CN102507444B - Auxiliary optical device of attenuation total reflection surface enhanced infrared spectrometer for DNA analysis - Google Patents

Abstract

The invention relates to an attenuated total reflection surface enhanced infrared optical device for DNA recognition and hybridization dynamics analysis. The device consists of an attenuated total reflection surface-enhanced infrared optical window coated with an island-shaped nano-gold film on the surface of a silicon hemisphere, an optical table base, a fixed bracket area, an infrared spectrum pool, and a PDMS sealing ring. The silicon hemisphere coated with a gold island film is placed on the base of the optical table, the gold film of the optical window faces the infrared spectrum pool, and the middle is sealed with a PDMS sealing ring; through the external thread of the fixing bracket and the internal thread of the upper bracket of the optical table base The spectral cell, PDMS sealing ring and silicon hemispherical optical window are screwed in and fixed to form a surface-enhanced infrared spectroscopy device for DNA analysis. The device is simple in structure, easy to operate, requires a small amount of sample, and can be used stably for a long time. It can conveniently detect infrared spectrum signals in the 1100cm ^-1 -4000cm ^-1 band in real time on-line, and can analyze DNA molecules with high sensitivity. Recognition and hybridization mechanism and kinetics research.

Description

An auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer

Technical field:

The invention belongs to the technical field of infrared spectroscopy, in particular to an attenuated total reflection surface enhanced infrared spectrometer auxiliary optical device for DNA analysis.

Background technique:

Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) is an important analytical tool for studying molecular structure information at interfaces. Combined with surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) in attenuated total reflection (ATR) mode, it has the advantages of strong surface signal, unimpeded mass transfer, and small interference from bulk solution. Research on recognition and hybridization kinetics. At the same time, the problems faced by traditional external reflection infrared absorption spectroscopy (IR Absorption Spectroscopy, IRAS) are avoided, such as insufficient surface signal, mass transfer supplementary hysteresis, and interference from solution background. The use of internal reflection surface enhanced infrared absorption spectroscopy technology as an extension of ordinary infrared spectroscopy has significantly improved the detection accuracy of infrared spectroscopy and greatly expanded the research scope of infrared spectroscopy, such as interface small molecule compounds, peptides, proteins, oligonucleotides and The dynamic behavior of biomolecules and biological tissues such as oligosaccharides, lipids, phages, viruses and cells.

There are two types of prism devices used to construct the internal reflection surface enhanced infrared absorption spectrum cell: Otto type and Kretschmann type. There are many kinds of prism shapes to choose from, among which Kretschmann hemispherical prism is used for research. The hemispherical prism can ensure that the incident light at any angle is perpendicular to the interface, the loss of reflected light is small, and the angle after entering the prism remains unchanged. Internationally, the liquid flow spectroelectrochemical cell designed by Osawa. and Ataka is the mainstream of application. The spectral pool devices of other groups are all modified on this basis. The Adzic group proposed the concept of combining ZnSe pillars and silicon wafer infrared windows. The micro-attenuated total reflection surface-enhanced infrared spectroscopy experimental technology developed by Sudo et al. can analyze small substances with a submillimeter size or nanometer layer thickness on the surface of the substance. According to "the theory of prism-film coupler for plane wave", Cai Wenbin's research group at Fudan University has developed a simple and easy auxiliary device for electrochemical internal reflection SEIRAS. The spectral assist device was patented in 2008. Professor Jiang Xiu'e from the Changchun Institute of Applied Chemistry combined the methods of directional assembly and membrane reorganization to innovatively build a platform that can be used to simulate the physiological existence of membrane proteins, and monitor the directional assembly and membrane reorganization of membrane proteins at the nano-interface through surface-enhanced infrared spectroscopy in situ The process has expanded the research of attenuated total reflection infrared spectroscopy on the structure and function of interface proteins. All of the above focuses on the expansion of the electrode system, the improvement of the film-making method, and the continuous improvement of the surface-enhanced infrared spectroscopy technology as a powerful auxiliary tool for on-site electrochemistry. In recent years, deoxyribonucleic acid (DNA) is the key to explain a variety of life phenomena, and has become one of the most important research fields in life science. DNA-based biosensors have been widely studied and applied in the fields of genetic engineering, medical diagnosis, new drug screening, drug action mechanism, environmental monitoring, forensic identification, etc. due to their simplicity, speed, sensitivity, and low price. DNA sensing technology expresses the process and results of hybridization with easily measurable physical signals such as light and electricity, so as to obtain information such as the concentration and sequence of target DNA molecules, which is an important means of current DNA research. However, almost no one at home and abroad pays attention to the development of a simple and easy-to-use micro DNA analysis and detection device for internal reflection surface enhanced infrared absorption spectroscopy. In addition, infrared spectroscopy can give the "fingerprint" characteristics of compounds, and is an important tool for analyzing DNA molecular structure and spatial orientation. Therefore, it is very important to develop a set of attenuated total reflection surface-enhanced infrared spectrometer auxiliary device that can achieve high sensitivity, high resolution, device miniaturization and low cost, and is suitable for DNA analysis and detection.

