CN101339135A - Method for promoting biological detection sensitivity by photon crystal - Google Patents
Method for promoting biological detection sensitivity by photon crystal Download PDFInfo
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- CN101339135A CN101339135A CNA200810117955XA CN200810117955A CN101339135A CN 101339135 A CN101339135 A CN 101339135A CN A200810117955X A CNA200810117955X A CN A200810117955XA CN 200810117955 A CN200810117955 A CN 200810117955A CN 101339135 A CN101339135 A CN 101339135A
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Abstract
The invention relates to a detection method of a variety of proteins and genes in a biological sample, in particular to a method which uses a photonic crystal to improve the sensitivity of biological detection. The method makes use of the characteristics of the photonic crystal to match the fluorescence labeled biological functional matter; the fluorescence labeled biological functional matter is used for detecting the specific response or specific affinity of the target biological matter; the target biological matter responds to the biological functional matter, to result in the luminous change of the biological functional matter which is labeled by fluorescence labeled molecules; a photon crystal is used for enhancing the reading and identification of fluorescence signals of the fluorescence labeled molecules; thus the method can effectively enhance the sensitivity of the detection method of the target biological matter, and further realize the high-sensitivity biological detection.
Description
Technical field
The invention belongs to the method that multiple proteins in the biological sample and gene are detected, particularly utilize photonic crystal to improve the method for biological detection sensitivity.
Background technology
The develop rapidly of life science has proposed a large amount of new problems to analytical chemistry, concentrates on the analysis of biomacromolecules such as polypeptide, protein, nucleic acid at present, biopharmaceutical analysis, and ultratrace, ultramicron bioactivator are analyzed, even microbiological analysis etc.Therefore, bioanalysis has become one of field, most important forward position of modern analysis chemical developer.In order to adapt to the requirement of this situation, numerous analytical chemistry workers are constantly making great efforts to develop new analytical approach and technology.Biology sensor is a kind of novel analytical instrument that grows up under multidisciplinary intersection background, and at present the total electricity of detection mechanism, light, heat, the amount used of biology sensor is 4 kinds, wherein with light signal as surveying the machine-processed optical biosensor that is called.Optical biosensor because of have fast, sensitive, accurately and characteristics such as high selectivity the most noticeable.Because optical biosensor has nondestructive operator scheme, higher signal produces and reading speed, add development of fiber technology and application in recent years, the test mode and the application of optical biosensor are all expanded greatly, become the most general biology sensor of application so far, have vast potential for future development.
With the fluorescence signal is read output signal, is a kind of topmost signals collecting form of optical biosensor.Fluorometry just occurs as far back as the sixties in 19th century as a kind of analytical approach.This method has many outstanding advantages: 1. selectivity is good; 2. has multiple location parameter (as fluorescence lifetime, fluorescence anisotropy, fluorescence quantum yield, fluorescence exciting wavelength, emission wavelength etc.); 3. highly sensitive; 4. have multiple detection technique and method (as synchronous fluorescence, derivative fluorescence, fluorescence polarization, fluorescence kinetics analytic approach, three-dimensional fluorescence spectrum method and resolution, time-resolved fluoroimmunoassay etc. mutually).So can be selected, thereby have that applicability is strong, selectivity good, the characteristics of range of linearity broad according to the difference of practical measurement condition.
Yet in real process, because the singularity of sample, the existing sensitivity of fluorescent technique still can not be satisfied the needs of all mensuration.Therefore, hope can further improve the sensitivity of fluoroscopic examination, and its range of application is enlarged more.Be used to improve the fluoroscopic examination sensitivity of method have multiple, for example: utilize new technology, new unit, further improve the detection sensitivity [CN 1602420A] of instrument; Fluorescence amplifies separation [CN 101082583]; [X.Gao, Y.Cui, R.M.Levenson, L.W.K.Chung, and S.Nie, Nat.Biotechnol.22,969 (2004) .] utilize enzyme linked immunoassay, [R.M.Lequin, Clin.Chem.51,2415 (2005) .] polymerase chain reaction, [R.K.Saiki, S.Scharf, F.Faloona, K.B.Mullis, G.T.Horn, H.A.Erlich, and N.Arnheim, Science 230,1350 (1985) .] many fluorescent chromophores probe [B.S.Gaylord, A.J.Heeger, G.C.Bazan, Proc.Natl.Acad.Sci.U.S.A.99,10954 (2002) .] etc. biochemical method improve the enlargement factor of fluorescence signal.
