CN103454253A - Organic phosphorus detection method based on surface plasmon resonance - Google Patents
Organic phosphorus detection method based on surface plasmon resonance Download PDFInfo
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- CN103454253A CN103454253A CN201310255808XA CN201310255808A CN103454253A CN 103454253 A CN103454253 A CN 103454253A CN 201310255808X A CN201310255808X A CN 201310255808XA CN 201310255808 A CN201310255808 A CN 201310255808A CN 103454253 A CN103454253 A CN 103454253A
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Abstract
The invention relates to the technical field of analytical chemistry and discloses an organic phosphorus detection method based on surface plasmon resonance (SPR). According to the organic phosphorus detection method, an organic phosphorus sample solution is combined with an immune sensing chip bonded with an OPs-Ab; OPs-Ag in the organic phosphorus sample solution is combined with the OPs-Ab to form a sample membrane on the immune sensing chip; visible light or infrared light penetrates through the sample membrane by a pre-set incidence angle to be transmitted to the immune sensing chip; light reflected from a photonic crystal structure on the immune sensing chip is detected to obtain a measured SPR map; the measured SPR map is compared with a standard SPR map to determine whether the organic phosphorus sample solution contains organic phosphorus or not according to whether an absorption peak is skewed or not; the concentration of the organic phosphorus is determined according to the displacement amount of the absorption peak. According to the organic phosphorus detection method, a prism does no need to be used for carrying out light splitting in organic phosphorus detection based on an SPR technology and a flowing cell does not need to be used for feeding a sample, so that the volume of an instrument is reduced and the miniaturization of the instrument is realized.
Description
Technical field
The present invention relates to field of biochemistry detection, particularly the organic phosphorus detection method based on surface plasma body resonant vibration.
Background technology
Biological chemistry detects at the numerous areas of the direct relation mankind survival and developments such as medical treatment, environmental protection, biology, food inspection important role all.In recent years, along with the continuous proposition of the detection demand to trace, micro-example, to the continuous expansion of the measurands such as particular matter, composition, state, traditional detection technique can't satisfy the demands.
Surface plasma body resonant vibration (Surface Plasmon Resonance, be called for short " SPR ") is a kind of physical optics phenomenon, by the free electron of incident light wave and metal surface, is interacted and is produced.When incident ray shines optically thinner medium from optically denser medium, when incident angle is greater than critical angle, total reflection phenomenon can occur.If there is the metallic film of tens nanometers between the two media interface, the P polarized component (P ripple) of the disappearance ripple produced during total reflection can enter golden film, with the free electron in golden film, interact, inspire the surface plasma wave (Surface Plasmon Wave, SPW) of propagating along golden film surface.When the angle of incident angle or wavelength are a certain particular value, Energy Coupling will occur and produce resonance in disappearance ripple and surface plasma, the energy of incident light will be transferred on surface plasma, cause energy of reflection light sharply to descend, produce resonance absorbing peak on reflectance spectrum, now the angle of incident light or wavelength are called SPR resonance angle or resonant wavelength.The refractive index of resonance angle or resonant wavelength and golden film surface dielectric is closely related, and the molecular mass that the variation of refractive index is combined with gold surface is directly proportional, and accordingly, can carry out determination and analysis to the determinand of golden film surface conjunction.
