CN102507444A - Auxiliary optical device of attenuation total reflection surface enhanced infrared spectrometer for DNA analysis - Google Patents
Auxiliary optical device of attenuation total reflection surface enhanced infrared spectrometer for DNA analysis Download PDFInfo
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- CN102507444A CN102507444A CN2011103841096A CN201110384109A CN102507444A CN 102507444 A CN102507444 A CN 102507444A CN 2011103841096 A CN2011103841096 A CN 2011103841096A CN 201110384109 A CN201110384109 A CN 201110384109A CN 102507444 A CN102507444 A CN 102507444A
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Abstract
The invention relates to an attenuation total reflection surface enhanced infrared optical device for DNA identification and hybridization dynamics analysis. The attenuation total reflection surface enhanced infrared optical device comprises an attenuation total reflection surface enhanced infrared optical window in which an island-shaped nanometer gold film is plated on the surface of a silicon hemisphere, an optical table base, a fixed support region, an infrared light spectrum pool, a PDMS (Polydimethylsiloxane) sealing ring and the like, wherein the silicon hemisphere plated with the gold island-shaped film is placed on the optical table base, a gold film surface of the optical window is toward the infrared light spectrum pool, and the middle of the optical window is sealed by the PDMS sealing ring; the light spectrum pool, the PDMS sealing ring and the silicon hemisphere optical window are screwed-in and fixed through an external thread of the fixed support and an internal thread of an upper support of the optical table base so as to form the surface enhanced infrared spectrometer for DNA analysis. The testing device, provided by the invention, has the advantages of simple structure, convenience for operation and small required sample amount, and can be stably used for a long time and convenient for real-time on-line detection of infrared spectrum signals at a waveband of 1100cm<-1> to 4000cm<-1>, and high-sensitivity analysis of DNA molecules, so that DNA identification and hybridization mechanisms and kinetics research can be realized.
Description
Technical field:
The invention belongs to the infrared spectrum technology field, be specially a kind of attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for DNA analysis.
Background technology:
The surface strengthens infrared absorption spectrum, and (Surface-Enhanced Infrared Absorption Spectroscopy SEIRAS) is a kind of important tools of analysis of studying the interface molecular structure information.Be equipped with attenuated total reflection (Attenuated Total Reflection; ATR) surface of pattern strengthens infrared absorption spectrum (ATR-SEIRAS); It is strong to have surface signal; Mass transfer is not obstructed and bulk solution disturbs advantages such as little, and mating surface choosing rule is convenient to carry out dna molecular identification and hybridization kinetics process study.(, mass transfer strong inadequately like surface signal replenishes and lags behind for IR Absorption Spectroscopy, the problem that IRAS) faces, the interference of solution background etc. to have avoided traditional external reflection infrared absorption spectrum simultaneously.Adopt inner reflection surface to strengthen the expansion of infrared absorption spectroscopies as common infrared spectrum; Significantly improved the precision that infrared spectrum detects; Greatly expanded the research range of infrared spectrum, like the dynamic behaviour of biomolecule such as interface micromolecular compound, polypeptide, protein, oligonucleotides and oligosaccharide, lipoid, bacteriophage, virus and cell and biological tissue's body.