But the detection sensitivity limitation that improves fluorescent techniques with above-mentioned these methods is very big, and the degree of raising is limited by the interference etc. of quantum yield, photodissociation and the background fluorescence of fluorescence species self.Consider from improving the instrument aspect, reach high-sensitive test request, need strict control experiment condition, reduce background fluorescence to greatest extent, need use complicated optical system, also high especially to the quality requirements of detecting device, therefore instrument costs an arm and a leg, experimentation is strict, this method be applied to have the reality difficulty, has seriously restricted the paces of its practicability.Therefore, the approach of the new raising fluoroscopic examination sensitivity of necessary searching.
Photonic crystal is a kind of periodic ordered structure of specific inductive capacity (or refractive index) that has, and can limit, controls and regulate and control photon.Photonic crystal can be used as a kind of good optical chamber, for emission and the propagation of controlling light provides good theory vision and test basis, especially in recent years in the research of the photonic crystal of visible-range, make it tempting application prospect be arranged at aspects such as photoelectric device and optical chips.
Summary of the invention
The object of the present invention is to provide a kind of combination property height, highly sensitive, cost is low, the photonic crystal that utilizes quick, convenient, that easily realize improves the method for biological detection sensitivity.
The present invention is a characteristic of utilizing photonic crystal, cooperate fluorescence labeling biological function material, utilize special response or the pathoklisis of fluorescence labeling biological function material to the target organism material, target organism material and biological function material respond, cause the luminous change of biological function material with the fluorescent tag molecule mark, strengthen the reading and distinguishing of fluorescence signal of fluorescent tag molecule by photonic crystal, can effectively strengthen the sensitivity of the detection architecture of target organism material, thereby realize highly sensitive biological detection.
The method that the present invention utilizes photonic crystal to improve biological detection sensitivity may further comprise the steps:
(1) in aqueous solution, utilize single fluorescent tag molecule or give the acceptor fluorescence mark right, the biological function material is carried out fluorescence labeling, obtain containing single fluorescent tag molecule or give the aqueous solution of acceptor fluorescence mark the biological function material of mark;
(2) photonic crystal of selecting the glow peak of the fluorescent tag molecule in the biological function material of the single fluorescent tag molecule mark in forbidden photon band band edge and the step (1) to be complementary, utilize the forbidden photon band band edge of photonic crystal to strengthen the reading and distinguishing of fluorescence signal of fluorescent tag molecule, thus the sensitivity that improves detection architecture; Or
Select the photonic crystal of the glow peak of giving body in the biological function material of mark being complementary for the acceptor fluorescence mark in forbidden photon band or forbidden photon band band edge and the step (1), utilize the forbidden photon band of photonic crystal or forbidden photon band band edge to strengthen to the reading and distinguishing of the right fluorescence signal of acceptor fluorescence mark, thus the sensitivity that improves detection architecture.Its principle be utilize photonic crystal forbidden photon band to giving luminous localization of body or forbidden photon band band edge to giving the luminous enhancing of body, promote to give the acceptor fluorescence mark between the fluorescence resonance energy transmission.
(3) adopt this area several different methods such as osmosis, investment, absorption method, covalent bond method or cross-linking method commonly used, contain single fluorescent tag molecule or be fixed on the photonic crystal of step (2) for the acceptor fluorescence mark to the single fluorescent tag molecule in the aqueous solution of the biological function material of mark or to the acceptor fluorescence mark what step (1) obtained, obtained filling the photonic crystal of fluorescence labeling biological function material; Described osmosis, investment, absorption method, covalent bond method or cross-linking method can be referring to (a) Li Yingxiu, Zhu Liande, Zhu Guoyi, analytical test journal, 2002,21,89; (b) money the army and the people, Li Xuxiang, sensor technology, 2001,20,6.
(4) the target organism substance solution is dripped on the resulting photonic crystal of filling fluorescence labeling biological function material of step (3), the target organism material is detected, by the variation that fluoroscopic examination instruments such as fluorescence spectrophotometer or fluorescence co-focusing microscope write down the fluorescence signal of fluorescence labeling biological function material, finish identification to the target organism material.
Describedly contain single fluorescent tag molecule or the volumetric molar concentration of the aqueous solution of the biological function material of mark is preferably 10 for the acceptor fluorescence mark
-16Mol～10 mol.
The volumetric molar concentration of described target organism substance solution is preferably 10
-16Mol～1 mol.
Described target organism material is selected from a kind of in nucleic acid, antigen-antibody, conjugated protein, lectins, the hormone receptor etc.
Described biological function material is selected from a kind of in enzyme, nucleic acid, antigen-antibody, conjugated protein, lectins, the hormone receptor etc.
Described single fluorescent tag molecule is selected from a kind of in chelate, fluorescin of 3 valency rare earth lanthanide etc. in organic dyestuff, luminescent quantum dot, compound fluorescent nano particles, the lanthanide chelate etc.