Invention content:

The purpose of the invention of this device is to solve the problems that current infrared devices are not suitable for online analysis of DNA hybridization, difficult to manufacture, low detection sensitivity, and large sample volume. Enhanced infrared spectrometer auxiliary optical device.

The island-shaped nano-noble metal film with surface infrared enhancement provides a platform for surface modification and DNA sensor construction, which can avoid the interference of background water and directly analyze and detect aqueous solution samples; it can study the structure and properties of functional surfaces in situ and monitor the interface in real time A molecular recognition reaction occurs; the activity of the molecule to be tested can be maintained, and the study of molecular reaction kinetics can be realized. Extend infrared spectroscopy to the application of DNA hybridization process, and realize real-time, in situ and online tracking of DNA recognition and hybridization process.

In order to achieve the above object, the present invention adopts the following technical solutions:

An auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer, as shown in Figure 1, is characterized in that the device includes a silicon hemisphere 1, and the surface of the silicon hemisphere 1 plane is coated with an island-shaped infrared enhancement effect The nano-gold film 2 constitutes an infrared light window, and an infrared spectrum pool 3 is sealed and fixed on the island-shaped nano-gold film on the silicon hemispheric plane surface of the infrared light window. The infrared spectrum pool is a cylinder with four axial transparent holes. The large hole is the reference electrode socket with an inner diameter of φ4-5mm; the three small holes are the sample inlet, sample outlet and counter electrode socket respectively; the inner diameter is φ2-3mm, and the bottom is a miniature columnar liquid storage pool, with a gold film As a working electrode, it can be used in infrared spectroelectrochemical research.

In the aforementioned attenuated total reflection surface-enhanced infrared spectrometer auxiliary optical device for DNA analysis, the infrared light window is sealed and fixed on the infrared spectrum pool, which can be equipped with the columns 4 on both sides of the silicon hemisphere and the light window bottom support 5 at the bottom of the silicon hemisphere , the fixed ring 6, the sealing ring 8 and the fixed pressure tube 7 link the infrared light window, the sealing ring 8, the fixed ring 6 and the fixed pressure tube 7 together, and the infrared spectrum pool can be a cylinder with a shoulder for fixing, the described There are two columns 4, and the column 4 is a cylinder with screw holes at both ends, and the light window bottom support 5 is a strip sheet, as shown in Figure 2, there is a disc with a hole in its center, which can hold the Silicon hemisphere, with holes at both ends, matches the screw holes at the lower end of the column 4, and can be fixed on the lower end of the column with screws. The fixed ring 6 is a ring with threads on the inner wall. There are two symmetrical rings on the fixed ring 6. Screw hole, matches with the screw hole of column 4 upper ends, can be fixed on column upper end, has a fixed pressure tube 7 in addition, and its internal diameter matches the cylinder diameter of infrared spectrum pool, and the outer wall of fixed pressure tube 7 is threaded, and The threads on the inner wall of the fixing ring 6 are matched, so that a sealing ring 8 is placed between the infrared light window and the infrared spectrum pool, and the fixing pressure tube 7 is screwed into the fixing ring 6 to be pressed tightly, thus forming an attenuated total reflection surface-enhanced infrared spectrometer for DNA analysis Auxiliary optics.

