collimated light is converged, so that an excitation beam which irradiates the micro flow channel groove 19 with extremely reduced irradiation area and extremely enlarged light intensity compared with the irradiation source beam is obtained, and the excitation beam can perform fluorescence excitation on the sample single molecules flowing through the micro flow channel groove 19, thereby being capable of obtaining a fluorescence signal of a single molecule with stronger light intensity and more accuracy.
Referring to fig. 1 and 2, a cap plate 20 is connected to the second low refractive index layer 15, and the cap plate 20 seals the first groove 16, the second groove 17, the third groove 18, and the micro flow channel groove 19.
Referring to fig. 4, the irradiation source beam first propagates in a non-destructive manner in the waveguide region including the straight waveguide core 161, then propagates to the graded region including the graded waveguide core 171, the beam is widened, then collimated by the first arc interface 182 and exits to the condensing waveguide core 181, and is converged by the second arc interface 183 onto the micro flow channel groove 19, so as to form an excitation beam, and the excitation beam is used for performing fluorescence excitation on the single molecules of the sample flowing in the micro flow channel groove 19 to generate fluorescent photons.
In the above technical solution, the straight waveguide core 161, the graded waveguide core 171, the third groove 18 and the light-gathering waveguide core 181 of the present invention form a photon nano jet structure, and the light field distribution of the graded region, the collimation region and the nano jet focusing region are respectively shown in fig. 8 to 10, so that the photon nano jet effect is formed in the micro-channel groove 19 by using the high effective refractive index of the waveguide core, so as to obtain optical focusing of less than 1 micron, and the energy density of the excitation light beam is increased to 5-10 times of the energy density of the irradiation source light beam, so that the excitation light intensity is amplified, and the signal-to-noise ratio of single molecule detection fluorescence signal is improved.
According to the invention, the photon nano jet technology and the micro-channel technology are combined, the photon nano jet effect is utilized to focus the excitation light beam, the excited area of the sample is reduced, stray light caused by scattering is reduced, the energy density of the excitation light beam is improved, the excitation light intensity is amplified, and the signal-to-noise ratio of single molecule detection fluorescence signals is improved; the invention can realize high-flux and high-accuracy single-molecule fluorescence detection on the sample by utilizing the micro-channel technology to carry out high-flux sample volume segmentation.
The photonic nanometer jet flow structure and the micro-channel are integrated on one chip smaller than 1 square centimeter, so that the function integration is realized. Meanwhile, the small volume and the integrated chip can bring system stability, and can be used for miniaturized portable instruments.
In addition, the chip of the invention is simple to prepare, can be reused, and can obviously reduce the cost of the chip.
Specifically, in one embodiment, the widths of the first grooves 16 and the second grooves 17 are 5 micrometers to 20 micrometers, respectively. The width of the straight waveguide core 161 is 5 micrometers to 20 micrometers. The width of the end of the graded waveguide core 171 facing away from the straight waveguide core 161 is 20 microns to 100 microns. The graded waveguide core 171 has a length of 100 microns to 1 mm. The radius of the arc of the first arc interface 182 is 100 micrometers to 400 micrometers. The radius of the arc of the second arcuate interface 183 is 20 microns to 100 microns.
In one embodiment, the thickness of the reflective layer 12 is 100nm to 800nm, the thicknesses of the first low refractive index layer 13 and the second low refractive index layer 15 are respectively 200 nm to 1 μm, the thickness of the high refractive index layer 14 is 200 nm to 10 μm, and the thickness of the substrate 11 is 200 μm to 3 mm.
In one embodiment, the refractive indexes of the first low refractive index layer 13 and the second low refractive index layer 15 are 1.47-1.51, respectively. The refractive index of the high refractive index layer 14 is 1.9 to 2.2.
In one embodiment, the reflective layer 12 is a non-transparent metallic reflective layer, and in particular, may be a chromium layer. The substrate 11 may be a glass sheet, a quartz sheet, a plastic sheet (e.g., PC, PS, etc.), a resin sheet (e.g., PDMS, etc.), or the like.
In one embodiment, the thickness of the cover 20 is 100 μm to 1mm, and the material of the cover 20 may be glass sheet, quartz sheet, plastic sheet (such as PC, PS, etc.), resin sheet (such as PDMS, etc.), etc.
In a specific embodiment, the bottom of the micro flow channel groove 19 at the excitation beam is provided with a groove 191 having a depth to the substrate 11, the substrate 11 is a transparent substrate 11, the groove 191 forms an optical aperture, and fluorescent photons are detected from the outside of the transparent substrate 11 facing away from the cover plate 20 through the groove 191. Since the rest positions all comprise the reflecting layer 12, the grooves 191 can filter stray light, only fluorescent photons can permeate, and fluorescent photons can be conveniently detected.