Described give the acceptor fluorescence mark right give a kind of in chelate that body and acceptor be selected from 3 valency rare earth lanthanide in organic dyestuff, luminescent quantum dot, compound fluorescent nano particles, the lanthanide chelate etc. respectively, the fluorescin etc.
Described organic dyestuff is tetramethyl rhodamine, RB 200, TRITC, fluorescein isothiocynate, ethidium bromide, acridine, phenanthridines, fluorescein, TOTO[S.C.Benson, R.A.Mathie, A.N.Glazer et al.Nucleic Acids Research, 1993b, 21,5720] or YOYO[S.C.Benson, P.Singh, A.N.Glazer.Nucleic Acids Research, 1993a, 21,5727] etc.
The nano particle that described luminescent quantum dot is made up of II-VI family element and III-V family element, they are CdS, CdSe or CdTe etc.
Described compound fluorescent nano particles can be referring to J.R.Taylor, M.M.Fang, S.M.Nie, Anal Chem, 2000,72 (9), 1979.
The chelate of 3 valency rare earth lanthanide is europium (Eu3), terbium (Tb3) or cerium (Ce3) etc. in the described lanthanide chelate.
Described fluorescin is phycobiliprotein or green fluorescent protein etc.
Described photonic crystal is the periodicity dielectric structure with forbidden photon band, and the photonic crystal etc. have the photonic crystal of opal structural or to have counter opal structure is arranged in self assembly.
Described photonic crystal with opal structural is that to be the mono-dispersion microballoon self assembly of the polystyrene, polymethylmethacrylate of 150nm～1.5um, poly-(styrene-methyl methacrylate-acrylic acid) or silicon dioxide by particle diameter prepared.Described preparation method with photonic crystal of opal structural comprises template [O.D.Velev, E.W.Kaler, Adv.Mater.2000,12,531], gravity settling [H.Miguez, F.Meseguer, C.Lopez, et al.Langmuir, 1997,13,6009], centrifugation method [C.F.Blanford at a slow speed, H.Yan, R.C.Schroden, et al.Adv.Mater.2001,13,401], with capillary force self assembly in the plane [N.D.Denkov, O.D.Velev, Kralchevsky P A, et al.Nature 1993,361,26], vertical deposition method [P.Jiang, J.F.Bertone, K.S.Hwang, et al.Chem.Mater.1999,11,2132] etc.
Described photonic crystal with counter opal structure is to be template with above-mentioned photonic crystal with opal structural, utilizes template to prepare.
Utilize single fluorescent tag molecule or give the acceptor fluorescence mark right, the biological function material is carried out fluorescently-labeled method can be referring to (a) O.Seitz, Angew.Chem.2000,39,3249; (b) D.M.Hammond, A.Manetto, J.Gierlich, V.A.Azov, P.M.E.Gramlich, G.A.Burley, M.Maul, T.Carell, Angew.Chem.2007,119,4262; Angew.Chem.Int.Ed.2007,46,4184; (c) F.He, Y.L.Tang, M.H.Yu, S.Wang, Y.L.Li, D.B.Zhu, Adv.Func.Mater.2006,16,91; (d) W.C.W.Chan, S.M.Nie, Science 1998,281, and 2016.
The present invention utilizes photonic crystal can modulate the characteristics of luminescence of fluorescent material, cooperates fluorescence labeling biological function material, obtains high sensitivity and reaches bio-identification system efficiently.The luminous change with the caused surrounding environment of bio-identification of fluorescent tag molecule changes, luminous intensity and route of transmission via the forbidden photon band of photonic crystal or forbidden photon band band edge modulation fluorescent tag molecule, make the detection architecture light signal strengthen, thereby realize high-sensitivity detection the target organism material.
The present invention has mainly provided a kind of method of utilizing photonic crystal to improve biological detection sensitivity, other available fluorescent material is still arranged except above-mentioned fluorescent tag molecule, on behalf of the present invention, the fluorescent tag molecule described in the present invention only limit the use of above-mentioned fluorescent material, has also comprised other biomarker fluorescent material.According to method provided by the invention, all can realize utilizing photonic crystal to improve biological detection sensitivity.
Method of the present invention can be widely used in quarantine, gene order and the gene function analysis in fields such as clinical detection, drug screening, life science, medical science, chemistry, new drug development, biological weapons war, judicial expertise, food and environmental sanitary inspection and the detection of various microorganisms.
Method of the present invention has following advantage:
1. the present invention utilizes the characteristic to the light regulation and control of photonic crystal, and the intensity that can strengthen the light signal of detection architecture also can be controlled its route of transmission, thereby can improve the sensitivity of detection architecture.
