CN1246686C - High-sensitivity surface plasma resonance detector with multiple total reflection - Google Patents
High-sensitivity surface plasma resonance detector with multiple total reflection Download PDFInfo
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- CN1246686C CN1246686C CN 200310100368 CN200310100368A CN1246686C CN 1246686 C CN1246686 C CN 1246686C CN 200310100368 CN200310100368 CN 200310100368 CN 200310100368 A CN200310100368 A CN 200310100368A CN 1246686 C CN1246686 C CN 1246686C
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Abstract
The present invention discloses a high-sensitivity surface plasma resonance detector with multiple total reflection processes, which belongs to the field of optical detectors. The high-sensitivity surface plasma resonance detector is characterized in that the plane above a cylindrical mirror is coated with a metal film which is used as a sensitive film, a photodetector is arranged under the cylindrical mirror, and an incident light source is arranged on the left of the cylindrical mirror; compared with the traditional single-point total reflection, light emitted by the light source is irradiated on the space between the cylindrical mirror and the metal film through the cylindrical mirror, and multiple internal total reflection processes are generated; therefore, the refracted light path and the light intensity of the emergent light can be simultaneously changed, the refracted light can be absorbed by detection sample molecules on the metal film, and the sensitivity of the resonance detection of surface plasma oscillators and signal resolution are increased. With the present invention, the in-situ real-time monitoring of the intermolecular interaction can be carried out without any signs; therefore, the present invention can be used for researches of multimolecular interaction, which comprise chemical detection, biochemical detection, biotechnological detection, molecular biological detection and microbiological detection.
Description
Technical field
The invention belongs to the optical detecting instrument scope, particularly be applicable to a kind of highly sensitive surface plasma resonance detecting device of structural change in detection molecules in the fields such as chemical analysis, environmental monitoring, immunoassay, DNA detection, drug screening or the monitoring molecule with repeatedly total reflection.
Background technology
Surface plasma resonance (SPR) is a kind of physical optics phenomenon.When the polarized light that is parallel to the surface impinges upon with certain incident angle (surface plasma angle) on the intermembranous interface of metal and ligand molecular, in when, on the interface total reflection taking place, incident light is coupled into metal and the intermembranous interface of ligand molecular, the electromagnetic wave that formation is propagated along the interface, i.e. surface plasma (SP).Light with the incident of surface plasma angle will cause that owing to surface plasma resonance the boundary reflection rate significantly reduces, because SPR is to very responsive and can be detected attached to the refractive index of the ligand molecular film of metal surface, the surface plasma angle difference of different ligands molecular film: and commaterial, attached to the amount difference of metal surface, the response intensity difference of SPR then.According to above-mentioned principle, the SPR molecule sensor is fixed on known molecule (as ligand molecular) metal (gold, silver etc.) the film surface of tens nanometer thickness usually, add the target molecule complementary with it, both rise in conjunction with the refractive index that will make metal film and solution interface, thereby cause resonant angle to change, if fixing incident angle just can carry out detection by quantitative to the target molecule of complementation according to the change degree of resonant angle.Whole sensing process comprises: the interaction (coupling) of (1) molecule; (2) sensitive layer ligand molecular film changes (specific inductive capacity/refraction index changing); (3) sensor electrical changes of magnetic field (reflecting light decay); (4) photosignal detects; (5) continuous detecting of signal and analysis.
Spr sensor mainly comprises optical waveguide coupled device, metal film, three ingredients of ligand molecular film.
1. optical waveguide coupled device: with light wave and SP coupling and it is resonated, must use coupled apparatus.Coupled apparatus commonly used is mainly prism.Selectable prism geometries is right angled isosceles triangle or semisphere.Semisphere can guarantee that the incident light of any angle is all vertical with the interface, and the reflected light loss is little, and the angle that enters behind the prism is constant.For SPR research, the refractive index difference of prism and medium is big more, and measurement sensitivity is high more.In order to improve sensitivity, answer the higher optical material of selective refraction rate to make prism, as BK7 (refractive index 1.51650), SF12 (refractive index 1.64831), SF6 (refractive index 1.80518).