Be used to make up prism apparatus that inner reflection surface strengthens the infrared absorption spectrum pond two kinds on Otto type and Kretschmann type arranged.Prism shape has multiple available, wherein how to be used for research with Kretschmann dome-type prism.The semisphere prism can guarantee that any angle incident light is all vertical with the interface, and the reflected light loss is little, and the angle that gets into behind the prism is constant.Liquid flow spectrum electrochemical pool with Osawa. and Ataka design is an application mainstream in the world.The spectrum tool device of other groups all is to convert on this basis.Adzic group has proposed the notion of combination zinc selenide cylinder and silicon chip infrared window.The miniature attenuated total reflection surface-reinforced infrared spectrum experimental technique of development such as Sudo can be analyzed the subtitle substance of material surface submillimeter size or nanometer layer thickness.The refined research group of the Cai Wen of Fudan University is according to " prism of plane wave-film coupling principle " (" the theory of prism-film coupler for plane wave "); Develop a kind of simple servicing unit that is used for galvanochemistry internal reflection SEIRAS, this infrared spectrum servicing unit was applied for a patent in 2008.Changchun should change professor Jiang Xiue made up the platform that can be used for analogue membrane albumen physiology existence with combining method innovation property that directed assembling and film recombinate; At assembling of the orientation of nano-interface and film regrouping process, expanded the research of attenuated total reflection infrared spectrum protein structure and function aspects through surface-reinforced infrared spectrum in-situ monitoring memebrane protein at the interface.Below all lay particular emphasis on the expansion of electrode system, the improvement of film-forming method, with the surface-reinforced infrared spectrum technology as the strong aid of field electrochemical and constantly be able to perfect.In recent years, DNA (DNA) was a key of explaining multiple biological phenomena, had become one of most important research field of life science.Because of characteristics such as it are easy, quick, sensitive, valency is low, obtain broad research and application based on the biology sensor of DNA in fields such as genetic engineering, medical diagnosis, new medicament screen, drug mechanism, environmental monitoring, legal medical expert's evaluations.The DNA sensing technology is expressed process and the result of hybridization with the physical signalling that light, electricity etc. is easy to measure, thereby obtains the information such as concentration, sequence of target dna molecule, is the important means that current dna is studied.Yet almost nobody pays close attention to and develops the minim DNA analysis and detection device that inner reflection surface strengthens infrared absorption spectrum that is used for that is simple and easy to usefulness both at home and abroad.In addition, infrared spectrum energy provides " fingerprint " characteristic of compound, is the important tool of analyzing DNA molecular structure and spatial orientation.Therefore, develop a cover and can realize high sensitivity, high resolving power, device miniatureization and low cost, and it is most important to be suitable for the attenuated total reflection surface-reinforced infrared spectrum instrument auxiliary device that DNA analysis detects.
Summary of the invention:
The goal of the invention of this device is that a kind of making of developing in order to solve problems such as present infrared facility is not suitable for DNA hybridization on-line analysis, manufacture difficulty is big, detection sensitivity is low, required sample size is big is simple and easy, can realize the surface-reinforced infrared spectrum appearance secondary optic of high sensitivity infrared detection.
Island nano-noble metal film with the outer humidification of surface red is that finishing, constructed dna sensor provide platform, can avoid the interference of background water, and aqueous sample is carried out direct analyzing and testing; Structure and character that can original position research function surface, observation interface generation molecular recognition reaction in real time; The activity that can keep testing molecule realizes the research of molecular reaction dynamics.Infrared spectrum technology is expanded in the application of researching DNA crossover process, realize in real time, original position, follow the tracks of DNA identification and crossover process online.
To achieve these goals, the technical scheme below the present invention has adopted:
A kind of DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for; As shown in Figure 1; It is characterized in that described device comprises a silicon hemisphere 1, the island nano-gold film 2 that the surface on silicon hemisphere 1 plane is coated with infrared enhancement effect constitutes infrared window, and sealing and fixing has infrared spectrum pool 3 on the lip-deep island nano-gold film of infrared window silicon hemispherical plane; Infrared spectrum pool is a right cylinder; Have four axial penetrating holes, wherein macropore is the contrast electrode socket, internal diameter φ 4-5mm; Three apertures are respectively sample inlet, sample export and to the electrode socket; Internal diameter is φ 2-3mm, and the bottom is miniature column liquid storage tank, is working electrode with golden film, can be applicable to the infrared spectrum electrochemical research.
The above-mentioned attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for DNA analysis, sealing and fixing infrared spectrum pool on the described infrared window can link together infrared window, O-ring seal 8, set collar 6 and fixing pressure pipe 7 through column 4 and the optical window collet 5 at the bottom of the silicon hemisphere, set collar 6, O-ring seal 8 and the fixing pipe 7 of pressing of accessory silicon hemisphere both sides; It can be the right cylinder of a band shoulder that infrared spectrum pool is used for fixing, and described column 4 has two, and column 4 is cylinders that there is screw at two ends; Described optical window collet 5 is a strip-shaped blade, and is as shown in Figure 2, and there is a disk with holes at its center; Can hold silicon hemisphere, two ends are porose, are complementary with the screw of column 4 lower ends; Can be screwed at column lower end, described set collar 6 is an annulus, and inwall has screw thread; Two screw holes that symmetry is arranged on the set collar 6 are complementary with the screw of column 4 upper ends, can be fixed on the column upper end; Other has a fixing pipe 7 of pressing, and the column diameter of its internal diameter and infrared spectrum pool is complementary, and the fixing outer wall of pipe 7 of pressing has screw thread; Be complementary with the screw thread of set collar 6 inwalls, so, pad is gone up O-ring seal 8 between infrared window and the infrared spectrum pool; To fix and press pipe 7 screw-in set collars 6 to compress, promptly be configured for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic.