2. the porous road structure in the photonic crystal among the present invention helps single fluorescent tag molecule or gives the acceptor fluorescence mark the right dispersion of giving body or acceptor, reduce the fluorescent tag molecule concentration quenching to detecting the influence of stability and sensitivity, the stability and the sensitivity that improve light signal.
3. the photonic crystal of utilization of the present invention is to adopt the material with good bio-compatibility to prepare, can provide good immobilization carrier for biological functional mass, and there is porous road structure, help evenly distributing fast of biological function material molecule, can improve the problems such as stability, sensitivity and response speed of detection.
4. preparation procedure of the present invention is simple, and cost of manufacture is low, the near infrared of especially self assembly sub-micron colloidal solid preparation and the photonic crystal of visible region.Simultaneously, be beneficial to the immobilization of biological function material, and applied widely, can realize more high sensitivity, biological detection better optionally.
Embodiment
Embodiment 1.
1. water preparation mass concentration is that single particle diameter that disperses of 0.2% is poly-(styrene-methyl methacrylate-acrylic acid) emulsion particle solution of 210nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the wave carrier piece with cleaning is a substrate, in temperature is 50 ℃, humidity is under 50% the controlled condition, the latax that step 1 is obtained by vertical sedimentation is deposited on the substrate, self assembly is arranged and is prepared poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal on substrate, poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band, and the forbidden photon band of photonic crystal is positioned at 518～527nm.
3. the water compound concentration is 10
-5Bromine second pyridine (EB) solution of mol/L.
4. the dna probe molecule drips of solution of fluorescein (F1) mark is added in the EB solution of step 3 gained and obtains to the dna probe molecule solution of acceptor fluorescence mark to mark, the acceptor fluorescence mark of giving that wherein obtains is 10 to the concentration of the dna probe molecule solution of mark
-6Mol/L.
5. poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal that step 2 is obtained is immersed in giving in the dna probe molecule solution of acceptor fluorescence mark to mark of step 4 gained, obtains containing the solution of poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal of having filled dna probe molecule.
6. utilize fluorescence spectrophotometer, with wavelength 488nm is excitaton source, adopts transmitted light path, vertical incidence, poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal is perpendicular to light path, and the fluorescence spectrum of acceptor fluorescence mark to the dna probe molecule solution of mark given in record.
7. (volumetric molar concentration of target single strand dna solution to be measured is 10 dropwise to drip target single strand dna solution to be measured giving of obtaining of step 5 in the dna probe molecule solution of acceptor fluorescence mark to mark
-16Mol～1 mol), vibration is fully hybridized sample, obtains biological sample solution, and record is along with the increase of target single strand dna concentration, the variation of the fluorescence spectrum of biological sample solution.The emission light of F1 is 516～525nm, the absorbing light of EB is 507～527nm, when target single strand dna base sequence is complementary with the dna probe molecule base sequence of giving the acceptor fluorescence mark to mark, form double-stranded DNA, the EB molecule embeds the base-pair of double-stranded DNA, with F1 fluorescence probe generation FRET (fluorescence resonance energy transfer), along with the increase of target single stranded DNA concentration, the F1 fluorescence intensity weakens, and the EB fluorescence intensity strengthens.The forbidden photon band of poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal is positioned at 518～527nm, can be in poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal with the luminous local of F1, reduce the loss of F1 light, make F1 more effectively to give EB with NE BY ENERGY TRANSFER, thereby improved the sensitivity that detects, can detect 10
-13Mol/L target single strand dna.
Embodiment 2
1. water preparation mass concentration is that single particle diameter that disperses of 0.2% is poly-(styrene-methyl methacrylate-acrylic acid) emulsion particle solution of 255nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the wave carrier piece with cleaning is a substrate, in temperature is 50 ℃, humidity is under 50% the controlled condition, the latax that step 1 is obtained by vertical sedimentation is deposited on the substrate, self assembly is arranged and is prepared poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal on substrate, and poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band.
3. selecting for use commercially available is that the single stranded DNA of end group is a probe molecule with amino, and it is that obtaining EB concentration is 10 in the phosphate buffered solution of pH=7.4 of ssDNA probe molecule of end group that EB is joined with amino
-4Mol/L is that the ssDNA probe molecular conecentration of end group is 10 with amino
-5The probe molecule solution of mol.
4. poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal that step 2 is prepared is immersed in the resulting probe molecule solution of step 3, poly-(styrene-methyl methacrylate-acrylic acid) photon crystal surface carboxyl is fixed for the amino fully hydridization of the ssDNA probe molecule of end group with amino, repeatedly wash photonic crystal with the phosphate buffered solution of pH=7.4 then, remove the not dna molecular of hydridization, obtain poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal that ssDNA probe molecule and EB fill.
