CN102262052B - Laser confocal obliquely-incident ellipsometric high-throughput biomolecular reaction imaging detection device - Google Patents
Laser confocal obliquely-incident ellipsometric high-throughput biomolecular reaction imaging detection device Download PDFInfo
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Abstract
The invention relates to a laser confocal obliquely-incident ellipsometric high-throughput biomolecular reaction imaging detection device which is characterized in that a sample substrate loaded with a biological sample microarray is arranged on a sample platform, and a multi-channel constant-temperature flow cell which is provided with a liquid inlet and a liquid outlet connected with a multi-channel injection pump is arranged below the sample substrate; the multi-channel constant-temperature flow cell is placed in the position of an objective table of an inverse laser confocal microscope; the two corresponding edges of the sample platform are respectively provided with two flat plates respectively provided with a groove, which are symmetrically and vertically arranged; the insides of the grooves are respectively provided with a mechanical arm, wherein one mechanical arm is provided with a laser device, a polarizer, a photoelastic modulator, a phase shifter and a focusing lens which are sequentially arranged in the direction of an incident light path, and the other mechanical arm is provided with a focusing lens, a chopper, a polarization analyzer and a photoelectric detector which are sequentially arranged in the direction of a reflected light path; and the data inlet/outlet ends of a phaselocked amplifier are respectively connected with the data outlet end of the photoelectric detector and the data inlet end of a data acquisition card, and a computer is respectively connected with the data acquisition card and a controller of the multi-channel injection pump.
Description
Technical field
The invention belongs to the device of high throughput testing biomolecular reaction, particularly a kind of based on the real-time monitored of laser confocal microscope system and the oblique incidence ellipsometry imaging pick-up unit of high throughput testing biomolecule microarry (biochip) reaction.
Background technology
In life science; Interaction between the biomolecule is a kind of basic biological phenomena; It also is one of significant problem of modern life science research; Interactional classic method between the research biomolecule has multiple, links immune analysis method, mark trace method etc. like radioimmunoassay method, enzyme.Yet; Because these methods will relate to cell, the biomolecule of variety classes and content; And exist complex interactions between various materials; Thereby utilize these traditional research methods to be difficult to accurately to obtain the relevant transmission information between the biomolecule, simultaneously, new albumen that increases day by day and dna sequence data also press for the interactional method between can accurate, high-throughout Rapid identification biomolecule.At the eighties of last century initial stage eighties, the someone proposes to combine through microelectric technique and biotechnology, and the conception of producing the microstructure of biologically active realizes the interaction between the high throughput testing biomolecule, the prototype of Here it is biochip technology.But because the restriction of relevant technological means such as process technology, up to eighties of last century nineties, biochip technology is just obtained significant progress.The particularly two-dimentional biochip of biochip has been realized industrialization at present.The chip detection technology of following biochip technology to grow up mainly is divided into two kinds at present; The one, chemical method; Like isotope labeling, fluorescence labeling and electrochemical method etc., using maximum at present is fluorescence labeling method, mainly adopts fluorescence laser co-focusing system to carry out the high throughput testing micro-array biomolecule reaction; The sensitivity of this method is higher, but need carry out processing in early stage, detection by quantitative difficulty, have damage and the easy light that takes place to drift phenomenon to testing sample sample; The 2nd, physical method; Like surface plasmons microscope, atomic force microscope, mass spectroscopy, ellipsometer etc.; Wherein ellipsometer need not marked to determinand owing to it; Can not cause any disturbance and damage yet, have in real time and sensitivity is widely used than advantage such as height biomolecule activity to be measured.Delustring formula ellipsometer, luminosity formula ellipsometer, elliptical polarization spectroscopy, infrared elliptical polarization spectroscopy, imaging ellipsometer and broad sense ellipsometer etc. have appearred at present.Wherein being used between the chip biological molecule interacting, what detect mainly is imaging ellipsometer, can carry out biomolecule thickness, diameter and the measurement of three-dimensional appearance; The scanning of the real-time biochip of non-marked and the absorption of various biomolecule, desorption process are measured and dynamics research; Through it can the Real Time Observation molecule between situation of change in the interaction process, obtain interactional information between the