The above-mentioned attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for DNA analysis; The preparation method of described island gold film; It is the infrared enhanced surface that adopts chemical replacement reaction (Galvanic reaction) and tool high reinforced effects even in silicon hemispherical plane surface preparation; Its concrete operation method is referring to Hiroto Miyake, Shen Ye, Masatoshi Osawa; Electroless deposition of gold thin films on silicon for surface-enhanced infrared spectroelectrochemistry, Electrochemistry Communications 4 (2002) 973-977.
The above-mentioned attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for DNA analysis, the material of described infrared spectrum pool is glass or teflon.
The above-mentioned attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for DNA analysis, described O-ring seal 8 are dimethyl silicone polymer (PDMS) ring.
The above-mentioned attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for DNA analysis; Be applied to DNA analysis and detection; Be at golden film surface fixed trapped DNA, when having target dna in the sample, an end of target dna and capture dna hybridization; The other end and signal dna hybridization; Then simultaneously load signal DNA and infrared probe are taken golden film surface to the nanogold particle of mercaptobenzoic acid, through the infrared signal of observation infrared probe to mercaptobenzoic acid, and then realize the crossover process of on-line tracing DNA directly perceived.
Beneficial effect of the present invention is:
Utilize the biocompatibility of preparation nanometer island gold thin film; Research is in the DNA biochemical property of virgin state under the liquid environment; Detecting spatial orientation and the three-dimensional conformation of dna molecular on certain specific interface changes; Obtain the relevance between surface information and its reactivity, study the interaction of non-life solid surface and life elementary cell DNA, obtain the model of molecule DNA and solid surface effect; Utilize the surface of processing to increase infrared spectrometer secondary optic researching DNA crossover process.The testing result that shows high confidence level in real time, intuitively obtains real-time, online analysis result, and in conjunction with observation, experiment and scientific hypothesis, dynamic tracking, equivalence or approximate artificial model simulate life process.
The DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for of the present invention except that infrared window, does not need other any specialized equipment; Other all parts all can be processed voluntarily; The processing unit (plant) needed raw material is simple, and structure is reasonable, and simple installation is prone to row; The required sample size of DNA analysis process is little, is easy to that real-time monitoring, cost are low, its fabrication and installation technology is very easily promoted the use of.
Description of drawings
Fig. 1 is for being used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic synoptic diagram.
Fig. 2 is the synoptic diagram of each parts of Fig. 1 infrared spectrometer secondary optic.
Fig. 3 is the structure dissection figure that is used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic optical window collet.
Fig. 4 is the structure dissection figure that is used for the set collar of DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic.
Fig. 5 is the structure dissection figure that is used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic spectrum liquid pool.
Fig. 6 is for being used for island nano-gold film sem photograph on the DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic infrared window.
The DNA that Fig. 7 attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic is applied to sandwich structure is hybridized real-time test experience mechanism figure.
Fig. 8 is that the surface strengthens attenuated total reflection surface enhancing infrared (SEIRA-ATR) light path system synoptic diagram.
Figure is the surface-reinforced infrared spectrum figure of 9 control experiments: driftlessness DNA only exists mercaptobenzoic acid-gold size-signal dna (a); There is (b) simultaneously in gold size-signal dna, target dna; Self assembly is to mercaptobenzoic acid (c) on the gold film; There is formation sandwich structure (d) in target dna simultaneously to mercaptobenzoic acid-gold size-signal dna.
Figure 10 is the attenuated total reflection surface-reinforced infrared spectrum figure of different hybridization time.
Figure 11 is 1587cm
-1, 1666cm
-1Place's absorption peak integral area is to the hybridization time curve.