5. (volumetric molar concentration of target single strand dna solution to be measured is 10 with target single strand dna solution to be measured
-16Mol～1 mol) drips on poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal that the ssDNA probe molecule and the EB of step 4 gained fill, utilize the fluorescence co-focusing microscope then, focus on poly-(styrene-methyl methacrylate-acrylic acid) photonic crystal, detect its fluorescence intensity.
6. when target single strand dna base sequence and ssDNA probe molecule basic group sequence were complementary, the EB fluorescence intensity increased substantially, thereby realized the detection of target single strand dna base sequence.The luminous of EB is 620～625nm, the forbidden photon band of the photonic crystal of step 1 gained is positioned at 625～630nm, and the forbidden photon band blue zone limit of photonic crystal is consistent with the luminescence distribution of EB, utilizes photonic crystal forbidden photon band blue zone limit to strengthen the luminous of EB, improve the sensitivity that detects, can detect 10
-13Mol/L target single strand dna.
Embodiment 3
1. water preparation mass concentration is that single particle diameter that disperses of 0.2% is the polymethylmethacrylate emulsion particle solution of 354nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the wave carrier piece with cleaning is a substrate, is 80 ℃ in temperature, and humidity is that the latax that step 1 is obtained by vertical sedimentation is deposited on the substrate under 80% the controlled condition, and self assembly is arranged and prepared the polymethylmethacrylate photonic crystal on substrate.The polymethylmethacrylate photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band.
3. be solvent with ethanol, concentrated hydrochloric acid is catalyzer hydrolysis tetraethyl orthosilicate and improves and prepare SiO
2Colloidal sol.Concrete grammar is as follows: the tetraethyl orthosilicate 3.5ml of 28wt%, absolute ethyl alcohol 10ml mix back magnetic and stir the concentrated hydrochloric acid that adds the 37wt% of catalytic amount down, continue under the room temperature to stir 4 hours, promptly obtain the SiO that particle diameter is about 20～30nm
2Vitreosol.
4. the self assembly on substrate that step 2 the is obtained template of polymethylmethacrylate photonic crystal that is arranged with the opal structural of the periodicity dielectric structure with forbidden photon band is vertically placed, the SiO that step 3 is obtained
2Colloidal sol vertically drops in template surface and guarantees till its surperficial complete wetting, places under the room temperature then it to be dried in 2 hours.SiO
2Colloidal sol can be penetrated into by capillary force in the slit of polymethylmethacrylate photonic crystal of compact arranged opal structural, and forms the skeleton of solid around polymer microballoon.Drip and fill out SiO 2～5 times
2Colloidal sol guarantee opal structural polymethylmethacrylate photonic crystal template the slit complete filling SiO
2Nano particle.At last, sample is warmed up to 500 ℃ with the speed of about 2 ℃/min, keep then 500 ℃ about 3 hours, to guarantee to remove fully the polymethylmethacrylate photonic crystal template of opal structural, prepare counter opal structure SiO
2Photonic crystal.
5. the water compound concentration is 10
-3Mol/L heterogenetic antibody solution, the counter opal structure SiO that step 4 is obtained
2The photon crystal surface functionalization was soaked in heterogenetic antibody solution 24 hours, made counter opal structure SiO
2The abundant contact reaction of photonic crystal carrier and heterogenetic antibody is repeatedly washed with aqueous solution then, removes unconjugated heterogenetic antibody, air dry then, the counter opal structure SiO of formation specific antibody embedding
2The photonic crystal insolubilized antibody.Add proteantigen solution to be measured then, (volumetric molar concentration of target protein antigen solution to be measured is 10
-16Mol～1 mol), make wherein proteantigen and the counter opal structure SiO of specific antibody embedding
2Specific antibody in the photonic crystal insolubilized antibody forms the counter opal structure SiO of antigen antibody complex embedding
2Photonic crystal.
6. select for use commercially available fluorescein isothiocynate (FITC) fluorescently-labeled with proteantigen solution to be measured in the antibody of proteantigen with special response; The water compound concentration is 10
-4The FITC of mol/L fluorescently-labeled with proteantigen solution to be measured in proteantigen have the solution of the antibody of special correspondence, then this drips of solution is added in the counter opal structure SiO of the antigen antibody complex embedding that step 5 obtains
2On the photonic crystal, make FITC fluorescently-labeled with proteantigen solution to be measured in the antibody of proteantigen with special correspondence, with the counter opal structure SiO of antigen antibody complex embedding
2Proteantigen specificly-response in the proteantigen of the photonic crystal solution to be measured, form proteantigen-FITC in heterogenetic antibody-proteantigen solution to be measured fluorescently-labeled with proteantigen solution to be measured in proteantigen have the counter opal structure SiO of the antibody complex embedding of special correspondence
2Photonic crystal.