biomolecule that a lot of conventional arts are difficult to provide.But because the research in this field is in developing stage, the many problem in science in practical application wait further exploration, as imaging ellipsometer, all adopt polychromatic light or multiple wavelength laser light source, physical construction more complicated; Adopt the CCD device, disturbed sample polarization of reflected light attitude, and very strong background signal is arranged, image taking speed is slow, can't realize the online detection of original position high sensitivity, data processing complex, and accuracy is high inadequately; Have the precision measure of different biochemical reactions also to be difficult to realize that the most experiments data obtain under coarse experimental design for the array on the both same chip of complex array, and the degree of accuracy and the comfort level that detect all have certain shortcoming; Can't avoid obeying specific adsorption etc.Therefore ellipse inclined to one side system still need optimization, have much room for improvement on principle and the data processing method and improve.Wherein one of method is to unite use to improve instrumental sensitivity and reliability, like document (Wei-Liang Hsu, Shu-Sheng Lee, Chih-Kung Lee with Other Instruments; J.Biomed.Opt., introduced people such as Wei-Liang Hsu 2009,14,024036) itself and surface plasma resonance have been combined the sensitivity that improves biochip test and accuracy etc.; Document (Schuy S, Faiss S, Yoder NC, Kalsani V; Kumar K, Janshoff A, et al, J.Phys.Chem.B Biointerfaces; 2008,112,8250.) having introduced people such as Schuy S combines it and realizes highly sensitive detection etc. with total internal reflection spectrum.Two of method is exactly the use of new technology and efficient algorithm, as having introduced the use of light polarization modulator and the introducing of genetic algorithm in the document (Cormier G, BoudreauR.Opt.Soc.Am, 2000,1,129.).The oblique incidence ellipsometry is that very big dirigibility has been arranged on the basis of the ellipse inclined to one side technology of traditional optical.The ellipse inclined to one side incident light of traditional optical is near normal incidence; Only be applicable to the detection of the optical properties of material of surface anisotropy; The ellipse inclined to one side technology of oblique incidence differential optical has added the device of adjustment back of the body end intensity and has eliminated back of the body end signal in the oblique incidence light path; Make its measurement be not limited to have the anisotropic material of surface optics like this, adopt the method for difference to improve precision greatly simultaneously.If the laser co-focusing technology is combined with the ellipse inclined to one side technology of optics; Utilize the high sensitivity of the ellipse inclined to one side technology of oblique incidence; Can obtain the dynamic information of intermolecular interaction; And the high three-dimensional image resolution ratio of laser co-focusing technology and darker characteristics such as penetrability, be aided with the integrated advantage of microflow control technique economy, be expected to realize to the original position of biologic array reaction in real time, high flux, qualitative, quantitative polynary Sensitive Detection.And also do not have burnt technology of set laser copolymerization and the ellipse inclined to one side technology of optics at present both at home and abroad in the commercial prod of one.Device of the present invention can be applied to comprise micromolecule; Protein molecular; Big molecule, nanometer monomer, interaction between the cell and dynamic process realize that original position is real-time; Quantitatively, the application in the qualitative Sensitive Detection; Simultaneously also make research field extend to fields such as nanometer, information, material from living things system, thereby further enrich understanding and the understanding of people, for the basic physical chemistry problem that discloses in life science, nano science, information science, the material science research provides new research method and means microworld.
The ellipse higher flux biomolecular reaction imaging detection device of a cover laser co-focusing oblique incidence of setting up of the present invention; On the ellipse inclined to one side principle of tradition, introduced difference polarization method again; Characteristics in conjunction with the sensitive fluorescence imaging of laser co-focusing technology; Be aided with micro-fluidic and chemical means and avoided non-special absorption, can realize the accurate detection of the original position of biochip, real-time, quantitative, qualitative, high flux, multicomponent reaction.Adopt efficient mathematical physical method research canonical biometric reaction mechanism simultaneously, expanded the range of application of this system.The polynary biochemical reaction detection system that can develop China's independent intellectual property right of carrying out of the present invention improves the international competitiveness of China aspect the biochip supplies analysis device, and huge applications prospect and economic worth are arranged.The imaging system of development can be applied to different plant species.
