CN105784662A - Liquid-phase suspension biochip based on multi-optical trap encoding bead array and two-photon fluorescence detection - Google Patents
Liquid-phase suspension biochip based on multi-optical trap encoding bead array and two-photon fluorescence detection Download PDFInfo
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Abstract
The invention provides a liquid-phase suspension biochip based on multi-optical trap encoding bead array and two-photon fluorescence detection. The liquid-phase suspension biochip has a configuration as follows: a near-infrared laser beam emitted by a near-infrared laser is expanded by a beam expanding system, is sequentially reflected by a telescope system and a dichroscope by using a holographic technology or a time-share scanning technology, and is focused into a sample pool through a high numerical aperture objective lens, to form multi-optical-trap optical tweezers; the multi-optical-trap optical tweezers capture a plurality of encoding beads enriched with objects to be detected, to form a bead array in the solution; after an infrared laser signal is filtered by a band-pass filter, a two-photon fluorescence signal from each bead is focused to an image detector by a lens and is subjected to imaging detection. The liquid-phase suspension biochip can perform real-time quantitative analysis on nucleic acids, proteins, virus particles and a plurality of objects to be detected, and has the advantages of high sensitivity, strong anti-interference ability, simultaneous determination of a plurality of components and the like.
Description
Technical field
The invention belongs to the micro-manipulation technology field of ligh trap light tweezer, fall within microsphere coding techniques field, be specifically related to the liquid phase suspension formula analyzing biochips system detected based on many ligh traps coding microball array and two-photon fluorescence.
Background technology
Traditional solid phase biological chip is the principle according to interaction special between biomolecule, by biochemical analysis process integration in solid phase mediator surfaces such as silicon chip, sheet glass, nylon membranes, it is achieved the high flux of multiple biotic component is quickly detected.This technology is widely used, but owing to being react at solid phase surface, thus have that reaction efficiency is low, stability and the defect such as reliability is poor.Luminex company of the U.S. 20th century nineties Later development based on coloured microsphere, laser technology, the multi-functional liquid phase chip analysis platform of fluidics and high-speed digital video camera technology, the fluorescence-encoded micro-beads of each color represents a kind of detection mark, specific binding at suspension target and microsphere, the microsphere flowing in single file in the channel, same microsphere with the laser excitation detection zone of two kinds of different wave lengths, wherein beam of laser is for identifying the coding information (color of microsphere, for qualitative), another beam of laser is for exciting the fluorescence intensity (for detection by quantitative) of microsphere.But this technology needs to use the coding microball of a large amount of multiple color, and can only realize the mensuration one by one of single microsphere.
Light tweezer concept is proposed in 1970 by American scientist Ashkin the earliest, the gauss laser of a branch of high concentration produce sufficiently strong ligh trap gradient force at focal point, the particle of micron or even nano-scale is caught and handled.This technological means has been widely used in material science and life science.Recent two decades comes, and along with the fast development of photoelectron and microelectric technique, traditional single beam light tweezer has derived this kind of multiple light forceps technology that can simultaneously manipulate multiple microgranule such as holographic optical tweezer, Time share scanning light tweezer, greatly extends the range of application of optical tweezer technology.Holographic optical tweezer technology is to be split beam of laser by holographic optical elements (HOE)s such as diffraction beam splitter (DOE), spatial light modulators (SLM) to realize, owing to being subject to the restriction of the intrinsic diffraction efficiency of beam splitting device and transmitance, if to catch the microgranule of more than ten by Simultaneous Stabilization in many optical trap arrays, then the watt level of laser instrument is required harsh, it usually needs power more than watt level can be only achieved satisfied effect.Time share scanning optical tweezer technology is then the deflection being controlled laser by devices such as scanning galvanometer (GM), acousto-optic deflection devices (AOD), single beam laser is made quickly to scan on focal plane, only when hot spot in action time of microgranule more than its minimum time of staying, and microgranule make the Brownian movement time more than its time departure time, microgranule could be stably strapped on laser beam scan path, although Time share scanning technology is not high to laser power requirements, but the ligh trap limited amount (being typically smaller than 100) that theoretically, the method can produce.
