CN104237169A - Detection method of SPR detection system based on external field modulation - Google Patents

Abstract

The invention provides a detection method of an SPR detection system based on external field modulation, including: 1) Obtaining the SPR angle scanning curves of the first sample solution and the second sample solution, calculating the sensitivity of the SPR detection system under 0 external field, and calibrating the detection Dynamic range; 2) Select an angle within the dynamic range as the incident angle to scan the external field, and obtain the linear coefficient of the reflected light intensity changing with the external field intensity; 3) Introduce the solution to be tested, and select the same incident angle as step 2). Angle, adjust the applied external field intensity, so that the detected reflected light intensity is always kept within the dynamic range, and read the external field intensity and reflected light intensity; 4) Based on the obtained linear coefficient, the measured reflected light intensity It is converted into the equivalent reflected light intensity under 0 external field, and then based on the sensitivity of the SPR detection system, the refractive index or concentration of the solution to be tested is calculated by using the equivalent reflected light intensity. The invention can significantly expand the dynamic range of the tunable surface plasmon resonance sensor.

Description

A detection method of SPR detection system based on external field modulation

technical field

The invention relates to the field of sensors and sensing technology, in particular, the invention relates to a detection method based on a tunable surface plasmon resonance sensor.

Background technique

Surface plasmon resonance sensors are an optical sensing technology that can detect changes in the refractive index in regions near metal interfaces. Since the 1980s, SPR biosensors have been rapidly developed and widely used due to their outstanding label-free, fast and sensitive and real-time detection advantages. At present, it has been applied in the fields of protein interaction, nucleic acid interaction, food safety, pesticide residues, environmental safety, drug screening, rapid and sensitive detection of infectious diseases, etc.

The tunable SPR biosensor is based on the surface plasmon resonance excited by the combination of prism and waveguide. For the specific principle, please refer to the literature "J.Homola, Surface plasmonresonance sensors for detection of chemical and biological species, Chem. Rev., (2008 ) 108:462-493”. Since the tunable SPR biosensor has more stringent requirements on the coupling conditions, the tunable SPR reflectance curve (the curve of the surface reflectivity of the SPR chip changing with the incident angle) is sharper, so it has higher sensitivity. At present, the detection methods based on tunable SPR biosensors mainly include angle scanning method and intensity detection method. Among them, the angle scanning method needs to change the optical path through the mechanical structure, which is difficult to operate and is easy to introduce errors artificially. The intensity detection method is to fix the angle of the incident light and measure the intensity of the reflected light reflected by the surface of the SPR chip. Since the intensity of the incident light is known, the reflectivity of the surface of the SPR chip can be calculated, and then the refractive index of the substance to be detected can be obtained. characteristic. This method can avoid changing the optical path through the mechanical structure, and the operation is simple and the error is relatively small. Intensity detection generally selects a certain intensity of incident light, fixes the incident angle of the incident light, and then measures the intensity of the reflected light after passing the substance to be detected on the surface of the SPR chip, and calculates the refractive index of the substance to be detected according to the intensity of the reflected light. other properties. The principle is as follows: the change of the refractive index of the detection surface (referring to the surface of the SPR chip combined with the substance in the solution to be tested) will cause the shift of the position of the SPR reflectance curve. Figure 1(a) shows the reflectance curve under an intensity detection method As an example of movement, since the reflectance corresponds to the detected reflected light intensity in actual detection, replacing the reflectance with reflected light intensity in the figure will not change the shape of the reflectance curve. Referring to Figure 1(a), the solid line in the figure is the reflectance curve of the original SPR chip surface, and the dotted line is the reflectance curve of the SPR chip surface combined with the solution to be tested. It can be seen that after the surface of the SPR chip is passed into the solution to be tested, the refractive index of the surface of the SPR chip increases, and the reflectivity curve moves to the right. The reflectivity is detected at a fixed angle, and when the refractive index of the surface changes, the detected reflectivity changes. The increase of the refractive index causes the reflectance curve to shift to the right, and at a fixed incident angle, the detected reflectance signal increases. When the fixed incident angle is selected at a suitable position and the detected refractive index change is controlled within a certain range, the refractive index of the solution to be measured and the reflectance change on the surface of the SPR sensor chip have a linear relationship, as shown in Figure 1(b). The line segment in the figure can be expressed by the function Y=A+B*X, where B is the intensity sensitivity, and the unit is RU or RIU. In this way, the refractive index of the surface of the substance to be detected can be converted according to the change of the reflectance, so as to obtain parameters such as the concentration and affinity of the substance to be detected. In the process of quantitative analysis, the line segment in Figure 1(b) must maintain a good linearity, which requires that the incident angle and the moving range of the SPR reflectance curve in Figure 1(a) meet certain conditions: that is, regardless of the reflectance curve No matter where it appears, the incident angle should be in its linear region, and the detection range that satisfies the above conditions is the dynamic range corresponding to the incident angle. For the reflectivity curve of the original SPR chip surface (actually, the scanning curve of the reflected light intensity changing with the incident angle), the linear region is generally within the range of 20% to 80% between the lowest point and the highest point of the curve. When the solution to be tested is passed through, the refractive index of the surface of the SPR chip combined with the substance in the solution to be tested becomes larger, which will cause the SPR curve to shift to the right along the X-axis (X-axis represents the incident angle), and the linearity of the SPR reflectance curve The region is also shifted to the right. When the refractive index of the solution to be measured is too large, this shift may cause the original incident light angle to be outside the linear region of the SPR reflectance curve, that is, beyond the dynamics of intensity detection at the original angle. scope. For tunable SPR biosensors, the original SPR reflectance curve on the surface of the SPR chip itself is relatively sharp, so the range of allowed translation of the SPR reflectance curve is small, resulting in a small dynamic range for its intensity detection. Not conducive to practical use.