Summary of the invention
The objective of the invention is the laser co-focusing technology is combined with the ellipse inclined to one side technology of oblique incidence; Utilize the highly sensitive performance of ellipse inclined to one side detection technique to obtain the dynamic information of intermolecular interaction; And utilize the high three-dimensional image resolution ratio of laser co-focusing technology and darker characteristics such as penetrability; Be aided with microflow control technique; With realize to biomolecule high flux, original position in real time, quantitative Sensitive Detection, thereby provide a kind of can the original position qualitative and quantitative detection again can the real-time monitored biomolecular reaction the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence.
The ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence of the present invention is based on laser confocal microscope system, and the original paper that is adopted is the commercially available prod; Described device comprises example platform, armstrong's patent angle station, ellipse inclined to one side detecting unit, the burnt unit of copolymerization, data acquisition and data handling system.
One has on the described example platform of three-D electric governor motion the sample substrate that is loaded with the biological sample microarray is arranged, and hyperchannel constant temperature flow cell is arranged below described sample substrate;
The inlet of described hyperchannel constant temperature flow cell is connected with the liquid outlet of multichannel injection pump through pipeline; The liquid outlet of hyperchannel constant temperature flow cell is connected with the inlet of multichannel injection pump through pipeline, so that the liquid circulation flow in the hyperchannel constant temperature flow cell is logical;
The burnt unit of described copolymerization comprises that one is inverted laser confocal microscope, settles described hyperchannel constant temperature flow cell in the stage position of being inverted laser confocal microscope;
Described armstrong's patent angle station comprises the vertical flat board of placing that has groove of two symmetries that are fixed on the corresponding both sides of described example platform, and two are installed in two mechanical arms in the groove respectively; But described mechanical arm in the vertical direction carries out the regulated at will (through the rotation of manual control machine tool arm, can regulate the incident beam that focuses on the sample and the angle of outgoing beam) between angle 0-90 °;
Described ellipse inclined to one side detecting unit comprises the laser instrument, the polarizer, light ball modulator, phase-shifter and the condenser lens that are provided with in proper order along the input path direction that is installed on the described mechanical arm, is installed in the condenser lens, chopper, polaroid analyze and the photodetector that are provided with in proper order along the reflected light path direction on described another mechanical arm;
Described data acquisition and data handling system comprise lock-in amplifier, data collecting card and computing machine;
The data inlet end of described lock-in amplifier is connected with the data endpiece of described photodetector through data line; The data endpiece of lock-in amplifier is connected with the data inlet end of described data collecting card through data line; The data endpiece of described data collecting card is connected with a data inlet end of described computing machine through data line, and another data inlet end of computing machine is connected with the controller of described multichannel injection pump through data line.
Described hyperchannel constant temperature flow cell adopting quartz glass or frosted glass are done wall and bottom surface, realize sealing by rubber seal.
Described data collecting card has BNC adapter and data acquisition software, and data collecting card carries out data acquisition, and carries out simple data processing by the data processing software in the data collecting card.
Described BNC adapter receives the outputting data signals of described lock-in amplifier; Gather the data of BNC adapter output by data collecting card; And sending described computing machine to, computing machine carries out data storage, analysis and processing and imaging to the data that the data capture card sends.
Described laser instrument, the polarizer, light ball modulator, phase-shifter and the condenser lens that is provided with in proper order along direction on the input path; The condenser lens, chopper, polaroid analyze and the photodetector that are provided with in proper order along the reflected light path direction are integrated in respectively on two mechanical arms; Two mechanical arms are installed in respectively on the flat board that has groove of two symmetries; Through the hand rotation mechanical arm, can regulate the incident beam that focuses on the sample substrate and the angle of folded light beam.
Laser instrument in the described ellipse inclined to one side detecting unit is that wavelength is the He-Ne laser instrument of 632.8nm.
The modulating frequency O=50kHz of described light ball modulator.
Described sample substrate is common lid slide or microslide.
After the light of above-mentioned laser instrument output becomes linearly polarized light through the polarizer; The cycle that produces P polarized light and S polarized light through light ball modulator changes; Shine on the sample after getting into condenser lens focusing through phase-shifter again, after reflected light gets into condenser lens, again behind chopper and polaroid analyze; Receive by photodetector; Output to lock-in amplifier by data line again and carry out carrying out data acquisition by data collecting card again after amplitude is separated with the position, and carry out data processing by the data handling system in the computing machine, the data message after will handling then converts image to.