Different from traditional Single Photon Absorption process, two-photon excitation relates to a kind of nonlinear optics, namely absorb two or more photon simultaneously, only when the instantaneous photon density of laser focusing focal point is sufficiently high, be only possible to this special absorbing phenomenon of triggering.In order to reach the stimulation effect of the best, generally to use near infrared pulsed laser device, and be converged to a minimum diffraction pattern through object lens.Therefore, two-photon excitation has a lot of advantage, such as deeper of penetration depth, higher spatial resolution, less background fluorescence, less optical damage etc..
Summary of the invention
The technical problem to be solved is to provide a kind of liquid phase suspension formula analyzing biochips system detected based on many ligh traps coding microball array and two-photon fluorescence.
Many ligh traps optical tweezer technology, microsphere coding techniques are combined by the present invention with two-photon fluorescence detection technique, by holographic technique or Time share scanning technology, catch multiple coding microball complex simultaneously, and when two-photon excitation, it is achieved the various biomolecules such as determinand such as nucleic acid, protein etc. or virion in complex sample (such as whole serum, whole plasm) is carried out highly sensitive detection.
In order to achieve the above object, the liquid phase suspension formula analyzing biochips system of the present invention, including near infrared pulsed laser device, microscope and white light LEDs, described the sent out laser of near infrared pulsed laser device first passes around by two groups of different focal lens (L1, L2) beam-expanding system formed is to regulate spot size with pupil after full microscope objective, then this laser beam is after beam splitting or Time share scanning, sequentially pass through dichroic mirror (DM) reflection, after being focused on by microscope objective, optical trap array is formed in focal plane, each ligh trap all can catch a coding microball complex, two-photon fluorescence signal from each coding microball complex passes through dichroic mirror (DM), after bandpass filter (BPF), focus to image detector through lens (L5) and carry out image checking；Described white LED light source after lens (L6) focus on as the instruction light source of coding microball complex.
Described the sent out laser of near infrared pulsed laser device can be split by DOE or SLM element.
Described the sent out laser of near infrared pulsed laser device carries out Time share scanning by elements such as GM or AOD.
In order to ensure to catch trap position and microscope parfocalization, it is possible to the telescopic system that is made up of two parfocal lens is coupled in this detection system, described the sent out laser of near infrared pulsed laser device after beam splitting or Time share scanning through this telescopic system.
Above-mentioned near infrared pulsed laser device watt level is adjustable, and wavelength is positioned at 780～1080nm wave band.
Above-mentioned near infrared pulsed laser device outlet optical quality and standard fundamental-mode gaussian beam are close to (M2Close to 1).
Above-mentioned DM can reflect infrared band, through visible light wave range.
Described image detector is sensitive face battle array image device (such as CCD, cmos device etc.), all can convert optical signal into signal of telecommunication output.
Use the method that the detection of above-mentioned liquid phase suspension formula analyzing biochips system is biological, comprise the following steps:
Step one, the microsphere encoded is caught determinand by chemical coupling mode or immunity combination, then fluorescent probe is coupled to microsphere surface, ultimately form the coding microball complex of coding microball determinand fluorescent probe, above-mentioned coding microball complex is placed in sample cell, sample cell is placed in the microscopical automatically controlled two-dimensional stage of said detecting system；
Step 2, start near infrared pulsed laser device, near infrared pulsed laser after microscope objective focuses on forms many ligh traps light tweezer at focal plane place, by regulating laser power size, it is ensured that the instantaneous photon density at each ligh trap place enough inspires two-photon fluorescence signal；
Step 3, ligh trap position immobilizes, and accurately controls sample cell by automatically controlled two-dimensional stage and moves, and catches many coding microball complex；
Step 4, using white LED light source as instruction light source, locating and enriching has the coding microball complex of determinand；
Step 5, switch to fluorescence field, the coding microball complex being only enriched with corresponding determinand just can inspire two-photon fluorescence signal, by detecting the captured respective fluorescence signal of coding microball complex and microsphere coding information being decoded (Microsphere Size and color), each component is respectively adopted working curve method, it is achieved the real-time quantitative analysis to determinand.
Above-mentioned determinand can be nucleic acid, protein, virion etc., can be enriched on coding microball by chemical coupling mode or immunity combination.
