CN108776126B - Surface plasma enhanced fluorescence sensor and refractive index change measuring method - Google Patents
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Abstract
The invention discloses a surface plasma enhanced fluorescence sensor and a measuring method thereof, wherein the surface plasma enhanced fluorescence sensor comprises a laser, a polaroid, a lens, a prism, a surface plasma enhanced fluorescence sensing chip, an adjustable voltage output device, a probe and a sample layer; the surface plasma enhanced fluorescence sensing chip sequentially comprises a glass substrate, a WCSPR device, a buffer layer and a sample pool; the WCSPR device comprises an upper metal layer, a refractive index adjusting medium layer and a lower metal layer; the sample cell is arranged on the lower surface of the buffer layer, and a gap is formed between the sample cell and the lower surface of the buffer layer; the refractive index of the sample layer in the sample cell was 1.33. The invention can realize the electric field distribution of the surface plasma wave in the buffer layer by adjusting the refractive index through the external field to change the refractive index of the dielectric layer or the combined refractive index, thereby realizing the detection of the electric field distribution in the dielectric layer and the adjustment of the local field enhancement coefficient.
Description
Technical Field
The invention relates to the technical field of sensors and sensors, in particular to a surface plasma enhanced fluorescence sensor and a refractive index change measuring method.
Background
When light is irradiated to a fluorescent compound molecule, the energy of the light causes some electrons around some nuclei in the molecule to transition from a ground state to a singlet state such as a first excited singlet state or a second excited singlet state. Since a singlet state such as the singlet state is unstable, the electron returns to the ground state and releases energy in the form of light, thereby generating fluorescence.
The existing literature reports that the intensity of surface-enhanced fluorescence is proportional to the ratio of the electric field intensity on the medium side to the electric field intensity on the metal film side at the metal film-medium interface, i.e., the field enhancement coefficient. From the analysis of the average field enhancement coefficient within the SPW penetration depth range, the long-range surface plasmon resonance mode excited by SPW coupling generated by the upper medium surface and the lower medium surface of the metal film at the same time is 3.625 times higher than that of the traditional SPR mode, and the corresponding SPEF signal peak intensity is 4.4 times higher.
The limitations of the above method are found in the following two aspects:
first, LRSPR devices are complex in structure and have a narrow range of choice of materials to process. The LRSPR phenomenon can be excited only under the conditions that the refractive indexes of media on two sides of the metal are similar and the thickness of the metal film is close to the skin depth, so that when an LRSPR device for exciting SPEF is prepared, the thickness of the metal film needs to be controlled, a proper buffer layer needs to be selected to realize refractive index matching with the medium on the other side of the metal film, and the difficulty in preparing the device and selecting the buffer layer material is increased.
Second, it is difficult for the LRSPR device to achieve adjustment of the local field enhancement factor. The thickness of the fluorescent compound molecular layer is usually much smaller than the propagation distance of the surface plasma wave, so that after the structural parameters of the metal film and the dielectric layer in the conventional LRSPR device are fixed, the electric field distribution and the local field enhancement coefficient near the metal film-dielectric layer are constants, and the electric field distribution and the local field enhancement coefficient are difficult to adjust according to the thicknesses of different fluorescent compound molecular layers to obtain the optimal SPEF signal amplification effect.
In summary, it is necessary to design a surface plasmon enhanced fluorescence sensor to compensate the above-mentioned defects.
Disclosure of Invention
The present invention proposes a surface plasmon enhanced fluorescence sensor which solves the above mentioned drawbacks of the prior art. Compared with the traditional surface plasma enhanced fluorescence sensor, the surface plasma enhanced fluorescence sensor can adjust and detect the electric field distribution in the dielectric layer and optimize the local field enhancement coefficient.