7. utilize the fluorescence co-focusing microscope, focus on proteantigen-FITC in heterogenetic antibody-proteantigen solution to be measured fluorescently-labeled with proteantigen solution to be measured in proteantigen have the embedding counter opal structure SiO of the antibody complex of special correspondence
2On the photonic crystal,, can carry out quantitative test to the proteantigen in the proteantigen solution to be measured according to fluorescence intensity.FITC emission light 520～530nm, bright yellow-green fluorescence is by the SiO of step 1～4 resulting counter opal structures
2The forbidden photon band of photonic crystal is positioned at 525～535nm, the SiO of counter opal structure
2The forbidden photon band blue zone limit of photonic crystal is consistent with the luminescence distribution of FITC, utilizes photonic crystal forbidden photon band blue zone limit to strengthen the luminous of FITC, has improved the sensitivity that detects.
Embodiment 4
1. water preparation mass concentration is that single particle diameter that disperses of 0.2% is the polymethylmethacrylate emulsion particle solution of 306nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the wave carrier piece with cleaning is a substrate, is 80 ℃ in temperature, and humidity is that the latax that step 1 is obtained by vertical sedimentation is deposited on the substrate under 80% the controlled condition, and self assembly is arranged and prepared the polymethylmethacrylate photonic crystal on substrate.The polymethylmethacrylate photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band.
3. be solvent with ethanol, concentrated hydrochloric acid is catalyzer hydrolysis tetraethyl orthosilicate and improves and prepare SiO
2Colloidal sol.Concrete grammar is as follows: the tetraethyl orthosilicate 3.5ml of 28wt%, absolute ethyl alcohol 10ml mix back magnetic and stir the concentrated hydrochloric acid that adds the 37wt% of catalytic amount down, continue under the room temperature to stir 4 hours, promptly obtain the SiO that particle diameter is about 20～30nm
2Vitreosol.
4. the self assembly on substrate that step 2 the is obtained polymethylmethacrylate photonic crystal template that is arranged with the opal structural of the periodicity dielectric structure with forbidden photon band is vertically placed, the SiO that step 3 is obtained
2Colloidal sol vertically drops in template surface and guarantees till its surperficial complete wetting, places under the room temperature then it to be dried in 2 hours.SiO
2Colloidal sol can be penetrated in the slit of compact arranged opal structural polymethylmethacrylate photonic crystal by capillary force, and forms the skeleton of solid around polymer microballoon.Drip and fill out SiO 2～5 times
2Colloidal sol guarantee opal structural photonic crystal template the slit complete filling SiO
2Nano particle.Utilize photodegradative method to remove the polymethylmethacrylate photonic crystal template of opal structural, obtain the SiO of counter opal structure
2Photonic crystal.
5. the water compound concentration is 10
-3Mol/L specific antibody and 10
-5Mol/L green fluorescent protein solution, the SiO of the counter opal structure that step 4 is obtained then
2Photonic crystal in this solution, soaked 24 hours, take out the SiO that air dry obtains the counter opal structure of specific antibody and the embedding of green fluorescent protein labeled molecule
2Photonic crystal.
6. (volumetric molar concentration of target protein antigen solution to be measured is 10 with the target protein antigenic solution
-16Mol～1 mol) dropping is at the SiO of the counter opal structure of resulting specific antibody of step 5 and the embedding of green fluorescent protein labeled molecule
2On the photonic crystal, make wherein target protein antigen and the SiO of the counter opal structure of specific antibody and the embedding of green fluorescent protein labeled molecule
2Specific antibody in the photonic crystal forms antigen antibody complex, obtains the SiO of the counter opal structure of antigen-antibody and green fluorescent protein embedding
2Photonic crystal utilizes the fluorescence co-focusing microscope then, focuses on the SiO of the counter opal structure of antigen-antibody and green fluorescent protein embedding
2On the photonic crystal, the variation of the fluorescence intensity of monitoring green fluorescent protein according to the green fluorescent protein fluorescence intensity, can realize detection by quantitative to the target protein antigenic solution.SiO by step 1～4 resulting counter opal structures
2The long band edge of the forbidden photon band of photonic crystal is positioned at 506～515nm and the luminous 507～511nm of green fluorescent protein is complementary, and utilizes the long band edge of forbidden photon band of photonic crystal to strengthen the luminous of green fluorescent protein, improves the sensitivity that detects.
Embodiment 5.
1. water preparation mass concentration is that single particle diameter that disperses of 0.2% is the silicon dioxide emulsion particle solution of 210nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the wave carrier piece with cleaning is a substrate, is 50 ℃ in temperature, and humidity is that the latax that step 1 is obtained by vertical sedimentation is deposited on the substrate under 50% the controlled condition, and self assembly is arranged and prepared the silicon dioxide photonic crystal on substrate.The silicon dioxide photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band, and the photonic crystal forbidden photon band is positioned at 518～527nm.