Figure 12 is the reappearance based on attenuated total reflection surface-reinforced infrared spectrum identification DNA crossover process.
Figure 13 is the DNA hybridization kinetics curve of selectivity experiment: 100nM target dna (a); 100nM single base mismatch DNA (b); Blank buffer solution (c); The 100nM DNA (d) that do not match fully.
Embodiment
Embodiment 1
The preparation of infrared window: the silicon hemisphere 1 of cut-off footpath 35mm; On the silicon hemispherical plane, plate island nano-Au films 2, island nano-Au films preparation method is specifically referring to 1.Hiroto Miyake; Shen Ye; Masatoshi Osawa, Electroless deposition of gold thin films on silicon for surface-enhanced infrared spectroelectrochemistry, Electrochemistry Communications 4 (2002) 973-977.
Embodiment 2
Optical window collet 5 and set collar 6, O-ring seal 8 and the fixing pipe 7 of pressing at the bottom of column 4 through accessory silicon hemisphere both sides and the silicon hemisphere link together infrared window, O-ring seal 8, set collar 6, infrared spectrum pool 3 and the fixing pipe 7 of pressing, and infrared spectrum pool 3 is right cylinders of a band shoulder, and material is a teflon; Cylinder diameter is φ 28.7mm; The diameter of shoulder is φ 37mm, takes on thick 4.5mm, and four sockets are arranged on the spectrum tool; Contrast electrode socket internal diameter φ 4mm wherein; To electrode socket internal diameter φ 2mm, be working electrode with golden film, can be applicable to the infrared spectrum electrochemical research; Two other is imported and exported for fluid sample, and internal diameter is φ 2mm, and the bottom is miniature column liquid storage tank, and described column has two, and column is the cylinder that there is screw at two ends; Diameter phi 10mm, high 21mm, described optical window collet 5 is a strip-shaped blade, and is as shown in Figure 2, there is a disk with holes at its center; Can hold silicon hemisphere, two ends are porose, and center pit is apart from being 50mm, are complementary with the screw of column 4 lower ends, can be screwed at column lower end; Described set collar 6 is an annulus, and inwall has screw thread, and two screw holes of symmetry are arranged on the set collar 6, is complementary with the screw of column 4 upper ends, can be fixed on upper end on the column; Other has a fixing pipe 7 of pressing, and the column diameter of its internal diameter and infrared spectrum pool is complementary, internal diameter φ 29mm, external diameter φ 39mm; The fixing outer wall of pipe 7 of pressing has screw thread, is complementary with the screw thread of set collar inwall, and the spiral shell square is 3um, so; Pad is gone up O-ring seal 8 between infrared window and the infrared spectrum pool, and O-ring seal 8 internal diameters are φ 10mm, external diameter φ 35mm, thick 2mm; To fix and press pipe 7 screw-in set collars 6 to compress, promptly be configured for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic.
Embodiment 3
Being used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic is assemblied in the ATR annex according to attenuated total reflection surface-reinforced infrared spectrum appearance (ATR-SEIRA) light path system synoptic diagram.Turn set collar 6 can be regulated the position of optical window in the ATR annex; Simultaneously, through regulating the angle of two reflective mirrors in the ATR annex, change the incident angle of infrared beam; Make the infrared beam incident angle greater than critical angle, this moment, infrared beam was at optical window inside surface generation total reflection phenomenon.Adopt incident angle greater than 70 ℃ in this experiment.In the ATR-SEIRAS experiment, the infrared light of p-polarization gets into optical window in optical window inside surface generation total reflection with the incident angle greater than 70 ℃.(Bruke Germany) is used for the collection of infrared spectrum to FTIS Tensor 27, is equipped with cadmium mercury telluride (MCT) detecting device and the KBr beam splitter of cooled with liquid nitrogen.
Embodiment 4
Be used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic; Be applied to DNA detection; The steps include: at golden film surface fixed trapped DNA, when having target dna in the sample, an end of target dna and capture dna hybridization; The other end and signal dna hybridization; Then simultaneously load signal DNA and infrared probe are taken golden film surface to the nanogold particle of mercaptobenzoic acid, through observing the infrared signal of infrared probe to mercaptobenzoic acid, and then the online intuitively tracking DNA crossover process of realization.Its mechanism is seen Fig. 7.
