CN102072891B - Metal-modified photonic crystal biological detection film as well as preparation method and application thereof - Google Patents
Metal-modified photonic crystal biological detection film as well as preparation method and application thereof Download PDFInfo
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- CN102072891B CN102072891B CN2009102378907A CN200910237890A CN102072891B CN 102072891 B CN102072891 B CN 102072891B CN 2009102378907 A CN2009102378907 A CN 2009102378907A CN 200910237890 A CN200910237890 A CN 200910237890A CN 102072891 B CN102072891 B CN 102072891B
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Abstract
The invention relates to a metal-modified photonic crystal biological detection film as well as a preparation method and application thereof. The film is a metal-modified photonic crystal film marked of a biological function substance which is marked by a fluorescence labeling molecule, wherein the biological function substance marked by the fluorescence labeling molecule is a biological function substance marked by a single fluorescence labeling molecule or a receptor fluorescence labeling pair. By the utilization of the characteristics of photonic crystal enhancement and metal surface enhancement, the adaptation of the fluorescence labeling biological function substance, and the specific responsiveness and the specific affinity of the fluorescence labeling biological function substance on a target biological substance, the target biological substance and the biological function substance generate response, thereby the illumination change of the biological function substance marked by the fluorescence labeling molecule is caused. Moreover, the sensitivity of a system for detecting the target biological substance can be effectively enhanced by reading and identifying fluorescent signals of the fluorescence labeling molecule through photonic crystal enhancement and metal surface enhancement, consequently the high-sensitivity biological detection can be realized.
Description
Technical field
The invention belongs to the field that multiple proteins in the biological sample and gene are detected, particularly photonic crystal biological detection film of modifying of metal and preparation method thereof, and utilize photonic crystal that metal modifies detection applications to biological substance.
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 under multidisciplinary intersection background, grows up, 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.XRF 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 (like fluorescence lifetime, fluorescence anisotropy, fluorescence quantum yield, fluorescence exciting wavelength, emission wavelength etc.); 3. highly sensitive; 4. have multiple detection technique and method (like synchronous fluorescence, derivative fluorescence, fluorescence polarization, fluorescence kinetics analytic approach, three-dimensional fluorescence spectrum method and resolution, time-resolved fluoroimmunoassay etc. mutually).So can select, 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 property 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 is 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.
The fluorescent emission intensity that refers to be distributed near the fluorescence species metal surface such as gold and silver or its colloidal sol can strengthen greatly, and has begun the acquisition important application in fields such as DNA detection, FRET immunoassays.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; And the periodic structure of high-sequential can be provided; To the research of photon crystal optics and architectural characteristic, make it tempting application prospect arranged in recent years at aspects such as photoelectric device and optical chips.
Summary of the invention
The object of the present invention is to provide the photonic crystal biological detection film that a kind of combination property is high, highly sensitive, cost is low, the metal of quick, convenient, the easy photonic crystal raising biological detection sensitivity of realizing that utilizes metallics or metallic film modification is modified.
A purpose more of the present invention is to provide the preparation method of the photonic crystal biological detection film that a kind of metal modifies.
An also purpose of the present invention is that the photonic crystal biological detection film that provides metal to modify carries out the purposes of context of detection to biological substance.
The present invention is a characteristic of utilizing photonic crystal and metal surface to strengthen; 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, and cause with the luminous change of the biological function material of fluorescent tag molecule mark, strengthen the reading of fluorescence signal and the distinguishing fluorescence signal of fluorescent tag molecule through photonic crystal enhancing and metal surface; Can effectively strengthen the sensitivity of the detection architecture of target organism material, thereby realize highly sensitive biological detection.
The photonic crystal biological detection film that metal of the present invention is modified is a kind of film that is grown on the support base, and described film is the photon crystal film of being modified by the metal of the biological function material institute mark of fluorescent tag molecule mark; The biological function material of described fluorescent tag molecule mark is single fluorescent tag molecule or gives the biological function material of acceptor fluorescence mark to mark.
The biological function material of the fluorescent tag molecule mark in the photon crystal film that the metal of described biological function material institute mark by the fluorescent tag molecule mark is modified accounts for 0.0001%～1% of film general assembly (TW);
The thickness of the photonic crystal in the photonic crystal that described metal is modified is 1 μ m～5mm.
The photonic crystal that described metal is modified is the photonic crystal that metallic film is modified or metal nanoparticle mixes; Metal in the photonic crystal that described metallic film is modified is 5nm～200nm at the thickness of photon crystal surface; Metal nanoparticle in the photonic crystal that described metal nanoparticle mixes accounts for 0.001%～100% of photonic crystal general assembly (TW) that metal nanoparticle mixes.
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 organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide etc.
Described a kind of in chelate that body and acceptor be selected from organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide respectively, the fluorescin etc. of giving who gives acceptor fluorescence mark centering, and can not select above-mentioned identical material simultaneously with acceptor to body.Above-mentioned composite fluorescence silica fluorescent nanoparticle is a kind of (J.R.Taylor, M.M.Fang, the S.M.Nie in chelate, fluorescin of organic dyestuff, luminescent quantum dot, the 3 valency rare earth lanthanide of coated with silica etc.; Anal Chem; 2000,72 (9), 1979).