The technical scheme of the invention is realized as follows:
the invention provides a measuring method of a surface plasma enhanced fluorescence sensor, which is characterized by comprising the following steps:
(S01) preparing a WCSPR device: ultrasonically cleaning ZF3 glass substrate layer with ethanol-ether mixed solution at volume ratio of 1:4 for 2 hr, cleaning the surface, placing in electron beam evaporation apparatus, and vacuumizing to reduce air pressure to 10-6Millitorr; evaporating a metal layer on the gold material at an evaporation rate of 0.01nm per second, then rotationally coating the EO-FTC at a rotating speed of 3500 revolutions per minute, and taking 10% by mass of polycarbonate as a refractive index adjustable dielectric layer with the thickness of 3 microns; then putting the mixture into an electron beam evaporation instrument for vacuumizing so as to reduce the air pressure value to 10-6Millitorr, evaporating a metal layer on the gold material at an evaporation rate of 0.01nm per second; adopting polar plate polarization method, at 135 deg.C and 200VUnder the condition that the flow voltage is applied to the refractive index adjusting medium layer, the refractive index adjusting medium layer is polarized for 30 minutes to have the electro-optic effect; coating a Cytop material serving as a buffer layer in a rotating mode at the speed of 3500 rpm, wherein the thickness of the Cytop material is 200 nanometers; fixing a sodium yttrium fluoride single nanoparticle layer on the surface of the Cytop by adopting a physical adsorption method;
(S02) fixing the WCSPR device prepared in the step (S01) on a prism made of ZF3 glass, and filling the WCSPR device with a refractive index matching fluid with a refractive index of 1.711@814 nm;
(S03) the bottom of the sample cell is contacted with a collimator for collecting the fluorescence emitted by the sodium yttrium fluoride single nanoparticle layer, and all the devices are fixed on a rotary table;
(S04) transmitting the fluorescence signal collected in the step (S03) to a collimator through a single-mode optical fiber of a fluorescence detection device, focusing the fluorescence signal through an optical filter and a focusing lens, then entering a photomultiplier, and collecting the collected signal through a computer after being amplified by a preamplifier;
(S05) applying direct current voltage signals with different amplitudes on the refractive index adjusting medium layer, taking transverse magnetic polarized light with the wavelength of 814 nanometers as incident light, rotating the rotary table by taking 0.01 degree as step length and recording the intensity of reflected light of the probe, rotating the rotary table to the angle after calculating the incident angle corresponding to the WCSPR mode, and recording the SPEF signals recorded on the preamplifier, namely completing the measurement of fluorescence intensity.
Compared with the prior art, the invention has the following advantages:
the invention provides a novel surface plasma enhanced fluorescence sensor, which can realize the electric field distribution of surface plasma waves in a buffer layer by adjusting the refractive index through an external field to change the refractive index of a dielectric layer or a combined refractive index, thereby realizing the detection of the electric field distribution in the dielectric layer and the adjustment of a local field enhancement coefficient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a surface plasmon enhanced fluorescence sensor chip according to the present invention.
FIG. 2 is a graph showing the response curve of the resonance angle and intensity of the WCSPR structure of the present invention as a function of the refractive index of the sample layer.
FIG. 3a is a schematic diagram of the electric field intensity distribution of the LRSPR structure in the direction perpendicular to the interfaces of the layers under the resonance angle.
FIG. 3b is a schematic enlarged partial view of 3000-3300nm depth in FIG. 3 a.
FIG. 4 is a graph showing the variation of the intensity of the electric field in the distance layer with the distance, compared with the intensity of the electric field in the prism, with the average value of the distance.
FIG. 5 is a schematic structural diagram of a fluorescence detection device.
FIG. 6 is a schematic of a peak signal of the SPEF signal recorded on the preamplifier.
Wherein: 1-a laser; 2-a polarizing plate; 3-a lens; 4-a prism; 5-upper metal layer; 6-refractive index adjusting dielectric layer; 7-lower metal layer; 8-a sample cell; 9-a buffer layer; 10-an adjustable voltage output device; 11-a probe; 12-a sample layer; 13-a collimator; 14-a single mode optical fiber; 15-behind the collimator; 16-an optical filter; 17-a focusing lens; 19-a computer; 20-a photomultiplier tube; 21-preamplifier.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
To facilitate and clarify the description of the examples that follow, before explaining certain embodiments of the invention in detail, some terms are to be interpreted and the following interpretation applies to the specification and claims.