In the traditional intensity detection method, when the detection signal exceeds the dynamic detection range, it is necessary to move the SPR scanning angle before continuing the detection. This solution needs to change the optical path through the mechanical structure, which often affects the accuracy and consistency of the experiment, and also needs to interrupt the experiment and re-scan and calibrate the SPR curve.

Therefore, the small dynamic range seriously affects the practicability of tunable SPR, a new type of SPR sensor, and there is an urgent need for a detection scheme based on tunable SPR biosensors with a large dynamic range.

Contents of the invention

The present invention proposes a detection scheme based on a tunable SPR biosensor with a large dynamic range.

In order to achieve the purpose of the above invention, the present invention provides a detection method based on a tunable surface plasmon resonance sensor, the tunable surface plasmon resonance sensor is an SPR detection system using a tunable SPR biochip modulated by an external field, The detection method comprises the following steps:

1) Set the external field to 0, pass the first and second sample solutions with known refractive index or concentration into the sample pool on the tunable SPR biochip, and complete the angle scanning respectively to obtain the first sample solution and the SPR angle scan curve of the second sample solution; calculate the sensitivity of the SPR detection system under 0 external field, and calibrate the dynamic range of detection according to the linear range of the first and second sample solution SPR angle scan curves;

2) Select an angle within the dynamic range as the incident angle, and scan the external field to obtain the scanning curve of the reflected light intensity changing with the external field intensity, and then calculate the linear coefficient of the reflected light intensity changing with the external field intensity;

3) Pass the solution to be tested into the sample cell on the tunable SPR biochip, select the same incident angle as in step 2), when the change of the reflected light intensity with time exceeds or is about to exceed the dynamic range, Maintaining the same incident angle, adjusting the applied external field intensity so that the detected reflected light intensity remains within the dynamic range, and reading the external field intensity and reflected light intensity at this time;

4) Based on the linear coefficient obtained in step 2), the reflected light intensity under the current external field intensity is converted into the equivalent reflected light intensity under 0 external field, and then based on the SPR detection system under 0 external field obtained in step 1). Sensitivity, using the equivalent reflected light intensity to calculate the refractive index or concentration of the solution to be measured.

Wherein, in the step 1), the angle scans are performed under different intensities of external fields to obtain the SPR angle scan curves of the first sample solution and the second sample solution, according to the SPR angle scan curves of the first and second sample solutions The linear range calibration corresponds to the dynamic range corresponding to the external field of each intensity;

In the step 3), when the reflected light intensity of the solution to be tested exceeds the dynamic range under 0 external field, adjust the external field intensity so that the reflected light intensity of the detection signal returns to the dynamic range under the corresponding external field intensity, and then read out External field intensity and reflected light intensity.

Wherein, the linear region is a reflected light intensity region where the linearity is greater than 0.9995.