As shown in Figure 1, sessile antibody 103 (biological identification molecule) on sensor 101 gold medal film 102 surfaces, then make free antigen 1 04 slowly by flow cell, and incident light 106 sees through prism 105 and sensor 101 is got to golden film 102 surfaces, detection of reflected light 107, obtain the SPR collection of illustrative plates.The disappearance ripple 110 of light when substrate 111 interface experiences total internal reflection and the free electron of metal surface, form electronics dilatational wave (being surface plasma wave 109) on the surface of metal approach sample 108 in vibration, when the frequency of disappearance ripple 110 and surface plasma wave 109 equates with wave number, surface plasma body resonant vibration occurs, and light signal is enhanced.When antigen-antibody generation specific binding, the resonance angle of SPR will change thereupon, and along with the change resonance angle in reaction time also corresponding change can occur, until antigen-antibody reaction reaches balance, resonance angle can not change yet again, therefore the variation of the resonance peak by the SPR reflectance spectrum can be carried out highly sensitive detection to antigen.In reality detects, liquid to be measured is passed through to flow cell, analysis before antigen-antibody generation specific binding and in SPR collection of illustrative plates afterwards resonance peak whether be offset, can determine in liquid to be measured whether contain can with the antigen of antibody generation specific binding, and according to degrees of offset, can determine in liquid to be measured can with the concentration of the antigen of antibody generation specific binding.But the instrument that adopts at present the SPR technology to carry out biomolecule detection and analysis is acquisition high precision and robotization detectability, its optical system, automatic sample handling system are comparatively complicated, and have the shortcoming that volume is large, cost is high, have limited their popularization operation.And some miniature instruments are as biology sensor and microfluidic systems SensiQ, normally automanual, can't realize automatic detection, and the sensor of this instrument dismounting is inconvenient, fixing easy leakage when tight of flow cell.
Summary of the invention
The object of the present invention is to provide a kind of organic phosphorus detection method based on surface plasma body resonant vibration, make in the organophosphorus based on the SPR technology detects and carry out light splitting without prism, do not adopt flow cell to carry out sample introduction, thereby the reduction printer body is long-pending, realizes instrument miniaturization.
For solving the problems of the technologies described above, embodiments of the present invention provide a kind of organic phosphorus detection method based on surface plasma body resonant vibration, comprise following steps:
S1., one immune sensing chip is provided; Wherein, described immune sensing chip comprises the photon crystal structure substrate, covers described suprabasil metal film and is bonded in the organophosphorus antibody OPs-Ab on described metal film;
S2. the organophosphorus sample liquid is attached on described immune sensing chip, the organophosphorus antigen OPs-Ag in described organophosphorus sample liquid is combined with described OPs-Ab, on described immune sensing chip, forms sample film;
S3. visible or infrared light is seen through to described sample film with the incident angle of presetting and beat on described immune sensing chip, detect the light reflected by the photon crystal structure on described immune sensing chip, obtain and measure the SPR collection of illustrative plates;
S4. described measurement SPR collection of illustrative plates and benchmark SPR collection of illustrative plates are compared, according to absorption peak, whether skew is arranged, determine in described organophosphorus sample liquid whether contain organophosphorus; According to the displacement of absorption peak, determine the concentration of organophosphorus.
Embodiment of the present invention in terms of existing technologies, by on the organophosphorus sample liquid has been attached to the bonding immune sensing chip of organophosphorus antibody OPs-Ab, organophosphorus antigen OPs-Ag in the organophosphorus sample liquid is combined with OPs-Ab, on the immune sensing chip, forms sample film; Visible or infrared light is seen through to sample film with the incident angle of presetting and beat on the immune sensing chip, detect the light reflected by the photon crystal structure on the immune sensing chip, obtain and measure the SPR collection of illustrative plates; To measure the SPR collection of illustrative plates and benchmark SPR collection of illustrative plates compares, according to absorption peak, whether skew be arranged, and determine in the organophosphorus sample liquid and whether contain organophosphorus; According to the displacement of absorption peak, determine the concentration of organophosphorus.Make in the organophosphorus based on the SPR technology detects and carry out light splitting without prism, do not adopt flow cell to carry out sample introduction, thereby the reduction printer body is long-pending, realizes instrument miniaturization.
In addition, prepared by following steps by described photon crystal structure substrate:
One substrate is provided;
Beat the hole of periodic lattice structural arrangement on described substrate surface.
Photon crystal structure substrate with hole of periodic lattice structural arrangement can strengthen catoptrical signal intensity, thereby has improved the sensitivity detected, and has reduced detection limit.