Above-mentioned organic dyestuff is tetramethyl rhodamine, RB 200, TRITC, fluorescein isothiocynate, ethidium bromide, acridine, phenanthridines, luciferin, 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 above-mentioned luminescent quantum dot is made up of II-VI family element and III-V family element, they are CdS, CdSe or CdTe etc.
3 valency rare earth lanthanide in the chelate of 3 above-mentioned valency rare earth lanthanide are europium (Eu
3+), terbium (Tb
3+) or cerium (Ce
3+) etc.
Above-mentioned fluorescin is phycobiliprotein or green fluorescent protein etc.
Described photonic crystal is that the photonic crystal etc. have the photonic crystal of opal structural or to have counter opal structure is arranged in self assembly with periodicity dielectric structure of forbidden photon band.
Described photonic crystal with opal structural be by particle diameter be polystyrene, the polymethylmethacrylate of 150nm～1.5um, the mono-dispersion microballoon self assembly that gathers (styrene-methyl methacrylate-acrylic acid) or silicon dioxide prepares.
Described photonic crystal with counter opal structure is to be template with above-mentioned photonic crystal with opal structural, utilizes template to prepare.
Described metal comprises multiple metals with surperficial enhancement effect such as gold, silver, platinum, copper or aluminium.
Described support base material can for: glass (simple glass, quartz glass or organic glass etc.), metallic film (aluminium foil or copper sheet etc.), plastic sheeting (plasticon, polymethylacrylic acid film, polypropylene or PVC etc.), paper etc. have the transparent of supporting capacity or have high reflectance sheet material.
The preparation method of the photonic crystal biological detection film that metal of the present invention is modified may further comprise the steps:
(1) in the WS, 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 WS of acceptor fluorescence mark the biological function material of mark;
(2) utilizing this area methods such as magnetron sputtering plating, vacuum evaporation plated film or metal nanoparticle doping commonly used that photonic crystal on support base is carried out metal modifies; Prepare the photonic crystal that metallic film is modified or metal nanoparticle mixes; Wherein, the metal in the film modified photonic crystal of preferable alloy is 5nm～200nm at the thickness of photon crystal surface; Metal nanoparticle in the photonic crystal that the preferable alloy nano particle mixes accounts for 0.001%～100% of photonic crystal general assembly (TW) that metal nanoparticle mixes;
The metallic film that the step (2) that the glow peak of the fluorescent tag molecule in the forbidden photon band band edge of (3) selecting photonic crystal and the biological function material of the single fluorescent tag molecule mark in the step (1) is complementary obtains is modified or the photonic crystal of metal nanoparticle doping; Utilize reading of the common fluorescence signal that strengthens described fluorescent tag molecule of forbidden photon band band edge and the metal of described photonic crystal, thus the sensitivity that improves the biological substance detection architecture; Or
Select forbidden band or the forbidden photon band band edge of photonic crystal and the photonic crystal that metallic film is modified or metal nanoparticle mixes that the step (2) of the glow peak of giving body in the biological function material of mark being complementary for the acceptor fluorescence mark in the step (1) obtains of photonic crystal; Utilize common enhancing of forbidden photon band band edge and the metal of forbidden photon band or photonic crystal of described photonic crystal describedly to give reading of the right fluorescence signal of acceptor fluorescence mark, thus the sensitivity that improves the biological substance detection architecture;
(4) photonic crystal of step (3) being selected that metallic film is modified or metal nanoparticle mixes contains single fluorescent tag molecule or gives in the WS of acceptor fluorescence mark to the biological function material of mark together with what support base was immersed in that step (1) obtains; Make the described single fluorescent tag molecule of step (1) or the biological function material of mark is fixed on the photonic crystal that metallic film is modified or metal nanoparticle mixes of step (3) selection for the acceptor fluorescence mark; Take out support base; Washing, drying obtain on support base by single fluorescent tag molecule or to the photon crystal film of acceptor fluorescence mark to the metal modification of the biological function material institute mark of mark.
Described by single fluorescent tag molecule or give in the photon crystal film that the acceptor fluorescence mark modifies the metal of the biological function material institute mark of mark, single fluorescent tag molecule or the biological function material of mark is accounted for 0.0001%～1% of film general assembly (TW) for the acceptor fluorescence mark.
Step (1) is described to be contained single fluorescent tag molecule or gives the acceptor fluorescence mark to containing single fluorescent tag molecule or the volumetric molar concentration of the biological function material of mark being preferably 10 for the acceptor fluorescence mark in the WS of the biological function material of mark
-16Mol～10 mol.
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 organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide etc.
Described a kind of in chelate that body and acceptor be selected from organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide respectively, the fluorescin etc. of giving who gives acceptor fluorescence mark centering, and can not select above-mentioned identical material simultaneously with acceptor to body.
