CN103454253B - Based on the organic phosphorus detection method of surface plasma body resonant vibration - Google Patents

Abstract

The invention relates to the technical field of analytical chemistry, and discloses an organic phosphorus detection method based on surface plasmon resonance. In the present invention, by combining the organophosphorus sample liquid on the immunosensing chip bonded with OPs-Ab, the OPs-Ag in the organophosphorus sample liquid combines with the OPs-Ab to form a sample film on the immunosensing chip; Visible light or infrared light is shot through the sample film at a preset incident angle on the immunosensing chip, and the light reflected by the photonic crystal structure on the immunosensing chip is detected to obtain the measured SPR spectrum; the measured SPR spectrum is compared with the reference Compare the SPR spectra, and determine whether the organic phosphorus sample solution contains organic phosphorus according to whether the absorption peak is shifted; determine the concentration of the organic phosphorus according to the shift of the absorption peak. In the detection of organic phosphorus based on SPR technology, no prism is used for spectroscopic analysis, and no flow cell is used for sample injection, thereby reducing the volume of the instrument and realizing the miniaturization of the instrument.

Description

Organophosphorus detection method based on surface plasmon resonance

technical field

The invention relates to the field of biochemical detection, in particular to an organic phosphorus detection method based on surface plasmon resonance.

Background technique

Biochemical testing plays an important role in many fields that are directly related to human survival and development, such as medical treatment, environmental protection, biology, and food testing. In recent years, with the continuous increase of detection requirements for trace and micro samples, and the continuous expansion of measured objects such as special substances, components, and states, traditional detection technologies have been unable to meet the needs.

Surface plasmon resonance (Surface Plasmon Resonance, referred to as "SPR") is a physical optical phenomenon, which is generated by the interaction of incident light waves and free electrons on the metal surface. When the incident light irradiates from an optically denser medium to an optically rarer medium, total reflection occurs when the incident angle is greater than the critical angle. If there is a thin metal film of tens of nanometers between the two medium interfaces, the P polarization component (P wave) of the evanescent wave generated during total reflection will enter the gold film, interact with the free electrons in the gold film, and excite Surface plasmon wave (SurfacePlasmonWave, SPW) propagating on the surface of gold film. When the angle or wavelength of the incident angle is a certain value, the evanescent wave and the surface plasmon will undergo energy coupling and resonate, and the energy of the incident light will be transferred to the surface plasmon, resulting in a sharp drop in the energy of the reflected light. In the reflection spectrum A resonant absorption peak is generated on the surface, and the angle or wavelength of the incident light at this time is called the SPR resonant angle or resonant wavelength. The resonance angle or resonance wavelength is closely related to the refractive index of the medium on the surface of the gold film, and the change in the refractive index is proportional to the molecular mass bound to the gold surface. Based on this, the analytes bound to the surface of the gold film can be detected and analyzed.

As shown in Figure 1, antibody 103 (biological recognition molecule) is immobilized on the surface of sensor 101 gold film 102, then free antigen 104 is slowly passed through the flow cell, incident light 106 passes through prism 105 and sensor 101 and hits gold film 102 surface, detect the reflected light 107 to obtain the SPR spectrum. The evanescent wave 110 when total internal reflection occurs at the interface of the substrate 111 and the free electrons on the metal surface oscillate to form electron density waves (i.e. surface plasmon waves 109) on the surface of the metal close to the sample 108. When the evanescent wave 110 When the frequency and wavenumber of the surface plasmon wave 109 are equal, surface plasmon resonance occurs to enhance the optical signal. When the antigen-antibody is specifically combined, the resonance angle of SPR will change accordingly, and the resonance angle will also change accordingly with the change of reaction time, until the antigen-antibody reaction reaches equilibrium, and the resonance angle will no longer change. Therefore, the antigen can be detected with high sensitivity through the change of the resonant peak of the SPR reflection spectrum. In the actual detection, pass the liquid to be tested through the flow cell, and analyze whether the resonant peak in the SPR spectrum shifts before and after the specific binding of the antigen and antibody, so as to determine whether the liquid to be tested contains The antigen, and according to the degree of offset, the concentration of the antigen that can specifically bind to the antibody in the test solution can be determined. However, in order to obtain high-precision and automatic detection capabilities, the current instruments that use SPR technology for biomolecular detection and analysis have complex optical systems and automatic sampling systems, and have the disadvantages of large volume and high cost, which limits their popularization and operation. . However, some small instruments, such as biosensors and microfluidic system SensiQ, are usually semi-automatic and cannot realize automatic detection, and the sensor of this instrument is inconvenient to disassemble, and the flow cell is easy to leak when it is not fixed tightly.