Specifically, the width of the groove 191 is equal to the light field width of the excitation light beam, the length of the groove 191 is equal to the width of the micro flow channel groove 19, and the influence of the scattered light of the excitation light beam can be avoided as much as possible by providing the groove 191 with such a size. It is noted that "equal" in this paragraph is not strictly absolute equal, but means equal or substantially equal.
In a specific embodiment, the micro flow channel groove 19 includes a single flow channel groove portion located in the middle area, two buffer groove portions located at two ends of the single flow channel groove portion, and two gradual change flow channel groove portions connecting the buffer groove portion and the single flow channel groove portion, the width of the gradual change flow channel groove portion gradually becomes smaller along the direction from the buffer groove portion to the single flow channel groove portion, and the excitation light beam is used for fluorescence excitation of the single molecules of the sample flowing in the single flow channel groove portion.
Referring to fig. 1 and 2, the cover plate 20 is provided with a sample inlet 21 and a sample outlet 22 at positions corresponding to the two buffer groove portions, respectively, and the sample inlet 21 and the sample outlet 22 are communicated with the corresponding buffer groove portions, respectively. A sample inflow line 23 is connected to the sample inlet 21, and a sample outflow line 24 is connected to the sample outlet 22, so that the sample passes through the microchannel grooves 19.
The sample inflow pipe 23 and the sample outflow pipe 24 may be specifically flexible pipes with an inner diameter of 50 micrometers to 500 micrometers and an outer diameter of less than 2 millimeters, which are directly glued with the cover plate 20, and may be cured after being compressed with the cover plate 20 by coating photosensitive and thermosensitive glue on the cover plate 20, or by using quick-curing glue, and then directly compressed after being coated, and waiting for curing for a certain time.
In one embodiment, the width of the single molecule runner trench is 2-10 microns; the width of the buffer groove part is 20-100 micrometers; the length of the gradual flow channel groove part is 1-3 mm, and the diameters of the sample inlet 21 and the sample outlet 22 are 100 micrometers-1 mm respectively.
The invention also provides a preparation method of the single-molecule fluorescence detection chip 100, which comprises the following steps:
1) A substrate 11 is provided, and a reflective layer 12, a first low refractive index layer 13, a high refractive index layer 14, and a second low refractive index layer 15 are sequentially formed on the substrate 11 to obtain an initial chip substrate 10.
The reflective layer 12, the first low refractive index layer 13, the high refractive index layer 14, and the second low refractive index layer 15 may be formed by magnetron sputtering, chemical vapor deposition, ion-assisted deposition, or the like.
2) The initial chip substrate 10 is etched to form a first trench 16, a second trench 17, a third trench 18, and a micro flow channel trench 19, forming the chip substrate 10.
3) A cover plate 20 is provided, and the first groove 16, the second groove 17, the third groove 18, and the microchannel groove 19 are sealed with the cover plate 20.
The adhesion of the chip substrate 10 to the cover plate 20 may be achieved by means of surface bonding or gluing. Specifically, the surface bonding may be performed by modifying the surfaces of the chip substrate 10 and the cover plate 20 by means of acid treatment, oxygen plasma cleaning, or the like, and then pressing the chip substrate 10 and the cover plate 20. The bonding may be performed by applying a photo-sensitive or thermo-sensitive glue to the cover plate 20, pressing it against the chip substrate 10 and curing it, or by using a fast curing glue, pressing it directly after application and waiting for curing for a certain time.
Referring to fig. 11, the present invention further discloses a single-molecule fluorescence detector, including the single-molecule fluorescence detection chip 100, an irradiation source, a driving pump, a micro objective lens, a single-photon detector, a time-resolved single-photon counter and a data processing module, wherein, a light beam emitted by the irradiation source enters the single-molecule fluorescence detection chip 100, is sequentially transmitted along the straight waveguide core 161, the gradual waveguide core 171 and the light-gathering waveguide core 181 to generate a photon nanometer jet effect, a focused excitation light beam is obtained, the driving pump drives a sample to enter the micro-channel groove 19 from the sample inlet 21 and flow out from the sample outlet 22, the excitation light beam is used for performing fluorescence excitation on the single-molecule sample flowing in the micro-channel groove 19 to generate fluorescent photons, the generated fluorescent photons penetrate the single-molecule fluorescence detection chip 100 at the groove 191, the micro objective lens collects the fluorescent photons emitted from the single-molecule fluorescence detection chip 100, and transmits the fluorescent photons to the single-photon detector, the single-photon detector converts the single fluorescent photons collected by the micro objective lens into pulse electrical signals, and transmits the pulse electrical signals to the time-resolved single-photon counter, and the time-resolved single-photon counter receives the pulse electrical signals, and records the arrival time of the single pulse electrical signals; the data processing module processes the pulse electrical signal and the time of arrival of the single pulse electrical signal into information of the change of light intensity with time, and calculates the information of the fluorescent marker in the sample passing through the microchannel grooves 19 according to the information of the change of light intensity with time.