In addition, the spread geometry of described periodic lattice structure is square or hexagon.The spread geometry of the periodic lattice structure in hole is more regular, and resonance peak is stronger, and half-wave is wide narrower, thereby further makes signal intensity be enhanced.
In addition, the grating constant of described photon crystal structure is 0.8 to 1.2 micron, and dutycycle is 1: 4.Can at utmost excite SPR resonance, further be conducive to the enhancing of signal intensity.
In addition, described organophosphorus antibody is bonded on described metal film by following steps:
Modify amido-NH on described metal film
2;
Modify organophosphorus antibody OPs-Ab on described amido.
On the immune sensing chip, the organophosphorus antibody of bonding has determined the detected organophosphorus kind of energy, detected organophosphorus kind as required, can freely select organophosphorus antibody, thereby make the usable range of organic phosphorus detection method of the present invention extensive, be conducive to apply.
In addition, in described step S2, comprise following sub-step:
The organophosphorus sample drop, to described immune sensing chip, and is placed 30 minutes under 37 ℃;
Rinse described immune sensing chip with PBS;
Room temperature is dried or is dried up, and on described immune sensing chip, forms sample film.
By the organophosphorus sample drop, to the immune sensing chip, easy operating, need not adopt complicated sampling system, and further the reduction printer body is long-pending, realizes instrument miniaturization.
In addition, in described step S2, comprise following sub-step:
Described immune sensing chip is placed in the organophosphorus sample liquid of 37 ℃ and soaks 30 minutes;
Rinse described immune sensing chip with PBS;
Room temperature is dried or is dried up, and on described immune sensing chip, forms sample film.
The immune sensing chip is placed in the organophosphorus sample liquid and soaks, can make the abundant combination of antigen and antibody, thereby make testing result more accurate.
In addition, after described step S4, also comprise following steps:
The immune sensing chip that has formed sample film is placed in the 0.25 mg/ml fluorescein isothiocynate FITC organophosphorus antibody OPs-Ab solution of 37 ℃, soaks 30 minutes.
By at surface conjunction FITC-OPs-Ab, and measure the SPR collection of illustrative plates, can check antibody and antigen all to be attached in substrate, further guarantee the accuracy of organophosphorus testing result.
In addition, after described step S4, also comprise following steps:
Adopt the hydrochloric acid HCl solution of 5 mM/ls to clean described immune sensing chip, remove the sample film formed on described immune sensing chip;
Before again in conjunction with sample film, measure the SPR collection of illustrative plates.
Wash away the sample film on the immune sensing chip, can make chip regeneration, recycling, cost-saving; And, by measuring the SPR collection of illustrative plates, can guarantee to clean up.
The accompanying drawing explanation
Fig. 1 detects principle schematic according to the organophosphorus based on surface plasma body resonant vibration of prior art;
Fig. 2 is the process flow diagram of the organic phosphorus detection method based on surface plasma body resonant vibration of one better embodiment according to the present invention;
Fig. 3 A to Fig. 3 C is the structural representation of immune sensing chip;
Fig. 4 OPs-Ab that has been immune sensing chip on bonding, combine OPs-Ag, combines again the SPR collection of illustrative plates schematic diagram of OPs-Ab.
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.Yet, persons of ordinary skill in the art may appreciate that in each embodiment of the present invention, in order to make the reader understand the application better, many ins and outs have been proposed.But, even without these ins and outs and the many variations based on following each embodiment and modification, also can realize each claim of the application technical scheme required for protection.
A better embodiment of the present invention relates to a kind of organic phosphorus detection method based on surface plasma body resonant vibration, and idiographic flow as shown in Figure 2, comprises following steps:
Can prepare by following steps by the photon crystal structure substrate: a substrate is provided, and beats the hole of periodic lattice structural arrangement on substrate surface.Photon crystal structure substrate with hole of periodic lattice structural arrangement can strengthen catoptrical signal intensity, thereby has improved the sensitivity detected, and has reduced detection limit.