Above-mentioned composite fluorescence silica fluorescent nanoparticle is a kind of (J.R.Taylor, M.M.Fang, the S.M.Nie in chelate, fluorescin of organic dyestuff, luminescent quantum dot, the 3 valency rare earth lanthanide of coated with silica etc.; Anal Chem; 2000,72 (9), 1979).
Above-mentioned organic dyestuff is tetramethyl rhodamine, RB 200, TRITC, fluorescein isothiocynate, ethidium bromide, acridine, phenanthridines, luciferin, 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 above-mentioned luminescent quantum dot is made up of II-VI family element and III-V family element, they are CdS, CdSe or CdTe etc.
3 valency rare earth lanthanide in the chelate of 3 above-mentioned valency rare earth lanthanide are europium (Eu
3+), terbium (Tb
3+) or cerium (Ce
3+) etc.
Above-mentioned fluorescin is phycobiliprotein or green fluorescent protein etc.
Described photonic crystal is that the photonic crystal etc. that has the photonic crystal of opal structural or have counter opal structure is arranged in periodicity dielectric structure with forbidden photon band, self assembly.
Described photonic crystal with opal structural be by particle diameter be polystyrene, the polymethylmethacrylate of 150nm～1.5um, the mono-dispersion microballoon self assembly that gathers (styrene-methyl methacrylate-acrylic acid) or silicon dioxide prepares.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, H.Yan, R.C.Schroden, et al.Adv.Mater.2001 at a slow speed; 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.
Described metal comprises multiple metals with surperficial enhancement effect such as gold, silver, platinum, copper or aluminium.
Described metal is modified photonic crystal, is to be carrier with the photonic crystal, utilizes vacuum evaporation metallic film, magnetron sputtering metallic film, electrochemical deposition of metal nano particle (metal nanoparticle doping) [P.N.Bartlett, J.J.Baumberg, Peter R.Birkin; M.A.Ghanem, and M.C.Netti, Chem.Mater.2002,14; 2199], surface adsorption metal nanoparticle (metal nanoparticle doping) [Z.-Z.Gu, R.Horie, S.Kubo, Y.Yamada; A.Fujishima, O.Sato, Angew.Chem.Int.Ed.2002,41; 1153, J.H.Zhang, J.B.Liu, S.Z.Wang, P.Zhan; Z.L.Wang and N.B.Ming, Adv.Func.Mater.2004,14,1089.].
The photonic crystal biological detection film that metal of the present invention is modified can detect biological substance, and its detection method is:
The target organism substance solution is dripped on the photonic crystal biological detection film of metal modification; The target organism material is detected; Through fluoroscopic examination instrument (XRF or fluorescence co-focusing microscope etc.) the single fluorescent tag molecule of record or to of the variation of acceptor fluorescence mark to the fluorescence signal of the photonic crystal of the metal modification of the biological function material institute mark of mark; Thereby accomplish the detection to the target organism material, the volumetric molar concentration that can detect described target organism substance solution is 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.
Principle of the present invention is to utilize photonic crystal can modulate the characteristics of luminescence of fluorescent tag molecule; Effect with the collaborative metal pair fluorescence enhancing of photonic crystal; And combined with fluorescent mark biological function material obtains high sensitivity and reaches bio-identification system efficiently the recognition reaction of target organism material.For containing single fluorescent tag molecule biological substance detection architecture; Be forbidden photon band band edge and the luminous common enhancing of the single fluorescent tag molecule of metal pair that utilizes photonic crystal; The enhancing that final realization is read the fluorescence signal of fluorescent tag molecule, thus realize high-sensitivity detection to the target organism material; For giving the right biological substance detection architecture of acceptor fluorescence mark; Be to utilize the forbidden photon band of photonic crystal to give luminous localization of body or forbidden photon band band edge to giving the luminous enhancing of body to giving acceptor fluorescence mark centering; Promote to give the acceptor fluorescence mark between the fluorescence resonance energy transmission; Collaborative metal pair to receptor marker between the common enhancing of FRET; The enhancing that final realization is read the fluorescence signal of fluorescent tag molecule, thus realize high-sensitivity detection to the target organism material.
The present invention has prepared the photonic crystal biological detection film that metal is modified; And be applied to biological detection; Provide a kind of metal that utilizes to modify the method that photonic crystal improves biological detection sensitivity; Other available fluorescent material is still arranged except above-mentioned fluorescent tag molecule, and 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 the following advantages:
1. the present invention has prepared the photonic crystal biological detection film that metal is modified; And be applied to biological detection, this method has that cost is low, simple to operate, easy to carry, error is less, quick, convenient, sensitive, specificity advantages of higher.
2. the present invention utilizes the intensity of the light signal that the characteristic that light regulation and control and metal surface are strengthened of photonic crystal can the enhancing detection system also can control its route of transmission, thereby can improve the sensitivity of detection architecture.
3. 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.