Above-mentioned microsphere material is transparent inorganic material (such as silicon dioxide), or transparent macromolecular material (such as polystyrene), size uniformity, size 1～4 μm, coded system can adopt color coding (blueness, green, yellow, redness, colourless), size coding (1 μm, 2 μm, 3 μm, 4 μm) or color in conjunction with the assembly coding of size, group/cording quantity, for up to 20 kinds, namely can realize at most detecting while 20 kinds of compositions in same sample.
In coding microball complex, described fluorescent probe can be fluorescence quantum or dye molecule.
Apparatus of the present invention adopt holographic technique or Time share scanning technology to form many ligh traps light tweezer at microscope stage, adopt automatically controlled two-dimensional stage to realize the accurate of sample cell is moved.Meanwhile, this method adopts microsphere coding techniques that the microsphere being enriched with different determinand carries out indicative function, and using fluorescence quantum or dye molecule as marker material.Additionally, this method adopts imaging mode to obtain the fluorescence intensity of each microsphere in micro-sphere array.
The present invention can adopt multiple specific enrichment means to be enriched with determinand.The method that such as can adopt dibit point immuno-sandwich, utilizes coding microball and fluorescent probe specific recognition and catches determinand, forms coding microball-determinand-fluorescent probe coupled complex.Then, the many ligh traps light tweezer formed by near infrared pulsed laser is caught above-mentioned coding microball complex and is formed micro-sphere array, realize the two-photon excitation of micro-sphere array simultaneously, fluorescence signal from coding microball carries out intensity detection by image detector, thus realizing the real-time quantitative analysis to multiple determinand.
Compared with existing analysis means, the invention have the characteristics that and beneficial effect:
1, many ligh traps optical tweezer technology, microsphere coding techniques are combined by the present invention with two-photon fluorescence technology, it is proposed to a kind of novel analysis and detection device, and multiple determinand can be analyzed by this device in real time.
2, the present invention utilizes the coding microball with indicative function to be enriched with determinand, simplifies the enrichment flow process of multicomponent determinand detection.
3, the multiple light forceps system that the present invention builds can be caught multiple microsphere simultaneously and form two-dimensional array, realizes quickly simultaneously detecting multiple microspheres on this basis.
4, the present invention is by single beam laser simultaneously as catching light source and excitation source, simplifies the structure of device, reduces the cost of device.
5, the present invention is by the simple White-light LED illumination light source instruction light source simultaneously as coding microball.
6, apparatus of the present invention are owing to adopting near infrared pulsed laser to realize two-photon fluorescence excitation, only laser focusing focal point just can go out fluorescence signal, overcome the autofluorescence interference being difficult in conventional single photon fluorescence detection method overcome, therefore when this method is used for the analysis of complex sample system, without separable programming, both improve the capacity of resisting disturbance of method, in turn simplify operating procedure.
7, apparatus of the present invention adopt sensitive image detector, it is not necessary to adopt complicated signal to amplify mode and can obtain significantly high detection sensitivity, simplify the experimental procedure of enrichment determinand.
Accompanying drawing explanation
Fig. 1 is the structural representation of apparatus of the present invention；
Fig. 2 is working curve schematic diagram；
Fig. 3 is the schematic diagram on color coding microball surface by two-site sandwich method enrichment liver cancer marker.
Fig. 4 is enriched with the schematic diagram of bird flu virus particle at size coding microsphere surface by two-site sandwich method.
Detailed description of the invention
Apparatus of the present invention include near infrared pulsed laser light source, holography or Time share scanning system, inverted microscope system and fluorescence signal detection system.Near infrared pulsed laser device is used for producing to meet the laser beam of two-photon excitation condition, and in conjunction with holography or Time share scanning system for producing many ligh traps light tweezer；Microscopic system is used for the trapped state of Real Time Observation micro-sphere array, is wherein coupled with automatically controlled two-dimensional movement platform and carrys out steady implementation passive operation mode；Image detection detection system is used for detecting the two-photon fluorescence signal that coding microball complex produces；Fig. 1 is a kind of concrete structure schematic diagram of apparatus of the present invention, below in conjunction with Fig. 1, apparatus of the present invention is described further.