SP appearing in the present invention is an abbreviation of Surface plasma, wherein the text means Surface plasma; SPR, as presented herein, is an abbreviation for Surface Plasmon Resonance, where the text means Surface Plasmon Resonance; LRSPR presented in the present invention is an abbreviation for Long Range Surface Plasmon Resonance, where the text means Long-Range Surface Plasmon Resonance; LRSP appearing in the present invention is an abbreviation of Long range surface plasma, wherein the text means Long range surface plasma; SPW appearing in the present invention is an abbreviation of Surface plasmon wave, wherein the text means Surface plasmon wave; SPEF, as presented in the present invention, is an abbreviation for Surface plasma Enhanced Fluorescence, where the text means Surface Enhanced Fluorescence; WCSPR presented in the present invention is an abbreviation for Waveguide-coupled Surface Plasmon Resonance, where the text means Waveguide coupled Surface Plasmon Resonance; the Cytop appearing in the invention is a non-crystalline high-transparency fluorine-containing polymer, and the corresponding material name is perfluoro (1-butyl vinyl ether) polymer; the formula of EO-FTC presented in the present invention is 2- (3-cyano-4 ((E) -2- (5- (4- (diethylamino) styryl) -3, 4-dihexyloxythiophen-2-) ethylene) -5, 5-dimethylfuran-2 (5H) -ylidene) malononitrile. Other English words appearing in the invention are codes and do not represent any other meanings.
Referring to fig. 1-6, the invention provides a surface plasma enhanced fluorescence sensor which takes glass as a substrate and adopts an electro-optic effect material as a refractive index adjusting medium layer, and comprises a laser, a polaroid, a lens, a prism, a surface plasma enhanced fluorescence sensing chip, an adjustable voltage output device, a probe and a sample layer; the surface plasma enhanced fluorescence sensing chip sequentially comprises a glass substrate, a WCSPR device, a buffer layer and a sample pool; the WCSPR device comprises an upper metal layer, a refractive index adjusting medium layer and a lower metal layer; the sample cell is arranged on the lower surface of the buffer layer, and a gap is formed between the sample cell and the lower surface of the buffer layer; the refractive index of the sample layer in the sample cell is 1.33; the surface plasma enhanced fluorescence sensing chip is fixed on the prism through the refractive index matching fluid; of course, the surface plasmon enhanced fluorescence sensing chip can also be prepared on a glass substrate; the refractive index adjusting medium layer is made of a high polymer material with an electro-optic effect; the surface plasma enhanced fluorescence sensor is provided with a rotary table, and the prism and the surface plasma enhanced fluorescence sensing chip are fixed on the rotary table.
The prism is prepared from ZF3 glass material; the buffer layer is a distance buffer layer which is a Cytop fluoride macromolecule layer and is formed by spin coating.
The invention also provides a measuring method of the surface plasma enhanced fluorescence sensor, which comprises the following steps: (S01) preparing a WCSPR device; (S02) fixing the WCSPR device prepared in the step (S01) on a prism made of ZF3 glass, and filling the WCSPR device with a refractive index matching fluid with a refractive index of 1.711@814 nm; (S03) the bottom of the sample cell is contacted with a collimator for collecting the fluorescence emitted by the single nano particle layer of sodium yttrium fluoride, and all the devices are fixed on the rotary table 18; (S04) transmitting the fluorescence signal collected in the step (S03) to a collimator through a single-mode optical fiber of a fluorescence detection device, focusing the fluorescence signal through an optical filter and a focusing lens, then entering a photomultiplier, and collecting the collected signal through a computer after being amplified by a preamplifier; (S05) applying direct current voltage signals with different amplitudes on the refractive index adjusting medium layer, taking transverse magnetic polarized light with the wavelength of 814 nanometers as incident light, rotating the rotary table by taking 0.01 degree as step length and recording the intensity of reflected light of the probe, rotating the rotary table to the angle after calculating the incident angle corresponding to the WCSPR mode, and recording the SPEF signals recorded on the preamplifier, namely completing the measurement of fluorescence intensity.