4. the photonic crystal of the metal of utilization of the present invention modification is to adopt the material with good bio-compatibility to prepare; Can good immobilization carrier be provided for biological functional mass; And there is porous road structure; The rapid and uniform that helps the biological function material molecule distributes, and can improve the problems such as stability, sensitivity and response speed of detection.
5. 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, the metal surface is 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 0.1% is the polystyrene latex grain solution of 205nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.Take by weighing 8.0g NaCl, 3.23g Na
2HPO
412H
2O and 0.45g NaH
2PO
42H
2O is dissolved in the 800ml distilled water, and with the pH value to 7.4 of HCl regulator solution, last adding distil water is settled to 1L, obtains 0.01M phosphate buffer (PBS).
2. the wave carrier piece (simple glass) with cleaning is a substrate; In the solution that contains the polystyrene latex grain that the vertical inserting step 1 of substrate is obtained; In temperature is 50 ℃; Humidity is under 50% the controlled condition, through vertical sedimentation the polystyrene latex grain that step 1 obtains to be deposited on the substrate, and self assembly is arranged and prepared polystyrene photon crystal on substrate; Polystyrene photon crystal is the opal structural with periodicity dielectric structure of forbidden photon band, and the forbidden photon band of polystyrene photon crystal is positioned at the 510nm place approximately.
3. the polystyrene photon crystal surface vapor deposition 5nm thick golden film of the vacuum evaporation technology that utilizes on step 2 substrate, obtaining.
4. the golden film modified polystyrene photon crystal that step 3 is obtained is immersed in together with substrate that to contain concentration be 10
-6The end group of mol/L is in the WS of fluorescein-labeled dna probe molecule of sulfydryl; Take out substrate after 2 hours; Fully wash repeatedly with PBS buffer solution; Air dry obtains measuring the polystyrene photon crystal biological detection film that the gold thin film of DNA base sequence is modified on substrate, the thickness that wherein detects the polystyrene photon crystal in the polystyrene photon crystal that the gold thin film in the film modifies is 3 μ m; The thickness of gold thin film is 5nm, and fluorescein-labeled dna probe molecule accounts for and detects 0.001% of film general assembly (TW).
5. the polystyrene photon crystal of the dna solution dropping of luciferin (F1) mark being modified at the gold of the dna marker of the resulting luciferin of step 4 (F1) mark; When target single strand dna base sequence and luciferin (F1) labeled DNA probe molecule basic group sequence are complementary; Luciferin (F1) marker DNA and target single strand dna form double-stranded DNA, and are fixed on the polystyrene photon crystal of gold modification.
6. utilizing XRF, is excitaton source with wavelength 488nm, adopts transmitted light path, and vertical incidence, the polystyrene photon crystal that gold is modified write down the F1 fluorescence spectrum perpendicular to light path.Record is along with the increase of target single strand dna concentration, and the double-stranded DNA fluorescence intensity of solution that luciferin (F1) marker DNA and target single strand dna form strengthens, and the emission light of F1 is about 516nm.The forbidden photon band position of polystyrene photon crystal is positioned at the 510nm place approximately, can strengthen the luminous of F1, and collaborative surface with two-dimensionally periodic structure gold thin film strengthens, thereby has improved the sensitivity that detects, and can detect 10
-13Mol/L target single strand dna.
Embodiment 2
Water preparation mass concentration be 0.2% single disperse particle diameter be 210nm gather (styrene-methyl methacrylate-acrylic acid) emulsion particle solution, fully ultrasonic, emulsion particle is evenly disperseed.
2. the piezoid with cleaning is a substrate; Contain in the solution that gathers (styrene-methyl methacrylate-acrylic acid) emulsion particle what the vertical inserting step 1 of substrate obtained; In temperature is 50 ℃; Humidity is under 50% the controlled condition; The latax that step 1 is obtained through vertical sedimentation is deposited on the substrate, and self assembly is arranged to prepare and gathered (styrene-methyl methacrylate-acrylic acid) photonic crystal on substrate, and gathering (styrene-methyl methacrylate-acrylic acid) photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band.
3. that utilizes that magnetron sputtering obtains on step 2 substrate gathers (styrene-methyl methacrylate-acrylic acid) silverskin that photon crystal surface vapor deposition 200nm is thick, obtains (styrene-methyl methacrylate-acrylic acid) photonic crystal of modified by silver.
4. the single stranded DNA of selecting 5 '-(FAM)-CCTAGCGGGCGCACCTCTCTITACGCTAGG-(SH/NH2)-3 ' for use is a probe molecule, its 5 ' end mark fluorescent group 6-carboxyl-luciferin (FAM), the amino (NH of 3 ' end mark
2); (styrene-methyl methacrylate-acrylic acid) photonic crystal of the modified by silver that step 3 is prepared soaks in the probe molecule solution; The sulfydryl of dna probe molecule is assembled at silver surface; Repeatedly wash the photonic crystal of modified by silver then with the PBS buffer solution of pH=7.4, remove the not dna probe molecule of hydridization, obtain silver-colored film modified (styrene-methyl methacrylate-acrylic acid) photonic crystal biological detection film of fluorescein-labeled DNA base probe molecule mark; Wherein detecting in the film thickness that gathers (styrene-methyl methacrylate-acrylic acid) photon crystal film is 10 μ m, and the thickness of gold thin film is 200nm, and the content of fluorescently-labeled dna probe molecule is 0.01%.