The present invention adopts near infrared pulsed laser and in conjunction with holography or Time share scanning system for producing many ligh traps light tweezer, the laser beam that device produces is swashed by near-infrared pulse, through battery of lens L1, after the beam-expanding system of L2 composition expands, make its spot diameter size can be full of pupil after microscope objective, then utilize DOE (or SLM) element to be split or the element such as GM (or AOM) quickly scans, after finally being reflected by DM and focused on by object lens, produce many ligh traps light tweezer at sample cell place.Additionally, can ensure to catch trap position and microscope parfocalization by the telescopic system that adjustment is made up of parfocal battery of lens L3-L4.Two-photon fluorescence signal from coding microball complex passes through DM, filters out after iraser signal through BPF, is converged to image detector by lens L5 and carry out image checking.In order to indicate coding microball complex, the White-light LED illumination light source after lens L6 focuses on can be adopted as instruction light source.
Microsphere coded system in the present invention can adopt microsphere color coding, size coding, or microsphere color is in conjunction with the assembly coding mode of size.Color is encoded, commercial coloud coding microsphere (five kinds of colors: blueness, green, yellow, redness, colourless) can be adopted.For size coding, the microsphere (microsphere of four kinds of sizes: 1 μm, 2 μm, 3 μm, 4 μm) of different-grain diameter can be adopted as solid phase carrier.Combination by color and size, it is possible to obtain 20 kinds of different coding modes, namely can measure at most the content of 20 kinds of different components in same sample simultaneously.Two kinds of coded systems all can use White-light LED illumination light source as instruction light source, namely by the color of bright field image identification microsphere and size, thus realizing the qualitative analysis to composition to be measured；By the fluorescence intensity of microsphere, it is achieved the quantitative analysis of composition to be measured.
The present invention adopts high-NA objective focus on laser and form light tweezer.The airtight sample room that sample cell can be made up of two panels coverslip, or the small container of transparent polymer material composition, sample capacity is micro updating.
The present invention adopts automatically controlled two-dimensional movement platform to carry out steady implementation passive operation mode.
First, a number of coding microball complex is added, by accurate moving stage, when microsphere is near ligh trap in sample cell, the ligh trap gradient force produced due to the gauss laser of high concentration can overcome the solution drag interaction power to microsphere, and microsphere will be tied to beam waist place by stabilized beam.By holographic technique or Time share scanning technology, many microspheres can be caught simultaneously, form two dimension micro-sphere array in the solution.By bright field light source LED, get final product the formation state of Real Time Observation color coding microball array, the coding microball being only enriched with corresponding determinand could in conjunction with upper fluorescent probe, by measure from the two-photon fluorescence signal intensity of fluorescent probe on coding microball complex, adopt working curve method, can realize the determinand of coding microball surface enrichment is carried out quantitative analysis, shown in specific as follows:
First, adopt two-site sandwich method, using fluorescence quantum or dye molecule as fluorescence labeling material, prepare the immune coding microball being enriched with variable concentrations gradient determinand.Then, catch many coding microball complex with the device in the present invention, for each concentration group simultaneously, two-photon fluorescence intensity by 80～100 microspheres of parallel assay, taking its meansigma methods, then drawing curve, this curve is linear with fluorescence intensity within the scope of finite concentration.
Fig. 2 is the schematic diagram utilizing working curve method to do quantitative analysis, measures 8 groups of coding microball complex (including blank group) being enriched with variable concentrations determinand altogether.In figure, every bit represents the two-photon fluorescence intensity meansigma methods of 80～100 microspheres, and error bar represents the standard deviation of corresponding concentration group microsphere fluorescence intensity.Within the scope of finite concentration, along with the reduction of testing concentration, the corresponding fluorescence intensity meansigma methods measured is gradually reduced, and fluorescence intensity is linear with testing concentration.For the detection of multiple determinand, working curve should include a plurality of curve of corresponding determinand.
The detection method that embodiment 1～2 pair present invention proposition is set forth below is described further.
Embodiment 1
The detection of five kinds of liver cancer marker AFP, CEA, Monophosphoinositideproteoglycans proteoglycans-3 (GPC-3), abnormal prothrombin (DCP) and alpha-L-fucosidase (AFU)
Using the 3 of finishing carboxylic group μm of five kinds of color micro-spheres (blueness, green, yellow, redness, colourless) as color coding microball, AFP, CEA, GPC-3, DCP, AFU antigen it is enriched with respectively by two-site sandwich method, and to launch quantum dot that wavelength is 605nm as fluorescence labeling material, specifically as shown in Figure 3.