Wherein the step (S01) includes the steps of: (S11) ultrasonically cleaning the ZF3 glass substrate layer for 2 hours by using ethanol-ether mixed solution with the volume ratio of 1:4, cleaning the surface of the substrate layer, putting the substrate layer into an electron beam evaporation instrument, and vacuumizing to reduce the air pressure value to 10-6Millitorr; (S12) evaporating a metal layer on the gold material at an evaporation rate of 0.01nm per second, then rotationally coating the EO-FTC at a rotating speed of 3500 rpm, and taking 10% by mass of polycarbonate as a refractive index adjustable dielectric layer with the thickness of 3 microns; then putting the mixture into an electron beam evaporation instrument for vacuumizing so as to reduce the air pressure value to 10-6Millitorr, evaporating a metal layer on the gold material at an evaporation rate of 0.01nm per second; (S13) applying 200V DC voltage at 135 deg.C by polar plate polarization methodUnder the condition of the refractive index adjusting medium layer, polarizing for 30 minutes to enable the refractive index adjusting medium layer to have an electro-optic effect; coating a Cytop material serving as a buffer layer in a rotating mode at the speed of 3500 rpm, wherein the thickness of the Cytop material is 200 nanometers; (S14) fixing a sodium yttrium fluoride single nanoparticle layer on the surface of the Cytop by adopting a physical adsorption method.
The fluorescence detection device comprises a collimator, a single-mode fiber, a collimator rear part, an optical filter, a focusing lens, a computer, a photomultiplier, a preamplifier and a glass substrate.
The rotation of the turntable, the reflected light intensity acquisition of the probe and the control of the voltage output of the adjustable voltage output device are all completed by a computer.
When the WCSPR reflected light power is calculated, the wavelength is 814nm, and the refractive index of the prism is 1.711@814 nm; the thicknesses of the upper metal layer and the lower metal layer are both 30nm, and the refractive indexes are 0.185+5.11i @814 nm; the thickness of a refractive index variable medium layer made of a high polymer material with an electro-optic effect is 3 mu m, the refractive index is 1.63@814nm, and the electro-optic coefficient is 100 pm/V; the thickness of the buffer layer is 200nm, the refractive index is 1.38@814nm, and the surface fluorescent compound is a sodium yttrium fluoride single nanoparticle layer; the refractive index of the sample layer in the sample cell was 1.33.
The expression of the WCSPR reflected light power is 1-1 by Fresnel's theorem.
Wherein r isi,i+1Is the reflectivity at the interface of adjacent layers, diIs the thickness of each layer, k0xIs the wave vector component of the parallel interface in the prism, λ is the wavelength of the incident light, θ is the angle of incidence, k0z,iIs the wave vector component, n, of the perpendicular interface of each layeriIs the refractive index of each layer; when the subscript i is 0 to 5, the subscript i respectively represents the prism, the upper metal layer, the refractive index adjusting medium layer, the lower metal layer, the buffer layer and the sample layer in the sample cell in sequence; FIG. 2 shows the response curve of the resonance angle and intensity of the WCSPR structure of the present embodiment as a function of the refractive index of the sample layer.
When the incident angle is the resonance angle theta of the LRSPR structure0Then, the upper metal layer,The distribution expression of the electromagnetic field in the refractive index adjusting dielectric layer, the lower metal layer, the buffer layer and the sample layer in the sample cell is as shown in formula (1-2).
Wherein EiIs the electric field vector of each layer, HiIs the vector of magnetic field in each layer, xy plane is parallel to the interface of each layer, z direction is perpendicular to the interface of each layer, subscripts of x, i, y, i and z of the vector of electric field and magnetic field respectively represent the components of the vector of electric field and magnetic field in different layers in the x, y and z directions, rot represents the rotation degree of calculation vector, div represents the divergence degree of calculation vector, c is the propagation speed of light in vacuum,iis the dielectric constant, k, of each layer0xIs the wave vector component of the parallel interface in the prism, λ is the wavelength of the incident light, k0z,iIs the wave vector component, n, of the perpendicular interface of each layeriIs the refractive index of each layer. And when the subscript i is 0 to 5, the subscript i respectively represents the prism, the upper metal layer, the refractive index adjusting medium layer, the lower metal layer, the buffer layer and the sample layer in the sample cell in sequence.