The present invention combines to be inverted the advantage of laser confocal microscope and ellipse inclined to one side detection, can carry out carrying out fluorescence imaging when the ellipse number of believing one side only of quantitative and qualitative detects to the reaction between biological sample simultaneously and observe.
The present invention has:
(1) the present invention adopts light path motionless, and sample is done the mode that two dimension moves, and combines oblique incidence and modulation system mutually, has improved signal to noise ratio (S/N ratio) and detection sensitivity.
(3) the present invention can realize the high throughput testing of solid phase and liquid phase biological sample.
(3) combine microflow control technique in the device of the present invention, can really realize multiple reaction high throughput testing simultaneously.
(4) apparatus structure of the present invention is simple, and easy operating can be applied to a plurality of fields such as chemistry, biology, medical science, material, environment, safety.For the further investigation of the problem in science of nano science, material science, biological chemistry and crossing domain provides new high-level efficiency research means.
Description of drawings
Fig. 1. the structural representation of the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence of the present invention.
Fig. 2. the ellipse higher flux biomolecular reaction imaging detection device of the employing laser co-focusing oblique incidence of the embodiment of the invention 1 is to carrying out the result of different bioprotein Molecular Detection on same cover glass; Wherein, Fig. 2 a and Fig. 2 b are respectively ellipsometry imaging and confocal fluorescent imaging results.
Reference numeral
1.He-Ne laser instrument 2. polarizers 3. light ball modulators
4. phase-shifter 5. condenser lenses 6. hyperchannel constant temperature flow cells
7. be inverted laser confocal microscope 8. multichannel injection pumps 9. condenser lenses
10. chopper 11. polaroid analyzes 12. photodetectors
13. lock-in amplifier 14. data collecting cards 15. computing machines
16. be loaded with the cover glass of biological sample microarray
Embodiment
Come the present invention is done further explanation and explanation below in conjunction with specific embodiment.
Embodiment 1
See also Fig. 1, the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence comprises example platform, armstrong's patent angle station, ellipse inclined to one side detecting unit, the burnt unit of copolymerization, data acquisition and data handling system.
One has on the described example platform of three-D electric governor motion the cover glass 16 that is loaded with the biological sample microarray is arranged; The hyperchannel constant temperature flow cell 6 that is of a size of 15mm * 15mm * 3mm is arranged below described cover glass; Described hyperchannel constant temperature flow cell 6 is that the adopting quartz glass polytetrafluoroethylmaterial material is done wall and bottom surface, is sealed by rubber seal.
The inlet of described hyperchannel constant temperature flow cell 6 is connected through the liquid outlet of pipeline with multichannel injection pump 8; The liquid outlet of hyperchannel constant temperature flow cell 6 is connected through the inlet of pipeline with multichannel injection pump 8; Multichannel injection pump is as the circulation power source of liquid in the hyperchannel constant temperature flow cell, so that the liquid circulation flow in the hyperchannel constant temperature flow cell is logical.
The burnt unit of described copolymerization comprises that one is inverted laser confocal microscope 7, settles described hyperchannel constant temperature flow cell 6 in the stage position of being inverted laser confocal microscope 7.
Described armstrong's patent angle station comprises the vertical flat board of placing that has groove of two symmetries that are fixed on the corresponding both sides of described example platform, and two are installed in two mechanical arms in the groove respectively; But described mechanical arm in the vertical direction carries out the regulated at will between angle 0-90 °.
Described ellipse inclined to one side detecting unit comprises the laser instrument 1, the polarizer 2, light ball modulator 3, phase-shifter 4 and the condenser lens 5 that are provided with in proper order along the input path direction on the mechanical arm that is installed in above-mentioned two mechanical arms, is installed in the condenser lens 9, chopper 10, polaroid analyze 11 and the photodetector 12 that are provided with in proper order along the reflected light path direction on another mechanical arm in above-mentioned two mechanical arms; The light of laser instrument 1 output is through behind 2 one-tenth linearly polarized lights of the polarizer, behind light ball modulator 3 and phase-shifter 4, by inciding on the cover glass that is loaded with the biological sample microarray 16 on the sample stage after condenser lens 5 focusing; Through the reflection of biological sample, get into chopper 10 via convergent lens 9, after being received by photodetector 12 by polaroid analyze 11 backs again, export by data line again.
Described data acquisition and data handling system comprise lock-in amplifier 13, have the data collecting card 14 and the computing machine 15 of BNC adapter and data acquisition software.