Contents of the invention

The object of the present invention is to provide a method for detecting organic phosphorus based on surface plasmon resonance, so that in the detection of organic phosphorus based on SPR technology, no prism is used for spectroscopic analysis, and no flow cell is used for sample injection, thereby reducing the volume of the instrument and realizing the miniaturization of the instrument change.

In order to solve the above technical problems, an embodiment of the present invention provides a method for detecting organic phosphorus based on surface plasmon resonance, comprising the following steps:

S1. Provide an immune sensor chip; wherein, the immune sensor chip comprises a photonic crystal structure substrate, a metal film covering the substrate, and an organophosphorus antibody OPs-Ab bonded to the metal film;

S2. Binding the organophosphorus sample solution to the immunosensing chip, the organophosphorus antigen OPs-Ag in the organophosphorus sample solution combines with the OPs-Ab to form a sample film on the immunosensing chip ;

S3. Visible light or infrared light is shot through the sample film at a preset incident angle on the immune sensor chip, and the light reflected by the photonic crystal structure on the immune sensor chip is detected to obtain the measured SPR Atlas;

S4. Comparing the measured SPR spectrum with the reference SPR spectrum, determining whether the organophosphorus sample solution contains organic phosphorus according to whether the absorption peak shifts; determining the concentration of the organophosphorus according to the shift of the absorption peak.

Compared with the prior art, the embodiment of the present invention combines the organophosphorus sample liquid on the immunosensing chip bonded with the organophosphorus antibody OPs-Ab, the organophosphorus antigen OPs-Ag and OPs in the organophosphorus sample liquid -Ab combined to form a sample film on the immunosensing chip; visible light or infrared light is transmitted through the sample film at a preset incident angle and hits the immunosensing chip, and the detection is reflected back through the photonic crystal structure on the immunosensing chip Obtain the measured SPR spectrum; compare the measured SPR spectrum with the reference SPR spectrum, and determine whether the organic phosphorus sample solution contains organic phosphorus according to whether the absorption peak shifts; determine the concentration of the organic phosphorus according to the shift of the absorption peak . In the detection of organic phosphorus based on SPR technology, no prism is used for spectroscopic analysis, and no flow cell is used for sample injection, thereby reducing the volume of the instrument and realizing the miniaturization of the instrument.

In addition, the photonic crystal structure substrate is prepared through the following steps:

provide a base;

Holes arranged in a periodic lattice structure are punched on the surface of the substrate.

The photonic crystal structure substrate with holes arranged in a periodic lattice structure can enhance the signal intensity of reflected light, thereby improving the detection sensitivity and reducing the detection limit.

In addition, the arrangement shape of the periodic lattice structure is square or hexagonal. The more regular the arrangement shape of the periodic lattice structure of the holes, the stronger the resonance peak and the narrower the half-wave width, thereby further enhancing the signal strength.

In addition, the lattice constant of the photonic crystal structure is 0.8 to 1.2 microns, and the duty ratio is 1:4. The SPR resonance can be excited to the greatest extent, which is further conducive to the enhancement of signal strength.

In addition, the organophosphorus antibody is bound to the metal film through the following steps:

modifying the amine group-NH ₂ on the metal film;

The organophosphorus antibody OPs-Ab is modified on the amine group.

The organophosphorus antibody bonded on the immunosensing chip determines the type of organophosphorus that can be detected. According to the type of organophosphate detected as required, the organophosphate antibody can be freely selected, so that the organophosphorus detection method of the present invention can be used in a wide range of applications. , which is conducive to popularization and application.

In addition, in the step S2, the following sub-steps are included:

Dropping the organophosphorus sample liquid onto the immunosensing chip and placing it at 37°C for 30 minutes;

Rinse the immune sensor chip with PBS;

Air-dry or blow-dry at room temperature to form a sample film on the immunosensing chip.

The organophosphorus sample liquid is dropped onto the immunosensing chip, which is easy to operate and does not need to use a complicated sampling system, further reducing the volume of the instrument and realizing the miniaturization of the instrument.