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 in the gathering on (styrene-methyl methacrylate-acrylic acid) photonic crystal of the modified by silver of the dna probe molecule mark of step 4 gained, utilize the fluorescence co-focusing microscope then, detect its fluorescence intensity.
6. when target single strand dna base sequence and ssDNA probe molecule basic group sequence were complementary, the dna probe molecule was opened, and fluorescence intensity increases substantially, thereby realized the detection of target single strand dna base sequence.The luminous of FAM is～550nm; The forbidden photon band that gathers (styrene-methyl methacrylate-acrylic acid) photonic crystal of silver-colored film modified (styrene-methyl methacrylate-acrylic acid) photonic crystal biological detection film of the measured DNA base sequence of step 4 gained is positioned at the 550nm place approximately; The forbidden photon band of photonic crystal is consistent with the luminescence distribution of FAM; Utilize photonic crystal forbidden photon band direct reflection to combine silver-colored film surface to strengthen; The FAM luminous intensity that improves greatly improves the sensitivity that detects, and can detect 10
-16Mol/L target single strand dna.
Embodiment 3
1. water preparation mass concentration is that single particle diameter that disperses of 3% is the polymethylmethacrylate emulsion particle solution of 354nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the PVC sheets with cleaning is a substrate; In the solution that contains the polymethylmethacrylate emulsion particle that the vertical inserting step 1 of substrate is obtained; In temperature is 80 ℃; Humidity is that the latax that step 1 is obtained through 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 following: 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 is obtained is arranged with the template of polymethylmethacrylate photonic crystal of the opal structural of the periodicity dielectric structure with forbidden photon band and vertically places, the SiO that step 3 is obtained
2Colloidal sol vertically drops in template surface and guarantees that the room temperature held dried it in 2 hours then till its surperficial complete wetting.SiO
2Colloidal sol can be penetrated into through capillary force in the slit of polymethylmethacrylate photonic crystal of compact arranged opal structural, and around poly (methyl methacrylate) micro-sphere, forms the skeleton of solid.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. with the resulting counter opal structure SiO of step 4
2Photonic crystal is immersed in the silver sol solution of 0.001mol/L, slowly lifts with 2mm/min, prepares the counter opal structure SiO of modified by silver
2Photonic crystal, wherein counter opal structure SiO
2The thickness of photon crystal film is 0.01mm, and Nano silver grain content is 0.01%.
6. the water compound concentration is 10
-3Mol/L heterogenetic antibody solution, the counter opal structure SiO of the modified by silver that step 5 is obtained
2Photonic crystal 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 the WS then, removes unconjugated heterogenetic antibody, air dry then, the counter opal structure SiO of the modified by silver 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 the counter opal structure SiO of the modified by silver of wherein proteantigen and specific antibody embedding
2Specific antibody in the photonic crystal insolubilized antibody forms the counter opal structure SiO of the modified by silver of antigen antibody complex embedding
2The photonic crystal insolubilized antibody.
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 modified by silver of the antigen antibody complex embedding that step 5 obtains
2On the photonic crystal insolubilized antibody, 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 the modified by silver of antigen antibody complex embedding
2Proteantigen specificly-response in the proteantigen of the photonic crystal insolubilized antibody 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 modified by silver of the antibody complex embedding of special correspondence
2The photonic crystal insolubilized antibody obtains measuring the counter opal structure SiO that the Nano silver grain of virus protein is modified
2Photonic crystal biological detection film, wherein the content of fluorescently-labeled heterogenetic antibody is 1%.
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 counter opal structure SiO of embedding modified by silver of the antibody complex of special correspondence
2On the photonic crystal insolubilized antibody,, can carry out quantitative test to the proteantigen in the proteantigen solution to be measured according to fluorescence intensity.FITC emission light is about 530nm, and bright yellow-green fluorescence is by the counter opal structure SiO of step 1～4 resulting modified by silver
2The forbidden photon band of photonic crystal insolubilized antibody is positioned at the 525nm place approximately, the counter opal structure SiO of modified by silver
2The forbidden photon band blue zone limit of photonic crystal insolubilized antibody is consistent with the luminescence distribution of FITC; Utilize photonic crystal forbidden photon band blue zone limit to strengthen the luminous of FITC; And the silver surface humidification, improved the sensitivity that detects, can detect the proteantigen of 10-6 mol.
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 aluminium flake with cleaning is a substrate; In the solution that contains the polymethylmethacrylate emulsion particle that the vertical inserting step 1 of substrate is obtained; In temperature is 80 ℃; Humidity is that the latax that step 1 is obtained through vertical sedimentation is deposited on the substrate under 80% the controlled condition, and self assembly is arranged and prepared opal structural colloidal crystal masterplate on substrate.