The data inlet end of described lock-in amplifier 13 is connected with the data endpiece of described photodetector 12 through data line; The data endpiece of lock-in amplifier 13 is connected with the data inlet end of described data collecting card 14 through data line; The data endpiece of described data collecting card 14 is connected with a data inlet end of described computing machine 15 through data line, and another data inlet end of computing machine 15 is connected through the controller of data line with described multichannel injection pump 8.
Laser instrument in this device is that to adopt power be 10mW, wavelength be the He-Ne laser instrument of 632.8nm as light source, the bright dipping aperture is 3mm; , laser instrument 1 emitting laser bundle becomes the linearly polarized light that the polarization direction is parallel to the plane of incidence after proofreading and correct the polarization direction via the polarizer (NewFocus 5524); Behind PEM100 light ball modulator (modulating frequency O=50kHz), produce the cycle variation (be 50kHz modulation period) that P polarized light and S polarized light are built, behind phase-shifter compensation (multistage half slide) control phase, focus on the cover glass that is loaded with the biological sample microarray again; Method with chemistry on the described cover glass is modified with bio-sensitive film such as IgG antibody molecule array; Regulate the incident angle of light beam through manual angle station; Make the incident light incident angle between 80-90 °; Cover glass has that circulation has anti-IgG in the following hyperchannel constant temperature flow cell of biological sample one side, and contacts with biomolecule microarry and to react, through multichannel injection pump (74901-15:0.001ul/hr～147ml/hr flow velocity; Adaptive 10ul～140ml syringe; Minimum step is 9.5 * 10-6mm/min, can realize the suction automatically simultaneously of 10 passages) control anti-IgG reactant passes in and out hyperchannel constant temperature flow cell and the biomolecule microarry reaction causes refraction index changing, and the change of refractive index causes the variation of ellipse inclined to one side detection signal.The temperature range of said hyperchannel constant temperature flow cell is 20 ℃～45 ℃, precision ± 0.5 ℃, and the flow rates of anti-IgG reactant turnover hyperchannel constant temperature flow cell is 0.005～380ml/min, precision is ± 1%. The described K9 glass of buying on the cover glass 16 employing markets of biological sample that is loaded with, cover glass are fixed in the three-D electric that can buy on the market to be transferred on the objective table, objective table and the supporting use of inversion laser confocal microscope.Described inversion laser confocal microscope (Nikon C1 Si) resolution is 2um.
Behind the light line focus lens 9 of biological sample surface reflection, receive by photodetector 12 (silicon photoelectric diode 9863/100B) by chopper 10 (Stanford ResearchSystems SR540) copped wave and polaroid analyze 11 (CVI Laser CPAD-10.0-425-675) back; Lock-in amplifier 13 (StanfordResearch Systems SR830 DSP) is received the light signal of photodetector 12 through data line, and light signal is read by the data acquisition and processing (DAP) system after converting electric signal to.
Device of the present invention is the mode that adopts fixed light path sample two dimension to move; Specifically be with the side of two mechanical arms (aluminium alloy processes) that are integrated with input path and reflected light path element respectively through screw retention on the same flat board that has a groove; Two mechanical arms can carry out the rotation perpendicular to surface level in groove; Can be that axle rotates simultaneously with the same point; Thereby can form the armstrong's patent angle station thus according to the incident angle that need between 80 °～90 °, regulate light beam of experiment; Simultaneously sample place can the accurate electronic control sample stage that moves on, realized that like this sample moves the fixing pick-up unit of light path.
Described data acquisition and data handling system comprise lock-in amplifier 13, have the data collecting card 14 (PCI-6220) and the computing machine 15 of the data acquisition software that BNC adapter (BNC-2110) and Labview write.The electric signal of lock-in amplifier 13 outputs at first carries out subchannel via the BNC adapter to be handled; The signal of lock-in amplifier output is divided into amplitude information and two parts of phase information; Then the amplitude information and the phase information of lock-in amplifier are sent into the data collecting card collection respectively; Sending the data that collect to write with Labview data processor at last stores, analyzes and handle; Be inverted laser confocal microscope simultaneously course of reaction is carried out to picture, the result is input to computing machine 15 and ellipse inclined to one side detection signal data in real time contrast.