In addition, in the step S2, the following sub-steps are included:

Place the immune sensor chip in the organophosphate sample solution at 37°C and soak for 30 minutes;

Rinse the immune sensor chip with PBS;

Air-dry or blow-dry at room temperature to form a sample film on the immunosensing chip.

Soaking the immunosensing chip in the organic phosphorus sample solution can fully combine the antigen and antibody, so that the detection result is more accurate.

In addition, after the step S4, the following steps are also included:

Place the immunosensing chip with the sample film formed in 0.25 mg/ml fluorescein isothiocyanate FITC organophosphorus antibody OPs-Ab solution at 37° C. for 30 minutes.

By binding FITC-OPs-Ab on the surface and measuring the SPR spectrum, it can be verified that both the antibody and the antigen are bound to the substrate, further ensuring the accuracy of the organophosphate detection results.

In addition, after the step S4, the following steps are also included:

Using 5 mmol/L hydrochloric acid HCl solution to wash the immune sensor chip to remove the sample film formed on the immune sensor chip;

Before rebinding the sample membrane, the SPR profile was determined.

Washing off the sample film on the immunosensing chip can regenerate the chip, reuse it, and save costs; and, by measuring the SPR spectrum, it can be ensured that it is cleaned.

Description of drawings

1 is a schematic diagram of the detection principle of organic phosphorus based on surface plasmon resonance according to the prior art;

Fig. 2 is the flowchart of the organic phosphorus detection method based on surface plasmon resonance according to a preferred embodiment of the present invention;

3A to 3C are structural schematic diagrams of the immune sensor chip;

Fig. 4 is a schematic diagram of the SPR spectrum of the immunosensing chip where only OPs-Ab is bonded, OPs-Ag is combined, and OPs-Ab is combined.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, various embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, those of ordinary skill in the art can understand that, in each implementation manner of the present invention, many technical details are provided for readers to better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation modes, the technical solution claimed in each claim of the present application can be realized.

A preferred embodiment of the present invention relates to a method for detecting organic phosphorus based on surface plasmon resonance, the specific process is shown in Figure 2, including the following steps:

Step 201, providing an immune sensor chip; wherein, the immune sensor chip includes a photonic crystal structure substrate, a metal film covering the substrate, and an organophosphorus antibody OPs-Ab bonded to the metal film;

The photonic crystal structure substrate can be prepared through the following steps: providing a substrate, and drilling holes arranged in a periodic lattice structure on the surface of the substrate. The photonic crystal structure substrate with holes arranged in a periodic lattice structure can enhance the signal intensity of reflected light, thereby improving the detection sensitivity and reducing the detection limit.

3A is a cross-sectional view of a photonic crystal structure substrate, and FIGS. 3B and 3C are top views of a photonic crystal structure substrate. In the figure, 301 is a substrate, 302 is a metal layer, and 303 is a hole in a periodic lattice structure. The photolithography process in the semiconductor process can be used to drill holes on the substrate. The pattern on the photolithography mask has a periodic lattice structure. After photolithography, the substrate has holes with a periodic lattice structure. The period of the lattice structure is controllable, for example, the period T of the pores of the periodic lattice structure is 0.8 to 1.2 microns, and the duty ratio is 1:4.

It is worth noting that the periodic structure causes a change in the periodic dielectric constant. For different periods T, the resulting periodic potential field will also be different, resulting in a corresponding change in the position of the resonance peak. The circular hole is measured by simulation and experiment Arranged in a square arrangement, it is obtained that the ratio of the resonance peak position to the lattice constant (that is, the period of the hole in the periodic lattice structure) is 1:1, and the hexagonal arrangement is basically close to

Also, the duty ratio is the ratio of the hole diameter to the hole pitch, and as the duty ratio becomes larger, the intensity of the resonance peak becomes larger. According to the principle of optics, light propagation is divided into two propagation modes: transverse electric wave (TE) and transverse magnetic wave (TM). When the duty ratio is small, the energy band gap can be created and the surface plasmon (SPR) resonance can be formed mainly by TM propagation mode, the influence of TE propagation mode can be basically ignored at this time, and when the duty cycle becomes larger, the TE propagation mode will gradually form a forbidden band. When the duty cycle reaches a certain value, the two forbidden bands completely overlap. At this time Both TE and TM propagation modes can create energy bandgap and excite SPR resonance, so that the highest intensity SPR resonance peak can be obtained.