3. utilize method deposited copper in the opal structural colloidal crystal masterplate of step 2 gained of electro-deposition.Use tetrahydrofuran (THF) to remove the colloidal crystal masterplate then, obtain golden inverse opal photonic crystal.
4. the water compound concentration is 10
-3Mol/L specific antibody and 10
-5Mol/L green fluorescent protein solution; The copper inverse opal photonic crystal that then step 3 is obtained soaked 24 hours in this solution; Take out the copper inverse opal photonic crystal that air dry obtains specific antibody and the embedding of green fluorescent protein labeled molecule; Obtain measuring the copper inverse opal photonic crystal detection film of specific proteins antigen, wherein the thickness of the sub-crystal of bronzing is 1 μ m, and specific antibody and green fluorescent protein labeled molecule content are 0.001%.
5. target protein antigenic solution (volumetric molar concentration of target protein antigen solution to be measured is 10-16 mol～1 mol) is dripped on the copper inverse opal photonic crystal of resulting specific antibody of step 4 and the embedding of green fluorescent protein labeled molecule; Make the specific antibody in the copper inverse opal photonic crystal of wherein target protein antigen and specific antibody and the embedding of green fluorescent protein labeled molecule form antigen antibody complex; Obtain the copper inverse opal photonic crystal of antigen-antibody and green fluorescent protein embedding; Utilize the fluorescence co-focusing microscope then; Focus on the golden inverse opal photonic crystal of antigen-antibody and green fluorescent protein embedding; 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.Be positioned at 506～515nm and the luminous 507～511nm of green fluorescent protein is complementary by the long band edge of the forbidden photon band of the resulting copper inverse opal photonic crystal in step 1～3; Utilize the long band edge enhancing of forbidden photon band of copper inverse opal photonic crystal to strengthen the luminous of common enhancing green fluorescent protein with the copper surface; Improve the sensitivity that detects, can detect the antigen of 0.1mol/L.
Embodiment 5.
1. water preparation mass concentration is that single particle diameter that disperses of 5% is the silicon dioxide emulsion particle solution of 210nm, and is fully ultrasonic, and emulsion particle is evenly disperseed.
2. the plasticon with cleaning is a substrate; In the solution of the emulsion particle that contains silicon dioxide that the vertical inserting step 1 of substrate is obtained, be 50 ℃ in temperature, humidity is under 50% the controlled condition; The latax that step 1 is obtained through vertical sedimentation is deposited on the substrate; Self assembly is arranged and is prepared the silicon dioxide photonic crystal on substrate, and the photonic crystal forbidden photon band is positioned at 518～527nm, and the thickness of the thickness of silicon dioxide photon crystal film is 5mm.
3. the silicon dioxide photonic crystal that step 2 is obtained is immersed in the HPtCl of 0.1M
4In, through the C of 1M
6H
5O
7Na
32H
2The reduction of O citric acid three sodium solution, the photonic crystal of the opal structural of the periodicity dielectric structure that the preparation nano platinum particle coats with forbidden photon band, the content of metal nanoparticle is 0.001%.
4. the Tris-HCl buffer preparation with pH=7.4 contains 100mM dithiothreitol (DTT) (DTT) 10
-6Mol/L i-motifDNA solution.The i-motif dna sequence dna that pH is had structural response is:
5’-(SH)-TTTTTCCCTAACCCTAACCCTAACCC-BODIPY493/503-3’——ssDNA1；
To K
+Have the i-motifDNA sequence of structural response to be with concentration of thrombin:
5’-(SH)-TTTTTGGGTAAGGGTAAGGGTAAGGG-BODIPY493/503-3’-ssDNA2.
5. will be coated with the SiO of nanometer platinum
2The opal structural photonic crystal is dipped in the solution of step 4 gained, and ambient temperature overnight is preserved, and obtains being modified with the nanometer platinum coated Si O of i-motif DNA
2The opal structural photonic crystal.
6. with the nanometer platinum coated Si O that is modified with i-motif DNA that obtains in the step 5
2The opal structural photonic crystal is dipped in a series of to be measured have different pH values or different K
+Or in the solution to be measured of concentration of thrombin, under Laser Scanning Confocal Microscope, measure fluorescence spectrum after a period of time.
7. the silicon dioxide opal structural photonic crystal band band edge of nanometer platinum coating and the fluorophor BODIPY493/503 fluorescence peak of DNA marked are complementary, and the surface plasma of combining nano platinum strengthens fluorescence signal, H jointly
+Concentration increases back ssDNA1 conformation and takes place to fold, and it is folding that concentration of thrombin increases back ssDNA2 conformation, causes FITC fluorescence by the cancellation of nanometer platinum, can detect 10
-5The mol/L fibrin ferment.
Embodiment 6.
Water preparation mass concentration be 0.05% single disperse particle diameter be 210nm gather (styrene-methyl methacrylate-acrylic acid) emulsion particle solution, fully ultrasonic, emulsion particle is evenly disperseed.