2. light source: the SPR device that (1) fixed wave length changes the incident angle metering system adopts He-Ne laser instrument (wavelength 632.8nm) as light source more.As light source, monochromaticity is good with laser instrument, the intensity height.Light emitting diode (LED) in addition) also can be used as the SPR light source, wavelength mostly is near infrared (wavelength 760nm).The monochromaticity of LED is better, and volume is little, and price is low, long service life.(2) be fixed into the SPR device that firing angle changes the wavelength measurement mode, light source is to launch the continuous light source of various wavelength, and has enough intensity and stability.
3. metal film: Au film and Ag film are two kinds of the most frequently used in SPR metallic films.The Ag film has very high reflectivity and higher measurement sensitivity, and stability is higher than the Al film, is the first-selected metal film of SPR.Au film stable best has stronger chemical inertness, especially is suitable for the out of use system of Ag film.Three characteristic parameters (resonant wavelength or resonance angle resonance width and the resonance degree of depth) from SPR spectrum, under same condition, the resonant wavelength of Ag film or the variation of resonance angle are obviously than Au film sensitivity, and the resonance degree of depth is approximately greater than the Au film, and the resonance half-peak breadth is significantly less than Au.The thickness of metallic film directly influences the resonance degree of depth, and along with the increase of the thickness of metal film, the resonance degree of depth diminishes, and promptly the minimal reflection coefficient becomes big.
The method for making of metal film is vacuum evaporation, magnetron sputtering etc. normally, and film thickness is 50~90nm normally.
4. ligand molecular film: the film build method of the ligand molecular film in this research has: the direct absorption method of metal film, covalently bound method, LB become embrane method and molecule die technology.Directly the absorption method principle is all very simple with operation, and method itself just can be used as the detection technique of adsorbable molecule.During as the method for detector ligand molecule, the molecule that has can't form stable film on the metal surface, though molecule is easy to be adsorbed on the metal surface sometimes, but because active region spatial obstacle and orientation effect, or macromolecular mass gathers and make compatible reaction be difficult for taking place, and makes to use to be subjected to many restrictions.Covalently bound method normally covers one deck biotin or sephadex earlier on the surface of golden film, makes detector surface have homogeneity, and had specificity on function, has eliminated the non-specific affine problem of molecule.Glucose gel sensor surface film thickness is about 100~150nm.(3) LB becomes embrane method to be also referred to as the unimolecule composite membrane technology, can successively transfer to some organic compound of arranging in order on the liquid level on the fixed substrate, realizes that the high-sequential of on-chip specific molecular is arranged.Film thickness control can be as accurate as tens of dusts.(4) molecule die technology is to construct sensing surface on molecular level, and mating surface unimolecule self-organization layer technology (SAM), improves the stability and the sensitivity of the sensing surface of SPR.
5. response curve analytical approach: according to the exiting principle of SPR, analyzing incident angle-intensity of reflected light response curve has 4 kinds of methods: (1) angular modulation method: fixedly incident light wavelength, the change incident angle is observed catoptrical normalized intensity.(2) wavelength-modulated method: the fixing angle of incident light, change the incident light wavelength, observe catoptrical normalized intensity.(3) intensity modulated method: the fixedly angle of incident light and incident light wavelength rely on the measure of the change change of refractive of intensity.(4) phase modulation (PM) method: the fixedly angle of incident light and incident light wavelength, observation incident light and catoptrical differing.