The structure perforated on the substrate constitutes a photonic crystal structure, and a photonic crystal refers to an artificial periodic dielectric structure with a Photonic Band-Gap (PBG) characteristic, and is sometimes called a PBG photonic crystal structure. Photonic crystals have the function of wavelength selection, which can selectively allow light of a certain wavelength to pass through and prevent light of other wavelengths from passing through it. In the photonic crystal, the periodic change of the refractive index produces the optical bandgap structure, so the optical bandgap structure controls the movement of light in the photonic crystal. In this embodiment, a material with a low refractive index (such as holes in a periodic lattice structure) periodically appears in certain positions of a high refractive index material (such as a metal layer). In the present invention, since the photonic crystal structure is also covered with a metal layer, when light is irradiated on the metal layer, reflection will occur, and because the surface is not smooth, the existence of periodic holes makes some The wavelength of light cannot be reflected back, and it plays the role of light splitting, forming a super prism with extremely high wavelength resolution and extremely small volume, which can omit the prism in the spectrum acquisition system and use no prism for light splitting, thereby reducing the volume of the instrument and realizing the miniaturization of the instrument . Here, it is worth explaining that the depth of the hole should not be too deep, otherwise the light will not be reflected after it enters; the depth of the hole should not be too shallow, otherwise the purpose of light splitting will not be achieved. In addition, as the depth of the hole increases, the signal enhancement effect is more obvious, but after reaching about 12 microns, the signal enhancement effect will be weakened. Therefore, the depth of the hole in the periodic lattice structure is more suitable between 1 and 15 microns. It is further conducive to the enhancement of signal strength.

It is worth mentioning that the arrangement shape of the periodic lattice structure in Figure 3B is a square, and can also be arranged in a hexagonal shape, as shown in Figure 3C, however, the present invention is not limited thereto, any periodic lattice structure The arrangement shapes of the structures are all within the protection scope of the present invention. The more regular the arrangement shape of the periodic lattice structure, the stronger the resonance peak and the narrower the half-wave width. In actual experiments, it can be seen that the hexagonal arrangement has a stronger resonance peak and a narrower half-wave width than the square arrangement. This is caused by the different degrees of periodicity of the two arrangements. The hexagonal arrangement has better periodicity than the square arrangement, and its periods in six directions are equal, while the square arrangement is only in two directions. The period is equal, which makes the light wave less affected by the direction of transmission during the transmission process of the hexagonal substrate, so it can excite stronger and narrower resonance peaks.

The organophosphorus antibody (OPs-Ab) is bonded on the metal film through the following steps: first modify the amine group (-NH ₂ ) on the metal film, and then modify the OPs-Ab on the amine group. Specifically, the substrate covered with the metal film was soaked in 20 mmol/L mercaptoethylamine solution at 37° C. for 2 hours, and then washed with phosphate buffered saline (PBS). Next, the substrate modified with amine groups was placed in the OPs-Ab solution added with 0.2 mmol/L carbodiimide EDC and 0.5 mmol/L NHS aqueous solution of N-hydroxysuccinimide at 4 °C. 12 hours, then washed with phosphate buffered saline (PBS).

The organophosphorus antibody bonded on the immunosensing chip determines the type of organophosphorus that can be detected. According to the type of organophosphate detected as required, the organophosphate antibody can be freely selected, so that the organophosphorus detection method of the present invention can be used in a wide range of applications. , which is conducive to popularization and application. You can choose a general-purpose antibody that binds to a variety of organophosphate antigens. For example, there is an organophosphate monoclonal antibody on the market that can detect five drugs: methyl parathion, fenitrothion, fenthion, chlorthion, pectin Phosphophosphorus; single antibodies that bind only one organophosphate antigen can also be selected.

Step 202, binding the organophosphorus sample liquid to the immunosensing chip, and the organophosphorus antigen OPs-Ag in the organophosphorus sample liquid combines with OPs-Ab to form a sample film on the immunosensing chip.

Since the sample film formed on the immunosensing chip is solid, the organic phosphorus detection instrument does not need to use a flow cell, which can further reduce the volume of the instrument and realize the miniaturization of the instrument; moreover, compared with injecting the organic phosphorus solution in the flow cell , The solid sample film has less interference to the optical signal than the liquid, because the liquid absorbs certain wavebands, so that the detection result is more accurate.