2. with paper substrate; In the solution that contains (styrene-methyl methacrylate-acrylic acid) emulsion particle that the vertical inserting step 1 of substrate is obtained; In temperature is 50 ℃; Humidity is that the latax that step 1 is obtained through vertical czochralski method is deposited on the substrate under 50% the controlled condition, and white assembling is arranged to prepare and gathered (styrene-methyl methacrylate-acrylic acid) photonic crystal on substrate; Gathering (styrene-methyl methacrylate-acrylic acid) photonic crystal is the opal structural with periodicity dielectric structure of forbidden photon band, and the thickness that gathers (styrene-methyl methacrylate-acrylic acid) photon crystal film is 1 μ m.
3. utilize magnetron sputtering gathering the thick aluminium film of (styrene-methyl methacrylate-acrylic acid) photon crystal surface vapor deposition 30nm, obtain film modified the gathering of aluminium (styrene-methyl methacrylate-acrylic acid) photonic crystal.
4. the water compound concentration is 10
-5The probe molecule solution of the hormone molecular probe of the CdSe mark of mol/L.
5. with the PBS buffer preparation target hormone receptor molecule solution to be measured of suitable pH value.
6. film modified the gathering of aluminium (styrene-methyl methacrylate-acrylic acid) photonic crystal that forbidden photon band that step 3 is obtained and CdSe exciting light are complementary is immersed in the target hormone receptor molecule solution to be measured of step 5 gained.4 ℃ of refrigerations are spent the night.
7. film modified the gathering of the aluminium that is adsorbed with hormone receptor molecule to be measured (styrene-methyl methacrylate-acrylic acid) photonic crystal of step 6 gained encapsulates through 5% bovine serum albumin; Be dipped in film modified the gathering of aluminium (styrene-methyl methacrylate-acrylic acid) photon crystal film that obtains the hormone molecular probe mark of CdSe mark in the probe molecule solution of hormone molecular probe of CdSe mark of step 4 gained after the washing, wherein the hormone molecule content of CdSe mark is 1%.
8. utilize XRF; With wavelength 488nm is excitaton source, adopts transmitted light path, vertical incidence; Film modified the gathering of aluminium (styrene-methyl methacrylate-acrylic acid) photon crystal film of the hormone molecular probe mark of CdSe mark is vertically put into target hormone receptor molecular solution; Perpendicular to light path, use XRF, the luminescent spectrum of the probe molecule solution of the hormone molecular probe of record CdSe mark.
9. the hormone molecular probe of target hormone receptor molecule and CdSe mark is fully hybridized; The increase of target hormone receptor molecular conecentration, the variation of the fluorescence spectrum of CdSe in film modified the gathering of aluminium (styrene-methyl methacrylate-acrylic acid) photonic crystal of the hormone molecular probe mark of detection CdSe mark.Being used to excite the incident laser of CdSe quantum dot is that wavelength is 488nm; The forbidden photon band of aluminium is film modified to be gathered (styrene-methyl methacrylate-acrylic acid) photonic crystal is positioned at 503nm; Cooperate the common enhancing fluorescence CdSe fluorescence signal that is modified at the aluminium film that gathers in (styrene-methyl methacrylate-acrylic acid) photonic crystal; Thereby improved the sensitivity that detects, can detect 10
-10Mol/L target hormone receptor molecule.
Claims (10)
1. the photonic crystal biological detection film modified of a metal, it is a kind of film that is grown on the support base, it is characterized in that: described film is the photon crystal film of being modified by the metal of the biological function material institute mark of fluorescent tag molecule mark; The biological function material of described fluorescent tag molecule mark is single fluorescent tag molecule or gives the biological function material of acceptor fluorescence mark to mark;
Described biological function material is selected from a kind of in enzyme, nucleic acid, antigen-antibody, conjugated protein, lectins, the hormone receptor.
2. the photonic crystal biological detection film that metal according to claim 1 is modified is characterized in that: the biological function material of the fluorescent tag molecule mark in the photon crystal film that the metal of described biological function material institute mark by the fluorescent tag molecule mark is modified accounts for 0.0001%～1% of film general assembly (TW).
3. the photonic crystal biological detection film that metal according to claim 1 is modified is characterized in that: the thickness of the photonic crystal in the photonic crystal that described metal is modified is 1 μ m～5mm.
4. according to the photonic crystal biological detection film of claim 1 or 3 described metals modifications, it is characterized in that: the photonic crystal that described metal is modified is the photonic crystal that metallic film is modified or metal nanoparticle mixes; Metal in the photonic crystal that described metallic film is modified is 5nm～200nm at the thickness of photon crystal surface; Metal nanoparticle in the photonic crystal that described metal nanoparticle mixes accounts for 0.001%～0.01% of photonic crystal general assembly (TW) that metal nanoparticle mixes.