Be the cut-open view of photon crystal structure substrate as shown in Figure 3A, as Fig. 3 B and 3C are the vertical views of photon crystal structure substrate, in figure, 301 is substrates, the 302nd, and metal level, the 303rd, the hole of periodic lattice structure.Can adopt the photoetching process in semiconductor technology to punch in substrate, the pattern on mask has the periodic lattice structure, and after photoetching, substrate has just had the hole of periodic lattice structure.The cycle of lattice structure is controlled, such as, the cycle T in the hole of periodic lattice structure is 0.8 to 1.2 micron, dutycycle is 1: 4.
What deserves to be explained is, periodic structure causes the periodically variation of specific inductive capacity, for different cycle T, the Periodic Potential caused also can be different, thereby cause resonant positions that corresponding the variation occurs, measure circular hole by simulation and experiment and be arranged as square arrangement, show that basic and ratio grating constant (being the cycle in the hole of periodic lattice structure) of its resonant positions is 1: 1, hexagonal array substantially close to
In addition, dutycycle is the ratio of Circularhole diameter and circular hole spacing, and along with dutycycle becomes large, it is large that the intensity of resonance peak becomes.From optical principle, light is propagated and is divided into transverse electric wave (TE) and two kinds of communication modes of transverse magnetic wave (TM), and in dutycycle hour, can manufacture and can be with forbidden band, what form surface plasma (SPR) resonance is mainly the TM communication mode, now TE communication mode impact can be ignored substantially, become large and work as dutycycle, the TE communication mode also forms forbidden band gradually, when dutycycle reaches a certain value, both overlap in forbidden band fully, and now TE and TM communication mode can be manufactured and can be with forbidden band, excite SPR resonance, thereby can obtain the SPR resonance peak of maximum intensity.
The structure of punching in substrate has formed photon crystal structure, and photonic crystal refers to the artificial periodic dielectric structure of (Photonic Band-Gap, referred to as the PBG) characteristic that has photon band gap, sometimes also referred to as the PBG photon crystal structure.Photonic crystal has the function that wavelength is selected, and can make selectively the light of certain wave band pass through wherein by the light that stops other wavelength.The cyclical variation that in photonic crystal is refractive index has produced the optical band gap structure, thereby the motion of light in photonic crystal by the optical band gap structure control.Present embodiment is the material in the appearance low-refraction of some positional cycle of high-index material (as metal level) (as the hole of periodic lattice structure).In the present invention, owing to also having covered metal level on this photon crystal structure, when having illumination to be mapped on metal level, can reflect, and, because surface is not level and smooth, the periodically existence in hole, can not reflect the light of some wavelength, play the effect of light splitting, form that wavelength resolution is high and super prism that volume is minimum can omit the prism in the spectra collection system, without prism, carry out light splitting, thereby the reduction printer body is long-pending, realizes instrument miniaturization.At this, what deserves to be explained is, the degree of depth in hole should not be excessively dark, crosses and deeply can cause after light incident enters, can't reflecting; The degree of depth in hole should not be excessively shallow, crosses shallow meeting and do not reach the purpose of light splitting.In addition, along with the increase of the degree of depth in hole, it is more obvious that signal strengthens effect, but, after arriving 12 microns left and right, signal strengthens effect and can weaken, therefore, the degree of depth in the hole of periodic lattice structure is comparatively suitable between 1 to 15 micron, further is conducive to the enhancing of signal intensity.
It is worth mentioning that, in Fig. 3 B, the spread geometry of periodic lattice structure is square, can also line up hexagon, and as shown in Figure 3 C, still, the present invention is not as limit, and the spread geometry of any period lattice structure is all within protection scope of the present invention.The spread geometry of periodic lattice structure is more regular, and resonance peak is stronger, and half-wave is wide narrower.In actual experiment, can find out hexagonal array than square arrangement have stronger resonance peak and narrower half-wave wide.This is to arrange by two kinds the different cycles degree had to cause, hexagonal array has better periodicity than square arrangement, its cycle on six direction all equates, and positive square arrangement only equates in the cycle of both direction, this makes light wave impact that is subject to transmission direction still less in hexagonal array substrate transmitting procedure, so it can excite stronger and narrower resonance peak.