(1) base sequence of the synthetic oligonucleotide (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) of experiment use is as follows:
Capture dna: 5 '-SH-(CH2) 6-TCG TAC GAT CGA TCC-3 '
Signal dna: 5 '-TAT CGT GTG AGC GGC TTT TTT TT-(CH
2)
6-SH-3 '
Target dna: 5 '-GCC GCT CAC ACG ATA TTT TTT TTG GAT CGA TCG TAC GA-3 '
Single base mismatch DNA:5 '-GCC GCT CAC ACG ATA TTT TTT TTG GAT CGA TGG TAC GA-3 '
DNA:5 '-ACA TGC TTG GAC TGC TTT TTT TTC AGG CTC ATC GTA CG-3 ' does not fully match
Use solution formula following in the experiment:
Test used oligonucleotide and be dissolved in the storing solution that is mixed with 100uM in the TE damping fluid earlier, be distributed into volume required after, be stored in-20 ℃ the refrigerator subsequent use.
TE damping fluid: 10mM Tris-HCl, 1.0mM EDTA (pH 8.0); Hybridization buffer: sodium citrate buffer 150mM, NaCl solution 750mM (pH 7.0); DNA is damping fluid fixedly: 50mM PBS (pH7.0,0.3M NaCl).PBS solution is by NaH
2PO
4And Na
2HPO
4Preparation.
6-sulfydryl hexanol (Sigma), to mercaptobenzoic acid (Sigma), and other reagent be analyze pure.Experiment institute water be ultrapure water (Milli-Q Millipore, USA).
(2) modification of the preparation of Au glue nano particle and unimolecular layer bio-barcode
1) preparation golden nanometer particle method reference literature: 2.Katherine C.Grabar; GrGrWith Freeman; Michael B.Hommer, Michael J.Natan, Preparation and Characterization of Au colloid Monolayers; Analytical Chemistry 67 (4) (1995), and 735～743.
2) with 20uL 1.0 * 10
-4M to mercaptobenzoic acid and 5uL 1.0 * 10
-4The signal dna of M joins in the 2mL gold size solution, and slowly electromagnetic agitation is 16 hours, and places 48 hours at 4 ℃.Use 50mM then, PBS (pH 7.0, and 0.3MNaCl) centrifuge washing is three times, and to remove not the material with the gold size bonding, the gold size that will modify the unimolecular layer biological barcode at last is dispersed among the PBS of 1mL (pH7.0,0.3M NaCl), is stored in 4 ℃.
(3) structure of sandwich structure DNA sensor
The island nano-gold film electrode that is deposited on Si optical window surface is immersed in 1.0 * 10
-8Assembling is 24 hours in the capture dna solution of M sulfydryl modification (PBS, pH7.0,0.3M NaCl), removes capture dna solution after assembling is accomplished also with a large amount of ultrapure water flushings.Subsequently electrode is immersed middle the reaction 30 minutes of sulfydryl hexanol solution (PBS, pH7.0,0.3M NaCl) of 1mM, and with a large amount of ultrapure waters flushings.Fix the island nano-gold film electrode note of capture dna and made ssDNA/AuNps.
Crossover process for the first time: the ssDNA/AuNps electrode immersed contain 1 * 10
-7In the hybridization buffer solution of M target dna, hybridized 3 hours,, use the flushing of PBS (pH7.0,0.3M NaCl) buffer solution at last with a large amount of hybridization buffer flushings three times.Island nano-gold film electrode note after the hybridization is made dsDNA/AuNps for the first time.Subsequently dsDNA/AuNps is immersed the gold size solution that has biological barcode, the additional base of the other end of target dna is then hybridized with the signal dna that is modified at the gold size nanoparticle surface, promptly carries out the hybridization second time.Crossover process adopts ATR-SEIRA spectrum to monitor in real time for the second time.
(4) ATR-SEIRA spectral characterization
The setting of ATR-SEIRA light path such as Fig. 8 add PBS (pH7.0,0.3M NaCl) spectrum as a setting at the dsDNA/AuNps electrode surface.The gold size solution that will have biological barcode joins in the spectrum tool, and opening entry attenuated total reflection infrared absorption spectrum.Spectral resolution 4cm
-1, the result scans 128 times mean value.In the spectrogram gatherer process, feed in the instrument sample cavity and remove CO
2Dried compressed air, to avoid aqueous vapor and CO in the environment
2Influence to background signal.All experiments are all at room temperature carried out.