In the above technical solution, the positive pressure or the negative pressure of the driving pump drives, the flow rate of the sample is controlled by the pressure, the flow rate of the sample at the light field of the excitation light beam passing through the micro-channel groove 19 is 0.1mm/s to 10m/s, and the single molecules of the sample are dispersed at the single molecule channel groove part and pass through the light field of the excitation light beam separately in time, namely, the groove 191. The sample may be recovered or passed into a waste reservoir after passing through the single molecule fluorescence detection chip 100.
In one embodiment, the irradiation source is a laser, and the power of the laser is 1W-500 mW.
In a specific embodiment, the single-molecule fluorescence detector further includes a single-mode fiber and/or a spatial light focusing structure disposed between the irradiation source and the single-molecule fluorescence detection chip 100, and the irradiation source sequentially couples the irradiation source beam into the straight waveguide core 161, the graded waveguide core 171 and the light-gathering waveguide core 181 of the single-molecule fluorescence detection chip 100 through the single-mode fiber and/or the spatial light focusing structure, so as to obtain an excitation beam with photon nano jet phenomenon, and generate fluorescence.
In a specific embodiment, a first filtering member is further disposed in front of the single-molecule fluorescent detection chip 100, for filtering stray light in the irradiation source beam. The first filter member may be a bandpass or shortpass filter, to filter out stray light generated by the laser light.
In a specific embodiment, a second filter member is further included between the microscope objective and the single photon detector, the second filter member being configured to remove scattered excitation light from the fluorescent photons. In particular, the second filter member may be a bandpass or longpass filter.
In a specific embodiment, a multimode optical fiber connected to the single photon detector is further included between the second filter member and the single photon detector, the multimode optical fiber being configured to transmit the fluorescent photons passing through the second filter member to the single photon detector.
Since the sample single molecule passes through the photon nano jet excitation area separately from time, the fluorescence signal of the fluorescent marker on the single molecule is displayed as the light intensity envelope of the pulse in time. By counting the pulse envelopes in the light intensity, the number of fluorescent markers passing through the microchannel grooves 19 per unit time can be calculated, thereby calculating the target solution concentration. The calculation formula is as follows: c=n/Q, where c is the target solution concentration, N is the number of light intensity envelopes measured per unit time, and Q is the flow rate. The flow Q can be calculated by measuring or observing the time of a certain volume of liquid passing through the micro-flow channel through the flowmeter.
Specifically, the single photon detector may be a photon counting PMT, a silicon photomultiplier, a single photon counting APD, or a single photon counting MPPC, etc.
The time resolution single photon counter can be a high-speed FPGA real-time data transmission program module or a time digital conversion card.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The single-molecule fluorescence detection chip is characterized by comprising a chip substrate and a cover plate arranged on the chip substrate, wherein the chip substrate comprises a substrate, and a reflecting layer, a first low refractive index layer, a high refractive index layer and a second low refractive index layer which are sequentially laminated on the substrate, and the refractive indexes of the first low refractive index layer and the second low refractive index layer are respectively lower than that of the high refractive index layer;
the upper surface of the chip substrate, which is close to the cover plate, is provided with two first grooves which are parallel to each other, two second grooves which extend forwards and outwards along each first groove, a third groove which is connected with the end parts of the two second grooves, which are far away from the first grooves, and a micro-channel groove which is arranged at intervals with the third groove and extends in the same direction, wherein the micro-channel groove is used for allowing single molecules of a sample to flow through, and the depths of the first grooves, the second grooves, the third grooves and the micro-channel groove are respectively equal to the depth of the reflecting layer;
the structure between the two first grooves forms a straight waveguide core, the structure between the two second grooves and the third grooves forms a gradual change waveguide core, and the structure between the third grooves and the micro-channel grooves forms a light-gathering waveguide core;
the interface between the graded waveguide core and the third groove is a first arc-shaped interface protruding towards the direction of the third groove, and the interface between the concentrating waveguide core and the third groove is a second arc-shaped interface protruding towards the direction of the third groove;
the cover plate is connected with the second low-refractive-index layer, and seals the first groove, the second groove, the third groove and the micro-channel groove;
the irradiation source beam is transmitted to the gradual change waveguide core through the straight waveguide core to be diverged, then is collimated and emitted to the condensing waveguide core through the first arc-shaped interface, and is converged to the micro-channel groove through the second arc-shaped interface to form an excitation beam, and the excitation beam is used for carrying out fluorescence excitation on sample single molecules flowing in the micro-channel groove to generate fluorescent photons.