5. the photonic crystal biological detection film that metal according to claim 1 is modified is characterized in that: described single fluorescent tag molecule is selected from a kind of in the chelate, fluorescin of organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide;
Described a kind of in chelate that body and acceptor be selected from organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide respectively, the fluorescin of giving who gives acceptor fluorescence mark centering, and can not select above-mentioned identical material simultaneously with acceptor to body;
A kind of in the chelate of the organic dyestuff that described composite fluorescence silica fluorescent nanoparticle is a coated with silica, luminescent quantum dot, 3 valency rare earth lanthanide, the fluorescin.
6. the photonic crystal biological detection film that metal according to claim 4 is modified is characterized in that: described photonic crystal is that the photonic crystal that has the photonic crystal of opal structural or have counter opal structure is arranged in the self assembly with periodicity dielectric structure of forbidden photon band;
Described photonic crystal with opal structural be by particle diameter be polystyrene, the polymethylmethacrylate of 150nm～1.5 μ m, the mono-dispersion microballoon self assembly that gathers (styrene-methyl methacrylate-acrylic acid) or silicon dioxide prepares;
Described photonic crystal with counter opal structure is to be template with above-mentioned photonic crystal with opal structural, utilizes template to prepare;
Described metal comprises gold, silver, platinum, copper or aluminium.
7. the preparation method of a photonic crystal biological detection film of modifying according to any described metal of claim 1～6, it is characterized in that: this method may further comprise the steps:
(1) in the WS, 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 WS of acceptor fluorescence mark the biological function material of mark;
(2) photonic crystal on support base is carried out metal and modify, the photonic crystal that the preparation metallic film is modified or metal nanoparticle mixes;
The metallic film that the step (2) that the glow peak of the fluorescent tag molecule in the forbidden photon band band edge of (3) selecting photonic crystal and the biological function material of the single fluorescent tag molecule mark in the step (1) is complementary obtains is modified or the photonic crystal of metal nanoparticle doping; Or
Select forbidden band or the forbidden photon band band edge of photonic crystal and the photonic crystal that metallic film is modified or metal nanoparticle mixes that the step (2) of the glow peak of giving body in the biological function material of mark being complementary for the acceptor fluorescence mark in the step (1) obtains of photonic crystal;
(4) photonic crystal of step (3) being selected that metallic film is modified or metal nanoparticle mixes contains single fluorescent tag molecule or gives in the WS of acceptor fluorescence mark to the biological function material of mark together with what support base was immersed in that step (1) obtains; Make the described single fluorescent tag molecule of step (1) or the biological function material of mark is fixed on the photonic crystal that metallic film is modified or metal nanoparticle mixes of step (3) selection for the acceptor fluorescence mark; Take out support base; Washing, drying obtain on support base by single fluorescent tag molecule or to the photon crystal film of acceptor fluorescence mark to the metal modification of the biological function material institute mark of mark;
Described biological function material is selected from a kind of in enzyme, nucleic acid, antigen-antibody, conjugated protein, lectins, the hormone receptor.
8. preparation method according to claim 7; It is characterized in that: described by single fluorescent tag molecule or give in the photon crystal film that the acceptor fluorescence mark modifies the metal of the biological function material institute mark of mark, single fluorescent tag molecule or the biological function material of mark is accounted for 0.0001%～1% of film general assembly (TW) for the acceptor fluorescence mark.
9. preparation method according to claim 7 is characterized in that: step (1) is described to be contained single fluorescent tag molecule or give the acceptor fluorescence mark is 10 to containing single fluorescent tag molecule or giving the acceptor fluorescence mark in the WS of the biological function material of mark to the volumetric molar concentration of the biological function material of mark
-16Mol～10 mol;
The photonic crystal that the described metallic film of step (2) is modified or metal nanoparticle mixes, wherein, the metal in the photonic crystal that metallic film is modified is 5nm～200nm at the thickness of photon crystal surface; Metal nanoparticle in the photonic crystal that metal nanoparticle mixes accounts for 0.001%～0.01% of photonic crystal general assembly (TW) that metal nanoparticle mixes;
Described single fluorescent tag molecule is selected from a kind of in the chelate, fluorescin of organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide;
Described a kind of in chelate that body and acceptor be selected from organic dyestuff, luminescent quantum dot, composite fluorescence silica fluorescent nanoparticle, 3 valency rare earth lanthanide respectively, the fluorescin of giving who gives acceptor fluorescence mark centering, and can not select above-mentioned identical material simultaneously with acceptor to body;
A kind of in the chelate of the organic dyestuff that described composite fluorescence silica fluorescent nanoparticle is a coated with silica, luminescent quantum dot, 3 valency rare earth lanthanide, the fluorescin.
10. the purposes of a photonic crystal biological detection film of modifying according to any described metal of claim 1～6, it is characterized in that: the photonic crystal biological detection film that described metal is modified is used for biological substance is detected; The target organism substance solution is dripped on the photonic crystal biological detection film of metal modification; The target organism material is detected; Through a fluoroscopic examination instrument record fluorescent tag molecule or to of the variation of acceptor fluorescence mark, thereby accomplish detection to the target organism material to the fluorescence signal of the photonic crystal of the metal modification of the biological function material institute mark of mark.
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