3. the water compound concentration is 10
-3Bromine second pyridine (EB) solution of mol/L.
4. the dna probe molecule drips of solution of fluorescein (F1) mark is added in the EB solution of step 3 gained and obtains to the dna probe molecule solution of acceptor fluorescence mark to mark, the acceptor fluorescence mark of giving that wherein obtains is 10 to the concentration of the dna probe molecule solution of mark
-5Mol/L.
5. the silicon dioxide photonic crystal that step 2 is obtained is immersed in giving in the dna probe molecule solution of acceptor fluorescence mark to mark of step 4 gained, obtains containing the solution of the silicon dioxide photonic crystal of having filled dna probe molecule.
6. utilizing fluorescence spectrophotometer, is excitaton source with wavelength 488nm, adopts transmitted light path, vertical incidence, and the silicon dioxide photonic crystal is perpendicular to light path, and the fluorescence spectrum of acceptor fluorescence mark to the dna probe molecule solution of mark given in record.
7. (volumetric molar concentration of target single strand dna solution to be measured is 10 dropwise to drip target single strand dna solution to be measured giving of obtaining of step 5 in the dna probe molecule solution of acceptor fluorescence mark to mark
-16Mol～1 mol), vibration is fully hybridized sample, obtains test sample solution, and record is along with the increase of target single strand dna concentration, the variation of the fluorescence spectrum of test sample solution.The emission light of F1 is 516～525nm, the absorbing light of EB is 507～527nm, when target single strand dna base sequence is complementary with the dna probe molecule base sequence of giving the acceptor fluorescence mark to mark, form double-stranded DNA, the EB molecule embeds the base-pair of double-stranded DNA, with F1 fluorescence probe generation FRET (fluorescence resonance energy transfer), along with the increase of target single stranded DNA concentration, the F1 fluorescence intensity weakens, and the EB fluorescence intensity strengthens.The forbidden photon band of silicon dioxide photonic crystal is positioned at 518～527nm, can with the luminous local of F1 in the silicon dioxide photonic crystal, reduce the loss of F1 light, make F1 more effectively to give EB NE BY ENERGY TRANSFER, thereby improved the sensitivity that detects, can detect 10
-14Mol/L target single strand dna.
Embodiment 6.
1. water preparation mass concentration is that single particle diameter that disperses of 0.2% is the silicon dioxide emulsion particle solution of 210nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the wave carrier piece with cleaning is a substrate, is 50 ℃ in temperature, and humidity is that the latax that step 1 is obtained by vertical sedimentation is deposited on the substrate under 50% the controlled condition, and self assembly is arranged and prepared the silicon dioxide photonic crystal on substrate.The silicon dioxide photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band.
3. the water compound concentration is 10
-5The probe molecule solution of the hormone molecular probe of the CdSe mark of mol/L.
4. the silicon dioxide photonic crystal that is complementary of forbidden photon band that step 2 is obtained and CdSe glow peak is immersed in the probe molecule solution of hormone molecular probe of CdSe mark of step 3 gained, obtains containing the solution of the silicon dioxide photonic crystal of the hormone molecular probe of having filled the CdSe mark.
5. utilizing fluorescence spectrophotometer, is excitaton source with wavelength 488nm, adopts transmitted light path, vertical incidence, and the silicon dioxide photonic crystal is used fluorescence spectrophotometer perpendicular to light path, the luminescent spectrum of the probe molecule solution of the hormone molecular probe of record CdSe mark.
6. the water compound concentration is 10
-4Tetramethyl rhodamine (TMR) solution of mol/L is added in target hormone receptor molecule drips of solution to be measured that (volumetric molar concentration of target hormone receptor molecule solution to be measured is 10 in this solution
-16Mol～1 mol), obtain the hormone receptor molecular solution of TMR mark.
7. the target hormone receptor molecule drips of solution to be measured of the TMR mark of step 6 gained is added in the solution of the silicon dioxide photonic crystal that contains the hormone molecular probe of having filled the CdSe mark that step 4 obtains, vibration, sample is fully hybridized, obtain detecting solution, record detects the variation of the fluorescence spectrum of solution along with the increase of the target hormone receptor molecular conecentration of TMR mark.The emission light of CdSe is 540～543nm, the absorbing light of TMR is 539～545nm, FRET (fluorescence resonance energy transfer) takes place in the TMR of the target hormone receptor molecule of the CdSe of the hormone molecular probe of CdSe mark and TMR mark, increase along with the target hormone receptor molecular conecentration of TMR mark, the CdSe fluorescence intensity weakens, and the TMR fluorescence intensity strengthens.The forbidden photon band of silicon dioxide photonic crystal is positioned at 533～545nm can be with the luminous local of CdSe at the silicon dioxide photonic crystal, reduce the loss of CdSe light, make CdSe more effectively to give TMR, thereby improved the sensitivity that detects, can detect 10 NE BY ENERGY TRANSFER
-10Mol/L target hormone receptor molecule.