Adopt above-mentioned experimental provision can detect variations in refractive index 2 * 10
-4, sample detection sensitivity reaches 10
-11G/ml, and to reach fluoroscopic image resolution be 2 μ m, longitudinal frame is 0.05nm.
Fig. 2 is for adopting the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence on same cover glass, carrying out different bioprotein molecules and simple target results of interaction to be detected.3 * 4 biomolecule arrays are arranged on the cover glass, and first classifies bovine serum albumin (BSA) as, and second classifies prostate specific antibody (anti-PSA) as, and the 3rd classifies sheep anti-mouse igg (Goat Anti-Mouse IgG) as, and the 4th classifies cancer embryo antibody (anti-CEA) as.Through the four-way syringe pump is the Cy3 fluorescence labeling prostate characteristic antigen (PSA) that each passage injects same concentrations in the hyperchannel sample cell; Have only the anti-PSA of secondary series can interact with it after feeding PSA, Fig. 2 a and Fig. 2 b are respectively ellipse inclined to one side and the result that confocal fluorescent forms images of the oblique incidence of using device of the present invention to obtain.In Fig. 2 a, have only the oblique incidence ellipsometry imaging of secondary series before and after PSA feeds, obvious variation to be arranged, first row are basic not to be changed, and third and fourth row change all not obvious.Have only secondary series after feeding PSA, tangible fluorescence to be arranged among Fig. 2 b.Through the comparison of Fig. 2 a and Fig. 2 b, further confirm to have only anti-PSA specific reaction to take place with PSA.Above result shows: device of the present invention can be realized the laser co-focusing and the ellipsometry imaging of high throughput biological molecular reaction simultaneously; Can carry out quantitative and qualitative Sensitive Detection to biomolecular reaction; Fluoroscopic image can be got rid of the non-specific adsorption in the ellipsometry imaging, makes testing result more accurate.
The structure of device is identical with embodiment 1, and just silicon photoelectric diode 12 employing models are the photomultiplier of 9863/100B, and other is identical with embodiment 1, and the sensitivity of the device of present embodiment and accuracy of detection can improve 2 numbers of poles.
The structure of device is identical with embodiment 1; Just silicon photoelectric diode 12 employing photosensitive areas are square high-speed induction silicon photo diode (BPX-65) composition 30 * 30 two-dimensional arraies of 1.0mm2; Other is identical with embodiment 1, and the device of present embodiment can realize that to area be 9 (cm)
2The microarray biological sample detects.
The structure of device is identical with embodiment 1, and just silicon photoelectric diode 12 adopts 1300 * 1024 CCD face battle array (AM1300), and other is identical with embodiment 1, and the device of present embodiment can realize that to area be 9 (cm)
2The microarray biological sample detects.
Adopt the device of embodiment 1; Just adopt the method for chemical modification; With the antibody modification of AIDS virus 1 type (HIV-1) core protein p24 on the cover glass surface; Stream has the antigen of AIDS virus 1 type (HIV-1) the core protein p24 of variable concentrations in the hyperchannel constant temperature flow cell, and other is identical with embodiment 1, can carry out the dynamic analysis of multiple concentration diagnostic detection to AIDS virus 1 type simultaneously.
Adopt the device of embodiment 1; Just adopt the method for chemical modification; The cover glass finishing there is the fluorescence probe that to discern mercury ion; Stream has the biological cell albumen that is polluted by mercury ion in the hyperchannel constant temperature flow cell, and other is identical with embodiment 1, can the mercury ion in the biosome be detected.
Adopt the device of embodiment 1; Just adopt the method for chemical modification; The cover glass finishing there is the fluorescence probe that to discern zinc ion; Stream has the biological cell albumen that is polluted by zinc ion in the hyperchannel constant temperature flow cell, and other is identical with embodiment 1, can the zinc ion in the biosome be detected.
Adopt the device of embodiment 1; Just adopt the method for chemical modification; The cover glass finishing there is the fluorescence probe that to discern magnesium ion; Stream has the biological cell albumen that is polluted by magnesium ion in the hyperchannel constant temperature flow cell, and other is identical with embodiment 1, can the magnesium ion in the biosome be detected.
Have the cover glass of biomolecule to put upside down finishing, be about to sample towards last, execute under the multi-passage circulating pond of example in 1 and the multichannel injection pump situation high throughput testing of the solid protein matter chip that can realize point sample instrument is prepared not using.