In actual implementation, the sample film can be formed on the immunosensing chip by the following methods:

Drop the organophosphorus sample solution onto the immunosensing chip and place it at 37°C for 30 minutes;

Rinse the immune sensor chip with PBS;

Air-dry or blow-dry at room temperature to form a sample film on the immunosensing chip.

This method is easy to operate, does not need to use a complicated sample introduction system, can further reduce the volume of the instrument, and realize the miniaturization of the instrument.

The sample film can also be formed on the immunosensor chip by the following methods:

Place the immune sensor chip in the organophosphate sample solution at 37°C and soak for 30 minutes;

Rinse the immune sensor chip with PBS;

Air-dry or blow-dry at room temperature to form a sample film on the immunosensing chip.

In this method, the immunosensing chip is immersed in the organic phosphorus sample solution, which can fully combine the antigen and the antibody, so that the detection result is more accurate.

In step 203, visible light or infrared light is projected on the immune sensor chip through the sample film at a preset incident angle, and the light reflected by the photonic crystal structure on the immune sensor chip is detected to obtain a measured SPR spectrum. Specifically, the visible light or infrared light emitted by the light source passes through the incident optical fiber at a preset incident angle and hits the immune sensor chip through the sample film; the incident light is reflected by the photonic crystal structure on the immune sensor chip, and enters through the outgoing optical fiber. The spectrometer is used to obtain the measured SPR spectrum.

Step 204, compare the measured SPR spectrum with the reference SPR spectrum, determine whether the organic phosphorus sample solution contains organic phosphorus according to whether the absorption peak shifts; determine the concentration of the organic phosphorus according to the shift of the absorption peak.

Among them, the reference SPR spectrum is obtained through the following steps: the visible light or infrared light emitted by the light source hits the immune sensor chip at a preset incident angle through the incident optical fiber; the incident light is reflected by the photonic crystal structure on the immune sensor chip, and then emitted The optical fiber enters the spectrometer to obtain the reference SPR spectrum. The reference SPR spectrum can be determined during chip preparation or before each detection. In addition, the SPR spectrum can be measured and compared before and after the OPs-Ab is bonded to ensure that the OPs-Ab is bonded to the immune sensor chip, thereby ensuring the correctness of the subsequent organophosphate detection results.

After step S4, the following steps are also included:

Place the immunosensor chip with the sample film formed in 0.25 mg/ml fluorescein isothiocyanate FITC organophosphorus antibody OPs-Ab solution at 37°C, soak for 30 minutes, then rinse with PBS, dry or blow dry , Determination of SPR spectrum. By binding FITC-OPs-Ab on the surface and measuring the SPR spectrum, it can be verified that both the antibody and the antigen are bound to the substrate, further ensuring the accuracy of the organophosphate detection results. In other words, the combination of fluorescently labeled antibodies can prove that both the antibody and the antigen are bound to the substrate in the previous operation. It can be seen from the SPR spectrum obtained by scanning that if there is a resonance peak shift, it may not necessarily prove that the antibody and antigen are bound to the substrate. , or it may be other substances. By combining fluorescently labeled antibodies and then measuring the SPR spectrum, if the resonance peak shifts, it means that the substrate must be bound to the antibody and antigen, which can ensure the accuracy of the organophosphate detection results.

As shown in Figure 4, the immunosensing chip is only bonded with OPs-Ab (401 in the figure), combined with OPs-Ag (402 in the figure), and combined with a schematic diagram of OPs-Ab (403 in the figure). In the experiment, the incident angle was selected to be 30°, the spectral range was 400-1000nm, and the absorption peak was around 945nm. It can be seen from the figure that the absorption peak combined with OPs-Ag is shifted compared with the absorption peak combined with OPs-Ab only, indicating that the organophosphorus sample solution contains OPs-Ag that can be detected by OPs-Ab, If the OPs-Ab bonded on the immunosensing chip is an organophosphorus monoclonal antibody, the antibody can detect five drugs: methyl parathion, fenitrothion, fenthion, chlorthion, skin fly Phosphorus, then the organophosphorus sample solution contains one or several of these five drugs. By measuring the displacement of the absorption peak of the 402 curve compared with the absorption peak of the 401 curve, the concentration of the organic phosphorus in the organic phosphorus sample solution can be determined. It is worth noting that the linear relationship between the concentration of OPs-Ag and the displacement of the absorption peak needs to be established in advance, that is to say, the organophosphorus standard solution of known concentration is prepared respectively, and the organophosphorus standard solution is combined on the immunosensing chip, respectively. Measure the SPR spectrum corresponding to the organophosphorus standard solution with different concentrations, record the displacement of the absorption peak, and establish the corresponding relationship between the concentration of the organophosphorus and the displacement of the absorption peak.