(5) SEIRA-ATR detects DNA hybridization
Adopt the hybridization of the signal dna on ATR-SEIRA technical monitoring target dna and golden nanometer particle surface.With dsDNA/AuNps electrode and PBS (pH7.0,0.3M NaCl) is background, gathers the infrared signal of probe molecule fixing on the golden nanometer particle to mercaptobenzoic acid with the starting point that is incorporated as of bio-barcode gold nano-particles modified.Utilize attenuated total reflection surface-reinforced infrared spectrum technology to make infrared signal obtain tens times enhancing, the DNA sensor based on DNA-bio-barcode technique construction has carried out the secondary amplification to detection signal, has obtained infrared spectroscopy signals preferably.
Control experiment attenuated total reflection surface-reinforced infrared spectrum under the different condition is (see figure 9) as a result: inject bio-barcode gold nano-particles modified solution and driftlessness DNA when existing, (curve a) almost not observe infrared absorption peak to mercaptobenzoic acid; Directly will be assembled in island nano-gold film surface (curve c) and produce the surface enhancing infrared absorption peak that significantly belongs to the various vibration modes of mercaptobenzoic acid mercaptobenzoic acid; Relatively a and c curve can find out the island nano-gold film can directly strengthen be fixed on golden film surface to the infrared absorption of mercaptobenzoic acid molecule and in the WS to not response of mercaptobenzoic acid.When target dna and the coexistence of bio-barcode gold nano-particles modified, can form sandwich structure, this structure is taken the bio-barcode gold nano-particles modified golden film surface to and then is produced the important surface of mercaptobenzoic acid is strengthened infrared absorption peak (curve d). and curve d is identical with the infrared signature absorption peak position of curve c.Show that to mercaptobenzoic acid signal dna success mark is to gold nano grain.When the gold nano grain of marking signal DNA only and target dna coexistence, only show more weak signal dna surface and strengthen infrared absorption peak (curve b).
The attenuated total reflection surface of different hybridization time strengthens FTIR and sees Figure 10.At 1750cm
-1～1250cm
-1A plurality of infrared absorption peaks have appearred in the wavelength coverage.Along with the increase of hybridization time, the infrared absorption peak among the figure constantly strengthens, until reaching balance.The ownership of each absorption peak is listed in table 1 among Figure 10.To 1587cm
-1, 1666cm
-1The absorption peak at place carries out integration, integrated value can be used for representing being fixed on the surface to the amount of mercaptobenzoic acid so that reflect the amount of DNA hybridization indirectly.With integrated value the time mapping is obtained Figure 11.As can beappreciated from fig. 11, along with the increase of hybridization time, the amount to mercaptobenzoic acid on the surface increases, and the increase of the golden nanometer particle quantity that is fixed on the interface is described.Because the fixedly dependence of golden nanometer particle is the hybridization of target dna and signal dna,, when mercaptobenzoic acid is no longer changed, explains to hybridize and accomplish the carrying out that the increase of mercaptobenzoic acid amount reflects hybridization reaction.Figure 12 is the reappearance based on attenuated total reflection surface-reinforced infrared spectrum identification DNA crossover process.
Table 1. surface-reinforced infrared spectrum peak ownership
DNA sensor based on the attenuated total reflection surface-reinforced infrared spectrum can show good selectivity and good detectability aspect the recognition objective DNA; Identification dna single base mispairing that can be sensitive, and realized in real time on-line tracing (seeing Figure 13) to short chain oligonucleotide DNA crossover process.
The present invention is used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic, is not limited to the foregoing description, prepares the attenuated total reflection surface-reinforced infrared spectrum instrument auxiliary device of different specification size according to different DNA analysis demands; And all parts of surface-reinforced infrared spectrum instrument auxiliary device that are used for DNA analysis that make up according to the method for the invention are seen shown in the accompanying drawing 1～6; The DNA that is applied to sandwich structure is hybridized real-time test experience mechanism figure and is seen shown in the accompanying drawing 7.