Wherein, in the step 1), based on the linear region of the SPR angular scanning curve under each external field intensity, the maximum reflected light intensity and the minimum reflected light intensity of the linear region are taken as 1 and 0, and the SPR angular scanning curve is normalized and, using the normalized SPR angle scanning curves of the first sample solution and the second sample solution, calculate the normalized sensitivity of the SPR detection system under 0 external field;

In the step 2), the maximum reflected light intensity and the minimum reflected light intensity in the linear region are taken as 1 and 0, and the curve of the reflected light intensity obtained by scanning the external field with the external field intensity is normalized;

In the step 3), according to the read-out external field intensity, according to the normalized SPR angle scanning curve of the corresponding external field intensity obtained in step 1), the read-out reflected light intensity is normalized;

In the step 4), based on the linear coefficient of the curve of the normalized reflected light intensity varying with the external field intensity obtained in step 2), the normalized reflected light intensity under the current external field intensity is converted to 0 external field The normalized equivalent reflected light intensity below, and then based on the normalized sensitivity of the SPR detection system under the 0 external field obtained in step 1), the normalized equivalent reflected light intensity is used to calculate the solution to be tested. Refractive index or concentration.

Wherein, the first and second sample solutions are 1×PBS, 2×PBS, deionized water, 1:200 phosphate buffer or glycerin solution with known refractive index or concentration.

Wherein, the external field is an electric field, thermal field, magnetic field or sound field, and the external field strength is voltage, temperature, magnetic field strength or sound strength.

Compared with the prior art, the present invention has the following technical effects:

1. Significantly expand the dynamic range of the tunable SPR sensor.

2. While expanding the dynamic range of the intensity detection method, the detection error is suppressed.

3. While expanding the dynamic range of the intensity detection method, it maintains a high sensitivity.

Description of drawings

Figure 1(a) shows the reflectance curves of the original SPR chip surface and the surface of the SPR chip after passing through the solution to be tested;

Figure 1(b) shows the relationship curve of the reflectance with the change of the refractive index of the solution to be tested;

Fig. 2 shows the principle schematic diagram of the SPR detection system of the external field modulation adopted in one embodiment of the present invention;

Fig. 3 shows a schematic diagram of the reflectance curve used to calibrate the intensity sensitivity S in one embodiment of the present invention;

Fig. 4 shows a schematic diagram of a reflectance curve used to calibrate the linear coefficient v of the external field modulation voltage and the reflected light intensity in one embodiment of the present invention;

Fig. 5 shows a flowchart of a detection method based on a tunable surface plasmon resonance sensor in an embodiment of the present invention.

Detailed ways

Below, the present invention will be further described in conjunction with the accompanying drawings and specific embodiments.

According to a first embodiment of the present invention, a detection method based on a tunable SPR sensor is provided. The detection method is realized based on an electric field modulated SPR detection system. For ease of understanding, a brief introduction to the SPR detection system for external field modulation is firstly given.

Figure 2 shows a tunable SPR sensor in the prior art ----- SPR detection system with electric field modulation, the detection system includes: monochromatic light source 1, optical assembly 2, high refractive index prism 3, light detection device 4 , electric field tunable WCSPR biochip 5 , sample pool 6 , microfluidic system 7 , voltage source 8 and control system 9 . Wherein, the monochromatic light source 1 and the optical component 2 form a parallel light output device for providing monochromatic, linearly polarized (p-polarized) and collimated light beams. The optical assembly 2 includes lens groups, filters, polarizers, etc. in an unlimited sequence. The high refractive index prism 3 is used to couple the light beam provided by the parallel light output device into the electric field tunable WCSPR biochip 5 . The electric field tunable WCSPR biochip 5 is sequentially composed of a biological detection layer, an upper metal layer, an electric modulation layer, a lower metal layer and a substrate from top to bottom. The voltage source 8 applies a voltage to the tunable WCSPR biochip 5 through the upper metal layer and the lower layer metal to tune the electric field to tune the physical properties (such as refractive index and thickness) of the electrical modulation layer in the WCSPR biochip 5 . In this embodiment, a device for tuning the refractive index of the electrical modulation layer is used, and the electrical modulation layer is an electro-optic modulation layer. The sample cell 6 is a device that confines the detection substance to the detection surface of the electric field tunable WCSPR biochip 5, and its quantity, shape and size are determined by the detection requirements. The light detector 4 is used to detect the reflected or transmitted light intensity from the detection layer of the electric field tunable WCSPR biochip 5 . The control system 9 is a software and hardware system for determining the detection angle, completing the external field scanning, determining the intensity of the modulated external field, recording the detection signal, and completing data analysis and processing, including but not limited to the turntable controller, field generator controller, data collector, central control controller and control and analysis software. The microfluidic system 7 is used to replace the sample in the sample pool. The microfluidic system 7 includes a fluid controller for cleaning and regenerating the surface of the biochip. The fluid controller includes a fluid pump, a gate valve and a microfluidic pipeline.