Claims (10)
1. method of utilizing photonic crystal to improve biological detection sensitivity is characterized in that this method may further comprise the steps:
(1) in aqueous solution, utilize single fluorescent tag molecule or give the acceptor fluorescence mark right, the biological function material is carried out fluorescence labeling, obtain containing single fluorescent tag molecule or give the aqueous solution of acceptor fluorescence mark the biological function material of mark;
(2) photonic crystal of selecting the glow peak of the fluorescent tag molecule in the biological function material of the single fluorescent tag molecule mark in forbidden photon band band edge and the step (1) to be complementary, utilize the forbidden photon band band edge of photonic crystal to strengthen the reading and distinguishing of fluorescence signal of fluorescent tag molecule, thus the sensitivity that improves detection architecture; Or
Select the photonic crystal of the glow peak of giving body in the biological function material of mark being complementary for the acceptor fluorescence mark in forbidden photon band or forbidden photon band band edge and the step (1), utilize the forbidden photon band of photonic crystal or forbidden photon band band edge to strengthen to the reading and distinguishing of the right fluorescence signal of acceptor fluorescence mark, thus the sensitivity that improves detection architecture;
What (3) step (1) is obtained contains single fluorescent tag molecule or is fixed on the photonic crystal of step (2) for the acceptor fluorescence mark to the single fluorescent tag molecule in the aqueous solution of the biological function material of mark or to the acceptor fluorescence mark, has obtained filling the photonic crystal of fluorescence labeling biological function material;
(4) the target organism substance solution is dripped on the resulting photonic crystal of filling fluorescence labeling biological function material of step (3), the target organism material is detected, by the variation that the fluoroscopic examination instrument writes down the fluorescence signal of fluorescence labeling biological function material, finish identification to the target organism material.
2. method according to claim 1 is characterized in that: described to contain single fluorescent tag molecule or give the acceptor fluorescence mark be 10 to the volumetric molar concentration of the aqueous solution of the biological function material of mark
-16Mol～10 mol.
3. method according to claim 1 is characterized in that: the volumetric molar concentration of described target organism substance solution is 10
-16Mol～1 mol.
4. method according to claim 1 is characterized in that: described photonic crystal is the periodicity dielectric structure with forbidden photon band, and the photonic crystal that has the photonic crystal of opal structural or have counter opal structure is arranged in self assembly.
5. method according to claim 4 is characterized in that: described photonic crystal with counter opal structure is to be template with photonic crystal with opal structural, utilizes template to prepare.
6. according to claim 4 or 5 described methods, it is characterized in that: described photonic crystal with opal structural is that to be the mono-dispersion microballoon self assembly of the polystyrene, polymethylmethacrylate of 150nm～1.5um, poly-(styrene-methyl methacrylate-acrylic acid) or silicon dioxide by particle diameter prepared.
7. according to claim 1 or 3 described methods, it is characterized in that: described target organism material is selected from a kind of in nucleic acid, antigen-antibody, conjugated protein, lectins, the hormone receptor.
8. method according to claim 1 and 2 is characterized in that: described biological function material is selected from a kind of in enzyme, nucleic acid, antigen-antibody, conjugated protein, lectins, the hormone receptor.
9. method according to claim 1 and 2 is characterized in that: described single fluorescent tag molecule is selected from a kind of in the chelate, fluorescin of 3 valency rare earth lanthanide in organic dyestuff, luminescent quantum dot, compound fluorescent nano particles, the lanthanide chelate;
Described give the acceptor fluorescence mark right give a kind of in chelate that body and acceptor be selected from 3 valency rare earth lanthanide in organic dyestuff, luminescent quantum dot, compound fluorescent nano particles, the lanthanide chelate respectively, the fluorescin.
10. method according to claim 9 is characterized in that: described organic dyestuff is tetramethyl rhodamine, RB 200, TRITC, fluorescein isothiocynate, ethidium bromide, acridine, phenanthridines, fluorescein, TOTO or YOYO;
The nano particle that described luminescent quantum dot is made up of II-VI family element and III-V family element;
The chelate of 3 valency rare earth lanthanide is europium, terbium or cerium in the described lanthanide chelate;
Described fluorescin is phycobiliprotein or green fluorescent protein.
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