Adopt the device of embodiment 1; Just adopt the method for chemical modification; The cover glass finishing there is the fluorescence probe that to discern fluorine ion; Stream has the biological cell albumen that is polluted by fluorine ion in the hyperchannel constant temperature flow cell, and other is identical with embodiment 1, can the fluorine ion in the biosome be detected.
Adopt the device of embodiment 1; Just adopt the method for chemical modification; The cover glass finishing there is the fluorescence probe that to discern mercury ion, zinc ion, magnesium ion, fluorine ion and cadmium ion simultaneously; Stream has the biological cell albumen that is polluted by mercury ion, zinc ion, magnesium ion, fluorine ion and cadmium ion in the hyperchannel constant temperature flow cell, and other is identical with embodiment 1, can detect simultaneously the mercury ion in the biosome, zinc ion, magnesium ion, fluorine ion and cadmium ion.
Alpha-fetoprotein (AFP), carcinomebryonic antigen (CEA), carbohydrate antigen 19-19 (CA199), carbohydrate antigen 24-2 (CA242), carbohydrate antigen 125 (CA125), carbohydrate antigen 15-3 (CA153), free prostate gland specially property antigen (F-PSA), prostate specially property antigen (PSA), neuron enolase (NSE), human chorionic gonadtropin (HCG), human growth hormone (HGH), ferritin tumor markers such as (Ferritin) are modified with on a slice lid glass; Other is identical with embodiment 1, can realize simultaneously the real-time quantitative of kinds of tumors label is detected.
Claims (6)
1. ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence, described device comprise example platform, armstrong's patent angle station, ellipse inclined to one side detecting unit, the burnt unit of copolymerization, data acquisition and data handling system; It is characterized in that:
One has on the described example platform of three-D electric governor motion the sample substrate that is loaded with the biological sample microarray is arranged, and hyperchannel constant temperature flow cell is arranged below described sample substrate;
The inlet of described hyperchannel constant temperature flow cell is connected with the liquid outlet of multichannel injection pump through pipeline, and the liquid outlet of hyperchannel constant temperature flow cell is connected with the inlet of multichannel injection pump through pipeline;
The burnt unit of described copolymerization comprises that one is inverted laser confocal microscope, settles described hyperchannel constant temperature flow cell in the stage position of being inverted laser confocal microscope;
Described armstrong's patent angle station comprises the vertical flat board of placing that has groove of two symmetries that are fixed on the corresponding both sides of described example platform, and two are installed in two mechanical arms in the groove respectively; But described mechanical arm in the vertical direction carries out the regulated at will between angle 0-90 °;
Described ellipse inclined to one side detecting unit comprises the laser instrument, the polarizer, light ball modulator, phase-shifter and the condenser lens that are provided with in proper order along the input path direction that is installed on the described mechanical arm, is installed in the condenser lens, chopper, polaroid analyze and the photodetector that are provided with in proper order along the reflected light path direction on another described mechanical arm;
Described data acquisition and data handling system comprise lock-in amplifier, data collecting card and computing machine;
The data inlet end of described lock-in amplifier is connected with the data endpiece of described photodetector through data line; The data endpiece of lock-in amplifier is connected with the data inlet end of described data collecting card through data line; The data endpiece of described data collecting card is connected with a data inlet end of described computing machine through data line, and another data inlet end of computing machine is connected with the controller of described multichannel injection pump through data line;
Data line on the described inversion laser co-focusing mirror is connected with the data port of described computing machine.
2. the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence according to claim 1; It is characterized in that: described hyperchannel constant temperature flow cell is that adopting quartz glass or frosted glass are done wall and bottom surface, realizes sealing by rubber seal.
3. the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence according to claim 1, it is characterized in that: described data collecting card has BNC adapter and data acquisition software.
4. the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence according to claim 1 is characterized in that: described laser instrument is that wavelength is the He-Ne laser instrument of 632.8nm.
5. the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence according to claim 1 is characterized in that: the modulating frequency Ω of described light ball modulator=50kHz.
6. the ellipse higher flux biomolecular reaction imaging detection device of laser co-focusing oblique incidence according to claim 1, it is characterized in that: described sample substrate is cover glass or microslide.
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