The working mechanism of the electric field modulated SPR detection system is as follows: the light beam emitted by the monochromatic light source 1 is shaped, filtered and polarized by the optical component 2, and the monochromatic, parallel and p-polarized light is coupled into the electric field through the high refractive index prism 3 Tunable WCSPR Biochip5. The light detector 4 receives the reflected light from the electric field tunable WCSPR biochip 5 , and detects the information on the surface of the tunable WCSPR biochip 5 through the change of light intensity. The upper and lower metal layers of the electric field tunable WCSPR biochip 5 are used as electrodes, and the voltage source 8 applies an electric field to the electro-optic modulation layer. The electro-optic modulation layer adopts linear electro-optic materials, and the relationship between the change of its refractive index and the applied electric field is:

$\mathrm{<span\; class="notranslate">\; \&Delta;\; n</span>}<span\; class="notranslate">\; =</span><span\; class="notranslate">\; -</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}{\mathrm{<span\; class="notranslate">\; no</span>}}^{\mathrm{<span\; class="notranslate">\; 3</span>}}{\mathrm{<span\; class="notranslate">\; \&gamma;</span>}}_{\mathrm{<span\; class="notranslate">\; 33</span>}}\frac{\mathrm{<span\; class="notranslate">\; V</span>}}{\mathrm{<span\; class="notranslate">\; d</span>}}$

Where d is the thickness of the electro-optic modulation layer, V is the voltage applied to the electro-optic modulation layer, γ ₃₃ is the electro-optic coefficient of the electro-optic modulation layer, and n is the original refractive index of the electro-optic modulation layer. It can be seen from the above formula that the change of the refractive index of the electro-optic modulation layer has a linear relationship with the applied electric field. Based on this basic principle, the refractive index of the electro-optic modulation layer is modulated by the applied electric field. The microfluidic system 7 injects a solution into the sample pool 6 or performs sample replacement and cleaning through devices such as a microfluidic pump and a gating valve. All the above-mentioned monochromatic light sources 1, light detectors 4, microfluidic systems 7, and voltage sources 8 are coordinated through the control system 9, including the magnitude of light intensity, the frequency of reflected light collection, the positions of light sources and light detectors , the flow rate of the sample solution, the flow time, the magnitude and frequency of the applied electric field, the processing of the obtained data, etc., so as to obtain the surface information of the biochip and complete the detection of the interaction of biomolecules.

Next, based on the SPR detection system of the above-mentioned external field modulation, the detection method based on the tunable SPR sensor of this embodiment is introduced. Referring to FIG. 5, the method includes the following steps:

Step 101: Use the first and second sample solutions with known refractive indices to calibrate the intensity sensitivity S of the chip when the modulation electric field intensity is zero.

Select the first and second sample solutions with known refractive indices, and perform angular scanning on the first and second sample solutions respectively to obtain their SPR angular scanning curves. In this embodiment, 1×PBS and 2×PBS are used as the first and second sample solutions respectively, and the SPR angle scanning curves thereof are shown in FIG. 3 , and the scanning data are shown in Table 1.

Table 1

The selection scheme of the above-mentioned first and second sample solutions is only exemplary, and those skilled in the art can use known solutions such as 1×PBS, 2×PBS, deionized water, 1:200 phosphate buffer, glycerin, etc. Two of them were arbitrarily selected as the first and second sample solutions.

Based on the scanning curve of 1×PBS, select the area whose linearity is greater than 0.9995 as the dynamic detection range, such as the angular range θ _i ~ θ _j in Figure 3 . Among them, the specific method of linearity analysis can refer to the literature "J. Homola, Surface plasmon resonance sensors for detection of chemical and biological species, Chem. Rev., (2008) 108:462-493", which will not be repeated here.