The above-mentioned attenuated total reflection surface-enhanced infrared spectrometer auxiliary optical device for DNA analysis, the preparation method of the island-shaped gold film is to use a chemical replacement reaction (Galvanic reaction) to prepare a uniform and high-enhancement effect on the silicon hemisphere plane surface Infrared enhanced surface, its specific operation method see: Hiroto Miyake, Shen Ye, Masatoshi Osawa, Electroless deposition of gold thin films on silicon for surface-enhanced infraredspectroelectrochemistry, Electrochemistry Communications 4(2002) 973-977.

In the aforementioned attenuated total reflection surface-enhanced infrared spectrometer auxiliary optical device for DNA analysis, the material of the infrared spectrum pool is glass or polytetrafluoroethylene.

In the aforementioned attenuated total reflection surface-enhanced infrared spectrometer auxiliary optical device for DNA analysis, the sealing ring 8 is a polydimethylsiloxane (PDMS) ring.

The above-mentioned attenuated total reflection surface-enhanced infrared spectrometer auxiliary optical device for DNA analysis is applied to DNA analysis and detection. It fixes and captures DNA on the surface of the gold film. Hybridization, the other end is hybridized with the signal DNA, and then the gold nanoparticles loaded with the signal DNA and the infrared probe p-mercaptobenzoic acid are brought to the surface of the gold film. By observing the infrared signal of the infrared probe p-mercaptobenzoic acid, the intuitive online Track the DNA hybridization process.

The beneficial effects of the present invention are:

Utilize the biocompatibility of nano-island-shaped gold films to study the biochemical properties of DNA in the original state in a liquid environment, detect the spatial orientation and three-dimensional conformation changes of DNA molecules on a specific interface, and obtain the relationship between surface information and its reactivity. Relevance, to study the interaction between non-living solid surface and DNA, the basic unit of life, to obtain the model of the interaction between biomolecular DNA and solid surface; use the processed surface enhanced infrared spectrometer to assist optical devices to study the DNA hybridization process. Real-time and intuitive display of high-reliability detection results, real-time and online analysis results, combined with observation, experiment and scientific assumptions, dynamic tracking, equivalent or approximate artificial models to simulate life processes.

The auxiliary optical device for DNA analysis attenuated total reflection surface enhanced infrared spectrometer of the present invention does not need any other special equipment except the infrared light window, and all other parts can be processed by itself. The raw materials required for the processing device are simple, the structure is reasonable, and the installation is easy. It is simple and easy to operate, the sample volume required in the DNA analysis process is small, it is easy to monitor in real time, the cost is low, and its production and installation technology is very easy to promote and use.

Description of drawings

Figure 1 is a schematic diagram of an auxiliary optical device for an attenuated total reflection surface-enhanced infrared spectrometer for DNA analysis.

Fig. 2 is a schematic diagram of components of the auxiliary optical device of the infrared spectrometer in Fig. 1 .

Fig. 3 is an exploded view of the structure of the light window bottom support of the auxiliary optical device of the attenuated total reflection surface-enhanced infrared spectrometer used for DNA analysis.

Fig. 4 is an exploded view of the structure of the fixed ring used for the auxiliary optical device of the attenuated total reflection surface-enhanced infrared spectrometer for DNA analysis.

Fig. 5 is an exploded view of the structure of the spectral liquid pool of the auxiliary optical device of the attenuated total reflection surface-enhanced infrared spectrometer used for DNA analysis.

Fig. 6 is a scanning electron micrograph of an island-shaped nano-gold film on the infrared window of the auxiliary optical device of the attenuated total reflection surface-enhanced infrared spectrometer used for DNA analysis.