Claims (5)
1. one kind is used for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic; It is characterized in that: it comprises a silicon hemisphere (1); The island nano-gold film (2) that the surface on silicon hemisphere (1) plane is coated with infrared enhancement effect constitutes infrared window, and sealing and fixing has infrared spectrum pool (3) on the lip-deep island nano-gold film of the silicon hemispherical plane infrared window, and this infrared spectrum pool is a right cylinder; Have four axial penetrating holes; One big three is little, and wherein macropore is the contrast electrode socket, internal diameter φ 4-5mm; Three apertures are respectively sample inlet, sample export and to the electrode socket; Internal diameter is φ 2-3mm, and the bottom is miniature column liquid storage tank, is working electrode with golden film, can be applicable to the infrared spectrum electrochemical research.
2. attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic according to claim 1 is characterized in that: sealing and fixing infrared spectrum pool on the described infrared window is that column (4) and the optical window collet (5) at the bottom of the silicon hemisphere, set collar (6), O-ring seal (8) and fixing through accessory silicon hemisphere both sides presses pipe (7) that infrared window, O-ring seal (8), set collar (6) and fixing the pressure are managed (7) and linked together; Infrared spectrum pool is the right cylinder of a band shoulder, and described column (4) has two, and column (4) is the cylinder that there is screw at two ends; Described optical window collet (5) is a strip-shaped blade, and there is a disk with holes at its center, holds silicon hemisphere; Two ends are porose, are complementary with the screw of column (4) lower end, are screwed at column lower end; Described set collar (6) is an annulus, and inwall has screw thread, and two screw holes of symmetry are arranged on the set collar (6); Be complementary with the screw of column (4) upper end; Be fixed on the column upper end, other has a fixing pipe (7) of pressing, and the column diameter of its internal diameter and infrared spectrum pool is complementary; The fixing outer wall of pipe (7) of pressing has screw thread; Be complementary with the screw thread of set collar (6) inwall, so, pad is gone up O-ring seal (8) between infrared window and the infrared spectrum pool; To fix and press pipe (7) screw-in set collar (6) to compress, promptly be configured for DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic.
3. the DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for according to claim 1 and 2, it is characterized in that: the material of described infrared spectrum pool is glass or teflon.
4. the DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for according to claim 2, it is characterized in that: described O-ring seal (8) is the dimethyl silicone polymer ring.
5. the DNA analysis attenuated total reflection surface-reinforced infrared spectrum appearance secondary optic that is used for according to claim 1 is in DNA analysis and the application that detects.
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| CN103033474A (en) * | 2012-12-10 | 2013-04-10 | 中南大学 | Electrochemical-optical combined in-situ study spectral cell |
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| CN109632848A (en) * | 2018-12-28 | 2019-04-16 | 厦门大学 | A kind of spectrum tool suitable for electrochemical in-situ XRD characterization |
| CN110087544A (en) * | 2016-12-26 | 2019-08-02 | 三菱电机株式会社 | Organism substance-measuring device and organism substance-measuring method |
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| CN103033474A (en) * | 2012-12-10 | 2013-04-10 | 中南大学 | Electrochemical-optical combined in-situ study spectral cell |
| CN103033474B (en) * | 2012-12-10 | 2016-06-08 | 中南大学 | Electrochemistry optics coupling on-spot study spectrum tool |
| CN110087544A (en) * | 2016-12-26 | 2019-08-02 | 三菱电机株式会社 | Organism substance-measuring device and organism substance-measuring method |
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| CN108458986A (en) * | 2018-03-05 | 2018-08-28 | 中国农业大学 | DNA infrared spectrum analysis assay method |
| CN109060900A (en) * | 2018-08-10 | 2018-12-21 | 华中科技大学 | A kind of decaying total reflection chip, its preparation and the application of boron-doped diamond modification |
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| CN109632848B (en) * | 2018-12-28 | 2020-05-22 | 厦门大学 | Spectrum pool suitable for electrochemistry normal position XRD characterization |
| CN113447546A (en) * | 2021-06-28 | 2021-09-28 | 中国科学院长春应用化学研究所 | Vacuum-compatible surface-enhanced infrared spectroscopy electrochemical device and application |
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