In addition, it is worth mentioning that washing off the sample membrane on the immunosensing chip can regenerate the chip, reuse it, and save costs; and, by measuring the SPR spectrum, it can be ensured that it is cleaned. Specifically, the immunosensing chip was washed with 5 mmol/L hydrochloric acid HCl solution to remove the sample film formed on the immunosensing chip; before the sample film was recombined, the SPR spectrum was measured.

The division of steps in the above methods is only for the sake of clarity of description. During implementation, they can be combined into one step or some steps can be split and decomposed into multiple steps. As long as they contain the same logical relationship, they are all within the scope of protection of this patent. ; Adding insignificant modifications or introducing insignificant designs to the algorithm or process, but not changing the core design of the algorithm and process are all within the scope of protection of this patent.

Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes can be made to it in form and details without departing from the spirit and spirit of the present invention. scope.

Claims (12)

1. based on an organic phosphorus detection method for surface plasma body resonant vibration, it is characterized in that, comprise following steps:

S1., one immune sensing chip is provided; Wherein, described immune sensing chip comprises photon crystal structure substrate, covers described suprabasil metal film and is bonded in the organophosphorus antibody OPs-Ab on described metal film;

S2. organophosphorus sample liquid be attached on described immune sensing chip, the organophosphorus antigen OPs-Ag in described organophosphorus sample liquid is combined with described OPs-Ab, and described immune sensing chip forms sample film;

S3. visible or infrared light is beaten on described immune sensing chip with the incident angle preset through described sample film through incident optical, incident light is by the photon crystal structure reflection on described immune sensing chip, enter spectrometer through outgoing optical fiber, obtain and measure SPR collection of illustrative plates;

S4. described measurement SPR collection of illustrative plates and benchmark SPR collection of illustrative plates are compared, whether have skew according to absorption peak, whether determine in described organophosphorus sample liquid containing 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, described photon crystal structure substrate is prepared by following steps:

One substrate is provided;

Described substrate surface is beaten the hole of periodic lattice structural arrangement.

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:

Described metal film is modified amido-NH ₂;

Described amido is modified organophosphorus antibody OPs-Ab.

6. the organic phosphorus detection method based on surface plasma body resonant vibration according to claim 5, is characterized in that, described metal film is modified in the step of amido, comprises following sub-step:

The substrate covering metal film is placed in 20 mM/ls of mercaptoethylmaine solution of 37 DEG C 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, described amido is modified in the step of organophosphorus antibody, comprises following sub-step:

Described substrate of modifying amido is placed on 4 DEG C add in the OPs-Ab solution of the N-hydroxy-succinamide NHS aqueous solution of 0.2 mM/l carbodiimide EDC and 0.5 mM/l to 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:

By organophosphorus sample drop on described immune sensing chip, and place 30 minutes at 37 DEG C;

Described immune sensing chip is rinsed with PBS;

Room temperature is dried or is dried up, and 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 DEG C and soaks 30 minutes;

Described immune sensing chip is rinsed with PBS;

Room temperature is dried or is dried up, and 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, described benchmark SPR collection of illustrative plates is obtained by following steps:

The visible or infrared light that light source is launched is beaten through incident optical on described immune sensing chip with the incident angle preset;

Incident light, by the photon crystal structure reflection on described immune sensing chip, enters spectrometer through outgoing optical fiber, obtains benchmark SPR collection of illustrative plates.

11. organic phosphorus detection methods based on surface plasma body resonant vibration according to claim 1, is characterized in that, after described step S4, also comprise following steps:

The immune sensing chip defining sample film is placed in the 0.25 mg/ml fluorescein isothiocynate FITC organophosphorus antibody OPs-Ab solution of 37 DEG C, soaks 30 minutes, then rinse with phosphate buffer PBS, after drying or drying up, measure SPR collection of illustrative plates.

12. organic phosphorus detection methods based on surface plasma body resonant vibration according to claim 1, is characterized in that, 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 that described immune sensing chip is formed;

Before recombining sample film, measure SPR collection of illustrative plates.