Fig. 7 is a schematic diagram of the real-time detection experiment mechanism of DNA hybridization applied to the sandwich structure of the auxiliary optical device of the attenuated total reflection surface-enhanced infrared spectrometer.

Fig. 8 is a schematic diagram of a surface-enhanced attenuated total reflection surface-enhanced infrared (SEIRA-ATR) optical system.

Figure 9 is the surface-enhanced infrared spectrogram of the control experiment: no target DNA, only p-mercaptobenzoic acid-gold colloid-signal DNA (a); gold colloid-signal DNA, target DNA exists simultaneously (b); Assembly of p-mercaptobenzoic acid (c); target DNA, p-mercaptobenzoic acid-gold colloid-signal DNA coexist to form a sandwich structure (d).

Fig. 10 is the attenuated total reflection surface-enhanced infrared spectrum at different hybridization times.

Figure 11 is the curves of the integrated area of the absorption peaks at 1587cm ^-1 and 1666cm ^-1 versus hybridization time.

Figure 12 shows the reproducibility of identification of DNA hybridization process based on attenuated total reflection surface-enhanced infrared spectroscopy.

Figure 13 is the DNA hybridization kinetic curve of the selectivity experiment: 100nM target DNA (a); 100nM single base mismatch DNA (b); blank buffer solution (c); 100nM complete mismatch DNA (d).

Detailed ways

Example 1

Preparation of infrared light window: take a silicon hemisphere 1 with a diameter of 35mm, and coat the island-shaped nano-gold film 2 on the plane of the silicon hemisphere. For the preparation method of the island-shaped nano-gold film, please refer 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.

Example 2

The infrared light window, the sealing ring 8, the fixing ring 6, the infrared spectrum pool 3 and the The fixed pressure tubes 7 are connected together, and the infrared spectrum pool 3 is a cylinder with a shoulder. Sockets, the inner diameter of the reference electrode socket is φ4mm, the internal diameter of the counter electrode socket is φ2mm, and the gold film is used as the working electrode, which can be applied to infrared spectroelectrochemical research; the other two are liquid sample inlets and outlets, the inner diameter is φ2mm, and the bottom is a miniature columnar storage Liquid pool, described upright post has two, and upright post is the cylinder that screw hole is arranged at both ends, diameter φ 10mm, height 21mm, described light window bottom bracket 5 is a strip sheet, as shown in Figure 2, there is a strip in its center The disc of the hole can support the silicon hemisphere, with holes at both ends, and the center hole distance is 50 mm, which matches the screw holes at the lower end of the column 4, and can be fixed at the lower end of the column with screws. The fixed ring 6 is a circular ring , the inner wall is threaded, and there are two symmetrical screw holes on the fixing ring 6, which match the screw holes on the upper end of the column 4 and can be fixed on the upper end of the column. The diameter of the body is matched, the inner diameter is φ29mm, the outer diameter is φ39mm, and the outer wall of the fixed pressure tube 7 is threaded, which matches the thread of the inner wall of the fixed ring, and the thread moment is 3um. In this way, a sealing ring 8 is placed between the infrared light window and the infrared spectrum pool. The sealing ring 8 has an inner diameter of φ10mm, an outer diameter of φ35mm, and a thickness of 2mm; the fixed pressure tube 7 is screwed into the fixed ring 6 and pressed tightly to form an auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer.

Example 3

The auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer (ATR-SEIRA) optical path system schematic diagram is assembled in the ATR attachment. Rotate the fixed ring 6 to adjust the position of the light window in the ATR attachment. At the same time, by adjusting the angles of the two mirrors in the ATR attachment, the incident angle of the infrared beam can be changed so that the incident angle of the infrared beam is greater than the critical angle. At this time, the infrared beam The light beam is totally reflected on the inner surface of the light window. In this experiment, the incident angle is greater than 70°C. In the ATR-SEIRAS experiment, the p-polarized infrared light enters the light window at an incident angle greater than 70°C and undergoes total reflection on the inner surface of the light window. Fourier transform infrared spectrometer Tensor 27 (Bruke, Germany) was used for the acquisition of infrared spectra, equipped with liquid nitrogen cooled mercury cadmium telluride (MCT) detector and KBr beam splitter.