Organophosphorus antibody (OPs-Ab) is bonded on metal film by following steps: first on metal film, modify amido (NH
2), then on amido, modify OPs-Ab.Specifically, the substrate that first will cover metal film is placed in 20 mM/ls of mercaptoethylmaine solution of 37 ℃ soaks 2 hours, then uses phosphate buffer (PBS) to wash.Then, by soaking 12 hours in the OPs-Ab solution of the N-hydroxy-succinamide NHS aqueous solution of the substrate of having modified amido be placed on 4 ℃ the add carbodiimide EDC of 0.2 mM/l and 0.5 mM/l, then use phosphate buffer (PBS) to wash.
On the immune sensing chip, the organophosphorus antibody of bonding has determined the detected organophosphorus kind of energy, detected organophosphorus kind as required, can freely select organophosphorus antibody, thereby make the usable range of organic phosphorus detection method of the present invention extensive, be conducive to apply.Can select the universal antibody of being combined with multiple organophosphorus antigen, such as, there is on the market a kind of organophosphorus monoclonal antibody can detect five kinds of medicines: parathion-methyl, fenifrothion, Entex, chlorothion, Nankor; Also can select the monospecific antibody of only being combined with a kind of organophosphorus antigen.
Be solid-state owing on the immune sensing chip, forming sample film, thereby make need not adopt flow cell in the organophosphorus detecting instrument, further the reduction printer body is long-pending, realizes instrument miniaturization; And, compared to inject organic phosphorus solution in flow cell, solid-state sample film is less to the interference of light signal than liquid, because liquid has absorption to some wave band, thereby makes testing result more accurate.
In reality realizes, can form by the following method sample film on the immune sensing chip:
The organophosphorus sample drop, to the immune sensing chip, and is placed 30 minutes under 37 ℃;
Rinse the immune sensing chip with PBS;
Room temperature is dried or is dried up, and on the immune sensing chip, forms sample film.
This method easy operating, need not adopt complicated sampling system, and further the reduction printer body is long-pending, realizes instrument miniaturization.
Also can form by the following method sample film on the immune sensing chip:
The immune sensing chip is placed in the organophosphorus sample liquid of 37 ℃ and soaks 30 minutes;
Rinse the immune sensing chip with PBS;
Room temperature is dried or is dried up, and on the immune sensing chip, forms sample film.
This method is placed on the immune sensing chip in the organophosphorus sample liquid and soaks, and can make the abundant combination of antigen and antibody, thereby make testing result more accurate.
Wherein, benchmark SPR collection of illustrative plates obtains by following steps: the visible or infrared light of light source emission is beaten on the immune sensing chip with the incident angle of presetting through incident optical; Incident light, by the reflection of the photon crystal structure on the immune sensing chip, enters spectrometer through outgoing optical fiber, obtains benchmark SPR collection of illustrative plates.Benchmark SPR collection of illustrative plates can be measured when prepared by chip, also can before each the detection, measure.In addition, can also be before bonding OPs-Ab and afterwards, measure the SPR collection of illustrative plates, compare, guarantee at immune sensing chip bonding OPs-Ab, thereby guarantee the correctness of the testing result of follow-up organophosphorus.