The first step, preparation immunity coding microball.Take the blue-coded microsphere 16 that one of which surface carboxyl groups is modified, first pass through the carboxyl of EDC/NHS reaction activation microsphere surface, then coupling one of which AFP monoclonal antibody 17 is at room temperature distinguished, obtain immune microsphere 18, the preparation method of all the other four kinds of immune microspheres is similar, finally mixes to buffer solution five kinds of immune coding microballs with standby.
Second step, prepares quantum dot probe.First activating, with SMCC, the quantum dot 19 that 605nm surface amino groups is modified, then covalent coupling another kind AFP monoclonal antibody 20, prepares quantum dot probe 21, and the preparation method of all the other four kinds of quantum dot probe is similar.
3rd step, is enriched with AFP, CEA, GPC-3, DCP, AFU tumor markers respectively.Enrichment for AFP, dibit point one-step method is adopted to catch antigen method, namely in centrifuge tube, it is separately added into a certain amount of above-mentioned five kinds of immune coding microball mixture, five kinds of quantum dot probe, and AFP antigen 22, only the microsphere of blue-coded could be enriched with AFP incorporating quantum point probe, finally gives coding microball complex 23.The enrichment principle of CEA, GPC-3, DCP, AFU is identical, simply changes the color coded system (i.e. green, yellow, redness, colourless) of its correspondence.
4th step, carries out detection by quantitative to AFP, CEA, GPC-3, DCP, AFU tumor markers respectively.Detection for AFP, many coding complex microspheres are caught respectively initially with this device, the micro-sphere array formed with the instruction of White-light LED illumination light source, only blue microsphere is just enriched with AFP antigen, it is then switched to fluorescence field, by the two-photon fluorescence signal of this coding microball surface quantum point of detection means measure.For the experimental group of variable concentrations determinand, often group 80～100 microspheres of parallel assay, take its fluorescence intensity meansigma methods, draw out working curve, can realize the detection by quantitative to AFP.According to the standard deviation of blank group intensity, the detection limit of the method can be calculated.The Cleaning Principle of CEA, GPC-3, DCP, AFU is identical with above-mentioned.
Five kinds of tumor markerses such as AFP, CEA, GPC-3, DCP, AFU are carried out detection by quantitative in whole serum (or blood plasma) by the 5th step respectively.Detecting step is similar with above-mentioned, is simply dispersed in serum (blood plasma) by coding microball complex and detects.
Embodiment 2
The detection by quantitative of tri-kinds of bird flu viruss of H1N1, H3N2, H9N2
Adopt size coding mode, respectively by finishing carboxyl, size is that the polystyrene microsphere of 1 μm, 2 μm, 3 μm is as solid phase carrier, H1N1, H3N2, H9N2 virus it is enriched with respectively by two-site sandwich method, and to launch wavelength for 605nm quantum dot as fluorescence labeling material, specifically as shown in Figure 4.
The first step, preparation immunity coding microball.Take one of which surface carboxyl groups to modify, size is the coding microball 24 of 1 μm, first pass through the carboxyl of EDC/NHS reaction activation microsphere surface, then at room temperature coupling one of which H1N1 monoclonal antibody 25, obtain immune microsphere 26, the preparation method of all the other two kinds of immune microspheres is similar, finally mixes to buffer solution three kinds of immune coding microballs with standby.
Second step, prepares quantum dot probe.First activating the quantum dot 27 that 605nm surface amino groups is modified, then covalent coupling another kind H1N1 monoclonal antibody 28 with SMCC, prepare three kinds of quantum dot probe 29, the preparation method of the sub-point probe of all the other two amounts is similar.
3rd step, is enriched with H1N1, H3N2, H9N2 virion respectively.Enrichment for H1N130, dibit point one-step method is adopted to catch virus, namely in centrifuge tube, it is separately added into a certain amount of above-mentioned three kinds of immune coding microball mixture, three kinds of quantum dot probe, and H1N1 virus, only particle diameter is that the coding microball of 1 μm could be enriched with H1N1 incorporating quantum point probe, finally gives coding microball complex 31.The enrichment principle of H3N2, H9N2 is identical, simply changes the size color coded system (namely 2 μm, 3 μm) of its correspondence.