Example 4

Auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer, applied to DNA detection, the steps are: immobilize the capture DNA on the surface of the gold film, when the target DNA exists in the tested sample, one end of the target DNA hybridizes with the capture DNA , the other end is hybridized with the signal DNA, and then the gold nanoparticles loaded with the signal DNA and the infrared probe p-mercaptobenzoic acid are brought to the surface of the gold film. By observing the infrared signal of the infrared probe p-mercaptobenzoic acid, an intuitive online The tracking of DNA hybridization process. Its mechanism can be seen in Figure 7.

(1) The base sequence of the artificially synthesized oligonucleotide (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) used in the experiment 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’

Total mismatch DNA: 5’-ACA TGC TTG GAC TGC TTT TTT TTC AGG CTC ATC GTA CG-3’

The solution formula used in the experiment is as follows:

The oligonucleotides used in the experiment were firstly dissolved in TE buffer to prepare a 100uM stock solution, which was aliquoted into required volumes and stored in a refrigerator at -20°C for future use.

TE buffer: 10mM Tris-HCl, 1.0mM EDTA (pH 8.0); hybridization buffer: sodium citrate buffer solution 150mM, NaCl solution 750mM (pH 7.0); DNA immobilization buffer: 50mM PBS (pH 7.0, 0.3M NaCl). The PBS solution was prepared by NaH ₂ PO ₄ and Na ₂ HPO ₄ .

6-Mercaptohexanol (Sigma), p-mercaptobenzoic acid (Sigma), and other reagents were of analytical grade. The water used in the experiment was ultrapure water (Milli-Q Millipore, USA).

(2) Preparation of Au colloidal nanoparticles and modification of monolayer biological barcodes

1) Reference literature for the method of preparing gold nanoparticles: 2. Katherine C.Grabar, GrGrWith Freeman, Michael B.Hommer, Michael J.Natan, Preparation and Characterization of Au colloid Monolayers, Analytical Chemistry 67(4)(1995), 735～ 743.

2) Add 20uL of 1.0×10 ^-4 M p-mercaptobenzoic acid and 5uL of 1.0×10 ^-4 M signal DNA into 2mL gold colloid solution, stir slowly for 16 hours, and place at 4°C for 48 hours. Then use 50mM, PBS (pH 7.0, 0.3MNaCl) to wash three times by centrifugation, to remove the material that is not bonded to the gold colloid, and finally disperse the gold colloid with the modified monolayer biological barcode in 1 mL of PBS (pH 7.0, 0.3M NaCl) and stored at 4°C.

(3) Construction of DNA sensor with sandwich structure

The island-shaped gold nano film electrode deposited on the surface of the Si light window was immersed in 1.0×10 ^-8 M mercapto-modified capture DNA solution (PBS, pH 7.0, 0.3M NaCl) for 24 hours, and the capture was removed after the assembly was completed. DNA solution and washed with plenty of ultrapure water. Subsequently, the electrode was immersed in 1 mM mercaptohexanol solution (PBS, pH7.0, 0.3M NaCl) for 30 minutes, and rinsed with a large amount of ultrapure water. The island-shaped gold nanofilm electrode immobilized with captured DNA is designated as ssDNA/AuNps.

The first hybridization process: immerse the ssDNA/AuNps electrode in the hybridization buffer solution containing 1×10 ^-7 M target DNA, hybridize for 3 hours, wash with a large amount of hybridization buffer three times, and finally wash with PBS (pH7.0, 0.3M NaCl ) buffer solution for washing. The island-shaped gold nanofilm electrode after the first hybridization is denoted as dsDNA/AuNps. Then dsDNA/AuNps is immersed in the gold colloid solution with biological barcode, and the extra bases at the other end of the target DNA are hybridized with the signal DNA modified on the surface of gold colloidal nanoparticles, that is, the second hybridization is performed. The second hybridization process was monitored in real time using ATR-SEIRA spectroscopy.