After step S4, also comprise following steps:
The immune sensing chip that has formed sample film is placed in the 0.25 mg/ml fluorescein isothiocynate FITC organophosphorus antibody OPs-Ab solution of 37 ℃, soaks 30 minutes, then rinse with PBS, after drying or drying up, measure the SPR collection of illustrative plates.By at surface conjunction FITC-OPs-Ab, and measure the SPR collection of illustrative plates, can check antibody and antigen all to be attached in substrate, further guarantee the accuracy of organophosphorus testing result.That is to say, the antibody of combined with fluorescent mark can prove in front operation that antibody and antigen all have been attached in substrate, the SPR collection of illustrative plates obtained by scanning can be found out, if the resonance peak drift is arranged, differing proves surely in substrate and combines antibody and antigen, it may be also other material, antibody by the combined with fluorescent mark, measure again the SPR collection of illustrative plates, if the resonance peak drift has occurred, illustrate in substrate and necessarily combine antibody and antigen, can guarantee like this accuracy of organophosphorus testing result.
The OPs-Ab that is immune sensing chip on bonding as shown in Figure 4 (in figure 401), combine OPs-Ag (in figure 402), combines again the schematic diagram of OPs-Ab (in figure 403), in experiment, select 30 ° of incident angles, spectral range 400-1000nm, absorption peak 945nm left and right.As seen from the figure, the absorption peak that combines OPs-Ag compared to a bonding absorption peak of OPs-Ab skew has occurred, illustrate in the organophosphorus sample liquid and contain the detected OPs-Ag of OPs-Ab energy, if on the immune sensing chip, the OPs-Ab of bonding is a kind of organophosphorus monoclonal antibody, this antibody can detect five kinds of medicines: parathion-methyl, fenifrothion, Entex, chlorothion, Nankor, contain one or more in these five kinds of medicines so in the organophosphorus sample liquid.Absorption peak by measuring 402 curves compared to the displacement of absorption peak of 401 curves, can determine the concentration of organophosphorus in the organophosphorus sample liquid.What deserves to be explained is, need to set up in advance the linear relationship of OPs-Ag concentration and absorption peak displacement, that is to say, prepare respectively the organophosphorus standard solution of concentration known, the organophosphorus standard solution is attached on the immune sensing chip, measure respectively the SPR collection of illustrative plates corresponding to organophosphorus standard solution of variable concentrations, record the absorption peak displacement, set up the corresponding relation of organophosphorus concentration and absorption peak displacement.
In addition, it is worth mentioning that, wash away the sample film on the immune sensing chip, can make chip regeneration, recycling, cost-saving; And, by measuring the SPR collection of illustrative plates, can guarantee to clean up.Specifically, adopt the hydrochloric acid HCl solution of 5 mM/ls to clean the immune sensing chip, remove the sample film formed on the immune sensing chip; Before again in conjunction with sample film, measure the SPR collection of illustrative plates.
The step of top the whole bag of tricks is divided, and just in order being described clearly, can to merge into a step while realizing or some step is split, and is decomposed into a plurality of steps, as long as comprise identical logical relation, all in the protection domain of this patent; To adding inessential modification in algorithm or in flow process or introducing inessential design, but the core design that does not change its algorithm and flow process is all in the protection domain of this patent.
Persons of ordinary skill in the art may appreciate that the respective embodiments described above are to realize specific embodiments of the invention, and in actual applications, can do various changes to it in the form and details, and without departing from the spirit and scope of the present invention.
Claims (13)
1. the organic phosphorus detection method based on surface plasma body resonant vibration, is characterized in that, comprises following steps:
S1., one immune sensing chip is provided; Wherein, described immune sensing chip comprises the photon crystal structure substrate, covers described suprabasil metal film and is bonded in the organophosphorus antibody OPs-Ab on described metal film;
S2. the organophosphorus sample liquid is attached on described immune sensing chip, the organophosphorus antigen OPs-Ag in described organophosphorus sample liquid is combined with described OPs-Ab, on described immune sensing chip, forms sample film;
S3. visible or infrared light is seen through to described sample film with the incident angle of presetting and beat on described immune sensing chip, detect the light reflected by the photon crystal structure on described immune sensing chip, obtain and measure the SPR collection of illustrative plates;
S4. described measurement SPR collection of illustrative plates and benchmark SPR collection of illustrative plates are compared, according to absorption peak, whether skew is arranged, determine in described organophosphorus sample liquid whether contain organophosphorus; According to the displacement of absorption peak, determine the concentration of organophosphorus.
2. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, by following steps, prepared by described photon crystal structure substrate:
One substrate is provided;
Beat the hole of periodic lattice structural arrangement on described substrate surface.
3. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 2, is characterized in that, the spread geometry of described periodic lattice structure is square or hexagon.
4. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, the grating constant of described photon crystal structure is 0.8 to 1.2 micron, and dutycycle is 1: 4.
5. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, described organophosphorus antibody is bonded on described metal film by following steps:
Modify amido-NH on described metal film
2;
Modify organophosphorus antibody OPs-Ab on described amido.
6. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 5, is characterized in that, on described metal film, modifies in the step of amido, comprises following sub-step:
The substrate that has covered metal film is placed in 20 mM/ls of mercaptoethylmaine solution of 37 ℃ and soaks 2 hours.
7. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 5, is characterized in that, on described amido, modifies in the step of organophosphorus antibody, comprises following sub-step:
To in the OPs-Ab solution of the N-hydroxy-succinamide NHS aqueous solution of described substrate of having modified amido be placed on 4 ℃ the add carbodiimide EDC of 0.2 mM/l and 0.5 mM/l, soak 12 hours.
8. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, in described step S2, comprises following sub-step:
The organophosphorus sample drop, to described immune sensing chip, and is placed 30 minutes under 37 ℃;
Rinse described immune sensing chip with PBS;
Room temperature is dried or is dried up, and on described immune sensing chip, forms sample film.
9. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, in described step S2, comprises following sub-step:
Described immune sensing chip is placed in the organophosphorus sample liquid of 37 ℃ and soaks 30 minutes;
Rinse described immune sensing chip with PBS;
Room temperature is dried or is dried up, and on described immune sensing chip, forms sample film.
10. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, in described step S3, comprises following sub-step:
The visible or infrared light of light source emission sees through described sample film through incident optical with the incident angle of presetting and beats on described immune sensing chip;
Incident light, by the reflection of the photon crystal structure on described immune sensing chip, enters spectrometer through outgoing optical fiber, obtains measuring the SPR collection of illustrative plates.
11. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, described benchmark SPR collection of illustrative plates obtains by following steps:
The visible or infrared light of light source emission is beaten with the incident angle of presetting on described immune sensing chip through incident optical;
Incident light, by the reflection of the photon crystal structure on described immune sensing chip, enters spectrometer through outgoing optical fiber, obtains benchmark SPR collection of illustrative plates.
12. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, after described step S4, also comprises following steps:
The immune sensing chip that has formed sample film is placed in the 0.25 mg/ml fluorescein isothiocynate FITC organophosphorus antibody OPs-Ab solution of 37 ℃, soaks 30 minutes.
13. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 1, is characterized in that, after described step S4, also comprises following steps:
Adopt the hydrochloric acid HCl solution of 5 mM/ls to clean described immune sensing chip, remove the sample film formed on described immune sensing chip;
Before again in conjunction with sample film, measure the SPR collection of illustrative plates.
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| CN109813685A (en) * | 2019-02-21 | 2019-05-28 | 中国科学技术大学 | A kind of single bacteria surface plasma mechanical oscillation imaging method measuring waste water acute toxicity |
| CN113466183A (en) * | 2020-03-31 | 2021-10-01 | 横河电机株式会社 | Spectral analysis apparatus, and operation method and program for spectral analysis apparatus |
| CN114562963A (en) * | 2022-03-02 | 2022-05-31 | 哈尔滨理工大学 | Surface topography measuring system for imaging nano material |
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| CN109813685B (en) * | 2019-02-21 | 2020-05-19 | 中国科学技术大学 | Single bacterium surface plasma mechanical vibration imaging method for determining acute toxicity of wastewater |
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| CN114562963A (en) * | 2022-03-02 | 2022-05-31 | 哈尔滨理工大学 | Surface topography measuring system for imaging nano material |
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