A point θ _k within the dynamic detection range θ _i ~ θ _j is selected as a fixed detection angle, and the corresponding reflected light intensities of the first and second sample solutions are I _k1 and I _k2 respectively. At the same time, it is known that the difference between the refractive index of 1×PBS and 2×PBS is 0.00154RIU (Refractive Index Unit, the refractive index unit), so the intensity sensitivity S of the chip can be calibrated when the modulation electric field intensity is zero:

$\mathrm{<span\; class="notranslate">\; S</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; k</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; k</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}}{\mathrm{<span\; class="notranslate">\; 0.00154</span>}}\mathrm{<span\; class="notranslate">\; RU</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; RIU</span>}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

Therefore, for the detection signal _Ix within the dynamic detection range, the refractive index _Rx can be expressed as:

${\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; PBS</span>}}<span\; class="notranslate">\; +</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; S</span>}}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; x</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; k</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; )</span>\mathrm{<span\; class="notranslate">\; RIU</span>}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 2</span>}<span\; class="notranslate">\; )</span>$

Step 102: Calibrate the linear coefficient v of the reflected light intensity varying with the modulation voltage of the external field.

For the above case where the modulation voltage is zero, the detection range of the reflection intensity corresponding to the dynamic detection range when the fixed detection angle is θ _k is I _j ~ I _i , and the range of the refractive index is R _j ~ R _i . During the detection process, if the reflected light intensity of the detected object exceeds this range, that is, it exceeds the linear detection range, the conversion relationship in formula (2) will not be established, and the calculated refractive index of the detected object (that is, the detection signal intensity) will also be inaccurate.

Fig. 4 shows the SPR reflectance curve obtained by scanning when the external field is 0 and applying a modulation voltage with an intensity of V _m to the chip, wherein, for the first sample solution, the angle θ _k when the modulation voltage is V _m The corresponding reflected light intensity value I _k1m .

Since the modulation voltage of the external field has a linear relationship with the change of the refractive index, that is, it has a linear relationship with the change of the reflected light intensity, so:

I _k1m -I _k1 =v(V _m -0) (3)

Right now,

v=(I _k1m -I _k1 )/V _m (4)

Therefore, the equivalent detection signal I _x of 0 external field corresponding to the detection signal I' _x under any external field modulation voltage V _x can be expressed as:

I _x =I' _x -vV _x (5)

It can be seen that after obtaining the linear coefficient v of the reflected light intensity changing with the modulation voltage of the external field, substituting I _x in the formula (5) into the formula (2), the refraction of the object under test can be calculated under any external field conditions rate size.

Step 103: Pass in the solution to be tested, and when the reflected light intensity of the solution to be tested exceeds the initial dynamic linear range of I _j ~ I _i , adjust the external field modulation voltage to make the reflected light intensity value of the detection signal return to I _j ~ I within the range of _i . Record the external field voltage V _x and the reflected light intensity value I _x at this time, and convert according to formulas (2) and (5) to obtain the refractive index of the solution to be tested. This scheme avoids adjusting the optical path or mechanical system, ensures the continuity of experiments and the accuracy and consistency of results, effectively expands the dynamic range of SPR intensity detection, and greatly improves the practical value of WCSPR sensors.

Table 2 shows the linearity of the SPR reflectivity curve of an SPR chip under different modulation voltages in the initial linear region (that is, the linear region of the SPR reflectivity curve when the modulation voltage is 0V) and their respective refractive index detection ranges.

Table 2

Voltage sensitivity Linearity Refractive index detection range 0V 25092.91 0.99979 1.3310-1.3324=0.0014 -10V 24403.88 0.99981 1.3317-1.3331=0.0014 -20V 23551.91 0.99982 1.3324-1.3338=0.0014 -30V 22438.69 0.99973 1.3331-1.3346=0.0015 -40V 21073.62 0.99947 1.3338-1.3355=0.0017 -50V 19648.24 0.99909 1.3346-1.3364=0.0018 -60V 19648.24 0.99757 1.3354-1.3372=0.0018 -70V 15969.09 0.99517 1.3362-1.3384=0.0022

It can be seen that when the modulation voltage range is 70V, the dynamic range of material refractive index detection is expanded from 1.3310-1.3324 to 1.3310-1.3384.

The present invention also provides a second embodiment, which, on the basis of the previous embodiment, takes into account the influence caused by the deformation of the angular scanning curve of the SPR chip itself after applying an external field, thereby further suppressing the error of the measurement result , improve measurement accuracy. The above-mentioned SPR detection system modulated by the external field is still taken as an example for illustration.