(4) ATR-SEIRA spectral characterization

The setting of the ATR-SEIRA optical path is shown in Figure 8, and PBS (pH7.0, 0.3M NaCl) was added to the surface of the dsDNA/AuNps electrode as the background spectrum. Add the gold colloid solution with the biological barcode into the spectral pool, and start recording the attenuated total reflection infrared absorption spectrum. The spectral resolution is 4cm ^-1 , and the result is the average value of 128 scans. During the spectrum acquisition process, dry compressed air with CO ₂ removed was introduced into the sample chamber of the instrument to avoid the influence of moisture and CO ₂ in the environment on the background signal. All experiments were performed at room temperature.

(5) SEIRA-ATR detects DNA hybridization

The ATR-SEIRA technique was used to monitor the hybridization of the target DNA to the signal DNA on the surface of the gold nanoparticles. With the dsDNA/AuNps electrode and PBS (pH7.0, 0.3M NaCl) as the background, the infrared signal of the probe molecule p-mercaptobenzoic acid immobilized on the gold nanoparticles was collected starting from the addition of biological barcode-modified gold nanoparticles. Using the attenuated total reflection surface-enhanced infrared spectroscopy technology, the infrared signal has been enhanced dozens of times. The DNA sensor based on the DNA-biological barcode technology has carried out secondary amplification on the detection signal and obtained a better infrared spectral signal.

The results of the attenuated total reflection surface-enhanced infrared spectroscopy (see Figure 9) of the control experiment under different conditions: when injecting the gold nanoparticle solution modified by the biological barcode without the presence of target DNA, almost no infrared absorption peak of p-mercaptobenzoic acid (curve a); Directly assembling p-mercaptobenzoic acid on the surface of the island-shaped gold nano-film (curve c) produced obvious surface-enhanced infrared absorption peaks attributable to various vibration modes of p-mercaptobenzoic acid; comparing a and c curves can be seen The island-shaped nano-gold film can directly enhance the infrared absorption of p-mercaptobenzoic acid molecules immobilized on the surface of the gold film, but has no response to p-mercaptobenzoic acid in aqueous solution. When the target DNA and biological barcode-modified gold nanoparticles coexist, a sandwich structure can be formed, which brings the biological barcode-modified gold nanoparticles to the surface of the gold film to generate the important surface-enhanced infrared absorption peak of p-mercaptobenzoic acid (curve d ). The positions of the infrared characteristic absorption peaks of curve d and curve c are the same. It indicated that p-mercaptobenzoic acid and signal DNA were successfully labeled on the gold nanoparticles. When the AuNPs that only label the signal DNA coexist with the target DNA, only a weak surface-enhanced infrared absorption peak of the signal DNA is shown (curve b).

The attenuated total reflection surface-enhanced infrared absorption spectra at different hybridization times are shown in Fig. 10 . Several infrared absorption peaks appeared in the wavelength range of 1750cm ^-1 ~ 1250cm ^-1 . With the increase of hybridization time, the infrared absorption peak in the figure is continuously enhanced until reaching equilibrium. The assignment of each absorption peak in Figure 10 is listed in Table 1. By integrating the absorption peaks at 1587cm ^-1 and 1666cm ^-1 , the integral value can be used to represent the amount of p-mercaptobenzoic acid immobilized on the surface and indirectly reflect the amount of DNA hybridization. Figure 11 is obtained by plotting the integral value versus time. It can be seen from Figure 11 that with the increase of hybridization time, the amount of p-mercaptobenzoic acid on the surface increases, indicating that the amount of gold nanoparticles immobilized on the interface increases. Since the fixation of gold nanoparticles depends on the hybridization of target DNA and signal DNA, the increase in the amount of p-mercaptobenzoic acid reflects the progress of the hybridization reaction. When the p-mercaptobenzoic acid does not change anymore, it means that the hybridization has been completed. Figure 12 shows the reproducibility of identification of DNA hybridization process based on attenuated total reflection surface-enhanced infrared spectroscopy.