At the boundary of adjacent layers, the above-mentioned electric and magnetic field components follow the theorem of continuity, expressed as (1-3).
The electric field intensity distribution of the LRSPR structure of the embodiment obtained by substituting the formula (1-3) for the formula (1-2) in the direction perpendicular to the interface of each layer at the resonance angle is shown in FIG. 3a, wherein the position with the depth of 0 is the interface between the prism or the glass substrate and the upper metal layer, different numbers are the same as those in the structure shown in FIG. 1, and FIG. 3b is a partial enlargement with the depth of 3000-3300nm in FIG. 3 a.
Based on the electro-optical effect, the refractive index of the refractive index adjusting medium layer is linearly adjusted by changing the output voltage of the adjustable voltage output device. The electro-optic effect is a nonlinear optical effect, and an optical material with the electro-optic effect can change the refractive index n of the optical material by delta n through applying an electric field, as shown in formulas (1-4)Where d is the thickness of the material, V is the voltage applied to the material, γ33Is the electro-optic coefficient.
When the refractive index of the refractive index adjusting medium layer changes, the electric field intensity distribution of the WCSPR structure in the direction perpendicular to the interface of each layer at the resonance angle changes as shown in the formulas (1-2) and (1-3), wherein the average value of the electric field intensity in the distance layer at different voltages compared with the electric field intensity enhancement coefficient in the prism along with the distance is shown in fig. 4.
The preparation of the surface plasma enhanced fluorescence sensor structure using glass as a substrate and an electro-optic effect material as a refractive index adjusting medium layer and the SPEF signal detection method are as follows.
In this embodiment, the WCSPR device is prepared as follows:
ultrasonically cleaning ZF3 glass substrate layer with ethanol-ether mixed solution at volume ratio of 1:4 for 2 hr, cleaning the surface, placing in electron beam evaporation apparatus, and vacuumizing to reduce air pressure to 10-6Millitorr; evaporating a metal layer on a gold material at an evaporation rate of 0.01nm per second, and then rotationally coating polycarbonate with the mass fraction of 10% of EO-FTC (2- (3-cyano-4 ((E) -2- (5- (4- (diethylamino) styryl) -3, 4-dihexylthiophene-2-) ethylene) -5, 5-dimethylfuran-2 (5H) -subunit) malononitrile) as a refractive index adjustable medium layer at a rotating speed of 3500 revolutions per minute, wherein the thickness of the polycarbonate is 3 micrometers; then putting the alloy material into an electron beam evaporation instrument for vacuumizing to reduce the air pressure value to 10-6 mTorr, and evaporating a metal layer on the gold material at the evaporation rate of 0.01nm per second; adopting a polar plate polarization method, under the condition that a direct current voltage of 200V is applied to the refractive index adjusting dielectric layer at the temperature of 135 ℃, polarizing for 30 minutes to enable the refractive index adjusting dielectric layer to have an electro-optic effect; coating a Cytop material serving as a buffer layer in a rotating mode at the speed of 3500 rpm, wherein the thickness of the Cytop material is 200 nanometers; and fixing a sodium yttrium fluoride single nanoparticle layer on the surface of the cell by adopting a physical adsorption method.
As shown in FIG. 5, the fluorescence detection device is fixed on a prism made of ZF3 glass, and the prism is filled with a refractive index matching fluid with the refractive index of 1.711@814 nm. The bottom of the sample cell 8 is in contact with a collimator 13 for collecting the fluorescence emitted by the single nanoparticle layer of sodium yttrium fluoride, all of which are fixed on a turntable 18. The collected fluorescence signal is transmitted to a collimator 15 through a single-mode fiber 14, then is focused through an optical filter 16 and a focusing lens 17 and then enters a photomultiplier 20, and the collected signal is amplified through a preamplifier 21 and then is collected through a computer 19. The rotation of the rotary table 18, the reflected light intensity acquisition of the probe 11 and the control of the voltage output of the adjustable voltage output device 10 are completed by a computer 19.