4th step, carries out detection by quantitative to H1N1, H3N2, H9N2 bird flu virus respectively.Detection for H1N1, many coding complex microspheres are caught respectively initially with this device, the micro-sphere array formed with the instruction of White-light LED illumination light source, only particle diameter is that the microsphere of 1 μm is just enriched with H1N1 virus particle, it is then switched to fluorescence field, by the two-photon fluorescence signal of this coding microball surface quantum point of detection means measure.For the experimental group of variable concentrations determinand, often group 80～100 microspheres of parallel assay, take its fluorescence intensity meansigma methods, draw out working curve, can realize the detection by quantitative to H1N1.According to the standard deviation of blank group intensity, the detection limit of the method can be calculated.The Cleaning Principle of H3N2, H9N2 is identical with above-mentioned.
5th step, carries out detection by quantitative to H1N1, H3N2, H9N2 bird flu virus in whole serum (or blood plasma) respectively.Detecting step is similar with above-mentioned, is dispersed in by coding microball complex in serum (blood plasma) and carries out detecting.
Claims (10)
1. a liquid phase suspension formula analyzing biochips system, including near infrared pulsed laser device, microscope and white light LEDs, described the sent out laser of near infrared pulsed laser device first passes around by two groups of different focal lens (L1, L2) beam-expanding system formed is to regulate spot size with pupil after full microscope objective, then this laser beam is after beam splitting or Time share scanning, sequentially pass through dichroic mirror (DM) reflection, after being focused on by microscope objective, optical trap array is formed in focal plane, each ligh trap all can catch a coding microball complex, two-photon fluorescence signal from each coding microball complex passes through dichroic mirror (DM), after bandpass filter (BPF), focus to image detector through lens (L5) and carry out image checking；Described white LED light source after lens (L6) focus on as the instruction light source of coding microball complex.
2. two-photon fluorescence according to claim 1 detection system, it is characterised in that described the sent out laser of near infrared pulsed laser device is split by DOE or SLM element.
3. analysis system according to claim 1, it is characterised in that described the sent out laser of near infrared pulsed laser device carries out Time share scanning by GM or AOD element.
4. analysis system according to claim 1, it is characterized in that, one telescopic system being made up of two parfocal lens coupled in this detection system, described the sent out laser of near infrared pulsed laser device after beam splitting or Time share scanning through described telescopic system.
5. analysis system according to claim 1, it is characterised in that described near infrared pulsed laser device wavelength is positioned at 780～1080nm wave band.
6. analysis system according to claim 1, it is characterised in that described image detector is CCD or CMOS.
7. use the method that liquid phase suspension formula analyzing biochips system detection described in claim 1 is biological, comprise the following steps:
Step one, the microsphere encoded is caught determinand by chemical coupling mode or immunity combination, then fluorescent probe is coupled to microsphere surface, ultimately form the coding microball complex of coding microball determinand fluorescent probe, above-mentioned coding microball complex is placed in sample cell, sample cell is placed in the microscopical automatically controlled two-dimensional stage of said detecting system；
Step 2, start near infrared pulsed laser device, near infrared pulsed laser after microscope objective focuses on forms many ligh traps light tweezer at focal plane place, by regulating laser power size, it is ensured that the instantaneous photon density at each ligh trap place enough inspires two-photon fluorescence signal；
Step 3, ligh trap position immobilizes, and accurately controls sample cell by automatically controlled two-dimensional stage and moves, and catches many coding microball complex；
Step 4, using white LED light source as instruction light source, locating and enriching has the coding microball complex of determinand；
Step 5, switch to fluorescence field, the coding microball complex being only enriched with corresponding determinand just can inspire two-photon fluorescence signal, by detecting the captured respective fluorescence signal of coding microball complex and microsphere coding information being decoded, each component is respectively adopted working curve method, it is achieved the real-time quantitative analysis to determinand.
8. detection method according to claim 7, it is characterised in that described determinand is nucleic acid, protein or virion.
9. detection method according to claim 7, it is characterised in that described microsphere coded system adopts color coding, size coding or color in conjunction with the assembly coding of size.
10. detection method according to claim 7, it is characterised in that in coding microball complex, described fluorescent probe is fluorescence quantum or dye molecule.
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