2. The single-molecule fluorescence detection chip of claim 1, wherein it satisfies at least one of the following technical features a-m:
a. the widths of the first groove and the second groove are respectively 5 micrometers to 20 micrometers;
b. the width of the straight waveguide core is 5-20 microns;
c. the width of the end part of the gradual change waveguide core, which is far away from the straight waveguide core, is 20-100 micrometers;
d. the length of the gradual change waveguide core is 100 micrometers to 1 millimeter;
e. the arc radius of the first arc interface is 100-400 microns;
f. the arc radius of the second arc interface is 20-100 microns;
g. the thickness of the reflecting layer is 100 nm-800 nm;
h. the refractive indexes of the first low refractive index layer and the second low refractive index layer are respectively 1.47-1.51;
i. the refractive index of the high refractive index layer is 1.9-2.2;
j. the thickness of the first low refractive index layer and the second low refractive index layer is 200 nanometers to 1 micrometer respectively;
k. the thickness of the high refractive index layer is 200 nanometers to 10 micrometers;
l, the reflecting layer is a non-transparent metal reflecting layer;
and m, the thickness of the cover plate is 100 micrometers to 1 millimeter.
3. The single-molecule fluorescent detection chip of claim 2, wherein the non-transparent metal reflective layer is a chromium layer;
the substrate is a glass sheet, a quartz sheet, a plastic sheet or a resin sheet.
4. The single-molecule fluorescence detection chip according to any one of claims 1 to 3, wherein a groove with a depth reaching the substrate is formed in the bottom of the microchannel groove at the excitation beam;
the substrate is a transparent substrate.
5. The single molecule fluorescent detection chip of claim 4, wherein the width of the groove is equal to the optical field width of the excitation beam;
the length of the groove is equal to the width of the micro-channel groove.
6. The single-molecule fluorescence detection chip according to claim 1, wherein the micro flow channel groove includes a single-molecule flow channel groove portion located in a middle region, two buffer groove portions located at both ends of the single-molecule flow channel groove portion, respectively, and two gradation flow channel groove portions connecting the buffer groove portion and the single-molecule flow channel groove portion, the width of the gradation flow channel groove portion gradually becoming smaller in a direction from the buffer groove portion to the single-molecule flow channel groove portion, the excitation light beam being for performing the fluorescence excitation on the single molecules of the sample flowing in the single-molecule flow channel groove portion.
7. The single-molecule fluorescence detection chip according to claim 6, wherein the cover plate is provided with a sample inlet and a sample outlet at positions corresponding to the two buffer groove portions, respectively, and the sample inlet and the sample outlet are respectively communicated with the corresponding buffer groove portions;
the width of the single-molecule flow channel groove part is 2-10 microns;
the width of the cache groove part is 20-100 microns;
the length of the gradual change flow channel groove part is 1-3 mm.
8. A method for manufacturing the single-molecule fluorescence detection chip according to any one of claims 1 to 7, comprising the steps of:
providing a substrate, and sequentially forming the reflecting layer, the first low refractive index layer, the high refractive index layer and the second low refractive index layer on the substrate to obtain an initial chip substrate;
etching the initial chip substrate to form the first groove, the second groove, the third groove and the micro-channel groove, so as to obtain the chip substrate;
and providing a cover plate, and sealing the first groove, the second groove, the third groove and the micro-channel groove by using the cover plate.
9. A single-molecule fluorescence detector, comprising a single-molecule fluorescence detection chip according to any one of claims 1 to 7.
10. The single molecule fluorescence detector of claim 9, further comprising:
the driving pump is used for driving the sample to flow in the micro-channel groove;
an illumination source;
a microobjective for collecting the fluorescent photons;
a single photon detector for converting the single fluorescent photons collected by the microscope objective into pulsed electrical signals;
a time-resolved single photon counter for receiving the pulsed electrical signal and recording the time of arrival of a single pulsed electrical signal; and
the data processing module is used for processing the pulse electric signals and the time of arrival of the single pulse electric signals into information of the change of light intensity along with time, and calculating the information of the fluorescent marker in the sample passing through the micro-channel groove according to the information of the change of light intensity along with time.
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