Direct current voltage signals with different amplitudes are applied to the refractive index adjusting medium layer, transverse magnetic polarized light with the wavelength of 814 nanometers is used as incident light, the rotary table 18 is rotated by taking 0.01 degree as a step length, the intensity of reflected light of the probe 11 is recorded, the rotary table 18 is rotated to the angle after the incident angle corresponding to the WCSPR mode is calculated, the SPEF signals recorded on the preamplifier 21 are recorded, and the peak value signals are shown in FIG. 6.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (1)
1. A measuring method of a surface plasma enhanced fluorescence sensor is characterized by comprising the following steps:
(S01) preparing a WCSPR device: ultrasonically cleaning ZF3 glass substrate layer with ethanol-ether mixed solution at volume ratio of 1:4 for 2 hr, cleaning the surface, placing in electron beam evaporation apparatus, and vacuumizing to reduce air pressure to 10-6Millitorr; evaporating a metal layer on the gold material at an evaporation rate of 0.01nm per second, then rotationally coating the EO-FTC at a rotating speed of 3500 revolutions per minute, and taking 10% by mass of polycarbonate as a refractive index adjustable dielectric layer with the thickness of 3 microns; then putting the mixture into an electron beam evaporation instrument for vacuumizing so as to reduce the air pressure value to 10-6Millitorr, evaporating a metal layer on the gold material at an evaporation rate of 0.01nm per second; by adopting a polar plate polarization method, under the condition that the direct current voltage of 200V is applied to the refractive index adjusting medium layer at the temperature of 135 ℃,polarizing for 30 minutes to enable the refractive index adjusting medium layer to have an electro-optic effect; coating a Cytop material serving as a buffer layer in a rotating mode at the speed of 3500 rpm, wherein the thickness of the Cytop material is 200 nanometers; fixing a sodium yttrium fluoride single nanoparticle layer on the surface of the Cytop by adopting a physical adsorption method;
(S02) fixing the WCSPR device prepared in the step (S01) on a prism made of ZF3 glass, and filling the WCSPR device with a refractive index matching fluid with a refractive index of 1.711@814 nm;
(S03) the bottom of the sample cell is contacted with a collimator for collecting the fluorescence emitted by the sodium yttrium fluoride single nanoparticle layer, and all the devices are fixed on a rotary table;
(S04) transmitting the fluorescence signal collected in the step (S03) to a collimator through a single-mode optical fiber of a fluorescence detection device, focusing the fluorescence signal through an optical filter and a focusing lens, then entering a photomultiplier, and collecting the collected signal through a computer after being amplified by a preamplifier;
(S05) applying direct current voltage signals with different amplitudes on the refractive index adjusting medium layer, taking transverse magnetic polarized light with the wavelength of 814 nanometers as incident light, rotating the rotary table by taking 0.01 degree as step length and recording the intensity of reflected light of the probe, rotating the rotary table to the angle after calculating the incident angle corresponding to the WCSPR mode, and recording the SPEF signals recorded on the preamplifier, namely completing the measurement of fluorescence intensity.
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| CN109827932A (en) * | 2019-01-31 | 2019-05-31 | 马佑桥 | One kind being based on Wave-guide coupling surface plasma resonance sensing chip |
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| WO2009030071A1 (en) * | 2007-09-06 | 2009-03-12 | National Center For Nanoscience And Technology, China | Wave-guide coupling spr sensor chip and sensor chip array thereof |
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| CN101349645A (en) * | 2007-07-20 | 2009-01-21 | 株式会社日立高新技术 | Nucleic acid analysis device and nucleic acid analyzer using the same |
| WO2009030071A1 (en) * | 2007-09-06 | 2009-03-12 | National Center For Nanoscience And Technology, China | Wave-guide coupling spr sensor chip and sensor chip array thereof |
| WO2011066667A1 (en) * | 2009-12-01 | 2011-06-09 | 国家纳米科学中心 | Surface plasmon resonance(spr) sensor based on measurement of resonance angle and measurement method thereof |
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