Table 1. Peak assignments of surface-enhanced infrared spectra

The DNA sensor based on attenuated total reflection surface-enhanced infrared spectroscopy can show good selectivity and excellent detection ability in identifying target DNA, and can sensitively identify DNA single base mismatches, and realize real-time short-chain oligomerization On-line tracking of nucleotide DNA hybridization process (see Figure 13).

The invention is used for the auxiliary optical device of the attenuated total reflection surface-enhanced infrared spectrometer for DNA analysis, and is not limited to the above-mentioned embodiments. Auxiliary devices for the attenuated total reflection surface-enhanced infrared spectrometer of different specifications and sizes are prepared according to different DNA analysis requirements; All components of the surface-enhanced infrared spectrometer auxiliary device for DNA analysis constructed by the above method are shown in Figures 1 to 6; the schematic diagram of the real-time detection experiment of DNA hybridization applied to a sandwich structure is shown in Figure 7.

Claims (4)

1. An auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer, characterized in that it includes a silicon hemisphere (1), and the surface of the silicon hemisphere (1) plane is coated with island-shaped nano-gold with infrared enhancement effect The film (2) constitutes an infrared light window, and the infrared spectrum pool (3) is sealed and fixed on the island-shaped nano-gold film infrared light window on the plane surface of the silicon hemisphere. The infrared spectrum pool is a cylinder with four axial holes. Transparent holes, one big and three small, the big hole is the reference electrode socket, the inner diameter is φ4-5mm; the three small holes are the sample inlet, the sample outlet and the counter electrode socket, the inner diameter is φ2mm, and the bottom is a miniature columnar reservoir. The liquid pool, with the gold film as the working electrode, can be applied to infrared spectroelectrochemical research. The infrared spectrum pool is sealed and fixed on the infrared light window, and the light through the columns (4) on both sides of the accessory silicon hemisphere and the bottom of the silicon hemisphere The window bottom support (5), the fixing ring (6), the sealing ring (8) and the fixing pressure tube (7) connect the infrared light window, the sealing ring (8), the fixing ring (6) and the fixing pressure tube (7) Together, the infrared spectrum pool is a cylinder with a shoulder, and there are two uprights (4), and the uprights (4) are cylinders with screw holes at both ends, and the bottom bracket of the light window (5) is a strip There is a disc with a hole in the center, which can support the silicon hemisphere. There are holes at both ends, which match the screw holes at the lower end of the column (4), and are fixed on the lower end of the column with screws. The fixed ring (6) is A circular ring with threads on the inner wall, and two symmetrical screw holes on the fixing ring, matching with the screw holes on the upper end of the column (4), fixed on the upper end of the column, and fixing the inner diameter of the pressure tube (7) to the column of the infrared spectrum pool The outer wall of the fixed pressure tube (7) is threaded, which matches the thread on the inner wall of the fixed ring (6). In this way, a sealing ring (8) is placed between the infrared light window and the infrared spectrum cell, and the fixed pressure tube ( 7) Screw into the fixing ring (6) and press it tightly, which constitutes an auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer. 2. the auxiliary optical device for DNA analysis attenuation total reflection surface enhanced infrared spectrometer according to claim 1, characterized in that: the material of the infrared spectrum pool is glass or polytetrafluoroethylene, and there are four sockets on the spectrum pool , the inner diameter of the reference electrode socket is φ4mm, the inner diameter of the counter electrode socket is φ2mm, and the gold film can be used as the working electrode for infrared spectroelectrochemical research; the other two are liquid sample inlets and outlets, the inner diameter is φ2mm, and the bottom is a micro-column reservoir. 3. The auxiliary optical device for attenuated total reflection surface-enhanced infrared spectrometer for DNA analysis according to claim 2, characterized in that: the sealing ring (8) is made of ring polydimethylsiloxane. 4. The auxiliary optical device for DNA analysis attenuated total reflection surface-enhanced infrared spectrometer according to claim 1, which is used for the detection of DNA.

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