The surface plasmon resonance detecting device is molecular biology and optics, galvanochemistry, the product that microelectronics etc. combine, has non-destructive, advantages such as high sensitivity and real-time online detection, can monitor intermolecular interaction in real time in position and need not any mark, thereby can be used for the research of the intermolecular interaction of multiple components, comprise chemistry, biological chemistry, biotechnology, molecular biology and microbiology detect, as environmental monitoring, blood analysis, antigen-antibody is analyzed, toxic gas detects, immunoassay, proteins interaction research, DNA duplicates and transcription analysis, drug screening, be with a wide range of applications in the association of continuous monitoring absorption and desorb and molecule and the many fields such as kinetic procedure research of dissociating.It can together use with polymerase chain reaction technology such as (PCR), is used to discern gene order, and can be used for analytical gas, fluid sample.
Optimize SPR biosensors structure, the sensitivity that improves SPR, the microminiaturization of realization detection robotization and measurement mechanism, low price etc. will be the emphasis of present research and development.
Summary of the invention
The objective of the invention is to propose a kind of highly sensitive surface plasma resonance detecting device with repeatedly total reflection, it is characterized in that: the cylindrical mirror 1 that adopts little radian is as refractive element, on the plane above the cylindrical mirror 1, make metallic film 4 as sensitive membrane, it can integrate or be deposited on the separable matrix with cylindrical mirror 1, the sensitive membrane top is a reaction tank, the below of cylindrical mirror 1 is an array photoelectric detector 5, the left side is an incident light source 2, light source 2, photoelectric detector 5, stepper motor 6 is connected with controller 7, and control step motor 6 drives light source 2, array photoelectric detector 5 moves around; With the variation of the light intensity that detects different wave length, diverse location incident light, controller 7 is connected with computing machine 8 interfaces.The length to height ratio of cylindrical mirror 1 is between 5 times~10 times, and 2~3 total reflections take place in device light.Be convenient and detect, add plating reflection horizon 3, make emergent light reflex to the same one side of incident light at the exit end of cylindrical mirror 1.
Described light source can be selected white light, ruddiness, green glow or blue light diode array for use, to adapt to the characteristic absorption wavelength of different measured matters.Control radiative beam size, can before array photoelectric detector 5, not add condenser, directly measure the spot size and the energy of irradiating light beam.
Described metallic film 4 is Au or Ag.
The invention has the beneficial effects as follows with traditional single-point total reflection and compare,
1. the light that sends of light source shines between post mirror and the metallic film by the post mirror, repeatedly inner full-reflection takes place, and by detection sample molecules absorption fixing on this film, the light intensity of refractive light paths and outgoing is changed simultaneously, thereby improved sensitivity and signal resolution that surface plasmon resonance detects.
2. emergent ray adopts array photoelectric detector 5 to detect, to distinguish the angle that surface plasmon resonance takes place.
3. control is automatically adopted in the position adjustment of light source and detecting device, and whole apparatus structure compactness is not subjected to the influence of bias light.
Description of drawings
Fig. 1 is repeatedly the structural representation of total reflection surface plasma resonance detecting device.
Fig. 2 is the top view and the hyperchannel sample cell synoptic diagram of post mirror.
Fig. 3 is the index path that adds behind the angle pencil of ray.
Embodiment
The present invention is a kind of highly sensitive surface plasma resonance detecting device with repeatedly total reflection.Its structure as shown in Figure 1, adopt the cylindrical mirror 1 of little radian to make Au or Ag metallic film 4 as refractive element, on the plane above the cylindrical mirror 1 as sensitive membrane, characteristic absorption wavelength for different measured matters can be made metallic film 4 (as shown in Figure 2) in the shunting, it is can be with cylindrical mirror 1 integrated or be deposited on the separable matrix, and the sensitive membrane top is a reaction tank.Two ends make metallic reflector 3 again, as thick aluminium or silver etc.The length to height ratio of cylindrical mirror 1 is between 5 times~10 times, and 2~3 total reflections take place in cylindrical mirror 1 light.The below that makes cylindrical mirror 1 is an array photoelectric detector 5, and the left side is an incident light source 2, and light source 2, photoelectric detector 5, stepper motor 6 are connected with controller 7; Adopt stepper motor 6 driving light sources 2 to move, thereby adjust the angle of incident light, with diverse location incident (as shown in Figure 3).And select white light, ruddiness, green glow or blue light diode array for use, to adapt to the characteristic absorption wavelength of different measured matters.Control radiative beam size, can before array photoelectric detector 5, not add condenser, directly measure the spot size and the energy of irradiating light beam, adopt stepper motor 6 driving array photoelectric detectors 5 to move, thereby follow the position of the angular setting array photoelectric detector 5 of emergent light.
Light source control, step motor control and array photoelectric detector detection signal are handled the back through microcontroller and are connected with computing machine by USB interface, and the receiving angle by computer software control light source (promptly selecting the light source of different emission wavelengths), incident angle of light, array photoelectric detector, realize detecting automatically, with the variation of the light intensity that detects different wave length, diverse location.The length to height ratio of refractive power device is between 5 times~10 times, and 2~3 total reflections take place in device light.Be convenient and detect, add plating reflection horizon 3, make emergent light reflex to the same one side of incident light at the exit end of refractive element.
The present invention can monitor intermolecular interaction in real time in position and need not any mark, thereby can be used for the polymolecular Study of Interaction, comprise chemistry, biological chemistry, biotechnology, molecular biology and microbiology detect, as in environmental monitoring, blood analysis, antigen-antibody analysis, toxic gas detection, immunoassay, proteins interaction research, DNA duplicate with the association of transcription analysis, drug screening, continuous monitoring absorption and desorb and molecule and many fields such as kinetic procedure research of dissociating in be with a wide range of applications.It can together use with polymerase chain reaction technology such as (PCR), is used to discern gene order, and can be used for analytical gas, fluid sample.
Claims (3)
1. highly sensitive surface plasma resonance detecting device with repeatedly total reflection, it is characterized in that, this detecting device comprises: the cylindrical mirror of little radian (1), on the plane above the cylindrical mirror (1) of little radian, make metallic film (4), the cylindrical mirror left side is incident light source (2), the cylindrical mirror two ends add plating reflection horizon (3), make emergent light reflex to the same one side of incident light; Cylindrical mirror right-hand member below is array photoelectric detector (5), light source (2), photoelectric detector (5), stepper motor (6) are connected with controller (7), control step motor (6) drives light source (2), array photoelectric detector (5) moves around, with the variation of the light intensity that detects different wave length, diverse location incident light, controller (7) is connected with the interface of computing machine (8); Wherein light source is white light, ruddiness, green glow or blue light diode array; The length to height ratio of cylindrical mirror is between 5~40 times.
2. according to the described highly sensitive surface plasma resonance detecting device with repeatedly total reflection of claim 1, it is characterized in that: described metallic film (4) is Au film or Ag film.
3. according to the described highly sensitive surface plasma resonance detecting device with repeatedly total reflection of claim 1, it is characterized in that: described exit end adds plating reflection horizon (3) and is thick Al film or thick Ag film.
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CN100343654C (en) * | 2004-11-30 | 2007-10-17 | 国家海洋环境监测中心 | Surface plasma resonance detection sample cell with repeatable response |
JP2007101241A (en) * | 2005-09-30 | 2007-04-19 | Fujifilm Corp | Sensing device |
CN102128809B (en) * | 2010-01-19 | 2013-01-02 | 北京金菩嘉医疗科技有限公司 | Surface plasma resonance sensor chip assembly and cylindrical prism chip |
CN103728271B (en) * | 2013-12-11 | 2017-01-04 | 王丽红 | Surface plasma resonance image-forming detection device and light source thereof |
CN106338470B (en) * | 2016-08-30 | 2019-01-01 | 上海理工大学 | A kind of light field travelling-wave cavity enhancing surface plasma resonance sensing equipment |
CN109030358B (en) * | 2018-07-31 | 2020-11-27 | 电子科技大学 | System and method for detecting weak infrared signal based on coaxial cavity microwave resonance principle |
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