In the second embodiment, before the step 103, re-scan the angle of the SPR curves under different voltages, and re-select its linear region (the region whose linearity is greater than 0.9995), that is, re-calibrate the corresponding to different voltage intensities. The detection range is used to obtain the dynamic linear region, and the obtained data are shown in Table 3.

table 3

Voltage sensitivity Linearity Refractive index detection range 0V 25092.91 0.99979 1.3310-1.3324=0.0014 -10V 24403.88 0.99981 1.3317-1.3331=0.0014 -20V 23551.91 0.99982 1.3324-1.3338=0.0014 -30V 22360.4 0.9998 1.3332-1.3347=0.0015 -40V 21314.11 0.99978 1.3341-1.3356=0.0015 -50V 19970.02 0.99974 1.335-1.3366=0.0016 -60V 18579.43 0.9998 1.3359-1.3376=0.0017 -70V 16995.87 0.99964 1.337-1.3389=0.0019

It can be seen from the attached table 3 that the linearity of the curve has been significantly improved, all above 0.9995. For ease of operation, the refractive index detection range at different voltages can be converted into a linear region of reflected light intensity at corresponding voltages.

In step 103, when the reflected light intensity of the solution to be tested exceeds the initial dynamic linear range of I _j ~ I _i , by adjusting the external field modulation voltage, the reflected light intensity value of the detection signal returns to the linear region under the corresponding external field modulation voltage , and then record the measured reflected light intensity value, and then calculate the refractive index of the solution to be tested according to the formula (2) (5).

The detection method of the above-mentioned second embodiment can ensure that the SPR reflectivity curve at any voltage has extremely high linearity, thereby suppressing detection errors while expanding the dynamic range of the intensity detection method.

The present invention also provides a third embodiment, which, on the basis of the foregoing embodiments, further improves the sensitivity of detection through a normalized method, and the detection method of this embodiment includes the following steps:

Step 201 : For the SPR chip that is not connected with the object to be detected, perform angular scanning under different modulation voltages to obtain corresponding SPR reflectivity curves (ie, SPR angular scanning curves).

Step 202: Perform normalization processing on the SPR reflectance curves under each modulation voltage to obtain a normalized conversion formula of its ordinate (representing reflected light intensity). Let the ordinate values of the data be y ₁ , y ₂ , y ₃ ... y _min ... y _max ..., then for any data point y _i in the linear region on the curve, y _min and y _max are respectively the curve The minimum and maximum values of the ordinate for the upper linear region. In this way, the normalized conversion formula can be expressed as:

${\mathrm{<span\; class="notranslate">\; the\; y</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; the\; y</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; the\; y</span>}}_{\mathrm{<span\; class="notranslate">\; min</span>}}}{{\mathrm{<span\; class="notranslate">\; the\; y</span>}}_{\mathrm{<span\; class="notranslate">\; max</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; the\; y</span>}}_{\mathrm{<span\; class="notranslate">\; min</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 6</span>}<span\; class="notranslate">\; )</span>$

203: Pass the first sample solution with known refractive index or concentration into the sample cell on the tunable SPR biochip to complete the angle scan, and obtain the SPR angle scan curve of the first sample solution.

204: Pass a second sample solution with known refractive index or concentration into the sample cell on the tunable SPR biochip to complete the angle scan, and obtain the SPR angle scan curve of the second sample solution.

205: Calculate the sensitivity of the detection system and the linear range of the SPR curves of the first and second sample solutions according to the SPR angle scanning curves obtained in steps 203 and 204 and the known refractive indices or concentrations of the first and second samples The corresponding reflected light intensity is used as its dynamic detection range.

206: Select the first sample solution in step 1), or an angle corresponding to the linear region in the angular scanning curve of the second sample solution in step 204 (such as the angle corresponding to the lowest point of the linear region), Carry out external field scanning, select the range of linearity greater than 0.9995 in the relationship curve between external field intensity and reflected light intensity as the linear area of external field scanning, and normalize the ordinate of the scanning result (i.e. reflected light intensity), and then get the normalized The linear coefficient v ₁ of the normalized reflected light intensity changing with the modulation voltage of the external field.

207: Pass the solution to be detected into the sample cell of the tunable SPR biochip, fix the scanning angle at the angle selected in step 206 for external field scanning, and detect the change of reflected light intensity. When the change of the reflected light intensity with time is about to or has exceeded the corresponding reflected light intensity range of the dynamic detection range, the fixed scanning angle is constant, and the external field intensity is adjusted according to the relationship between the external field intensity obtained in step 206 and the dynamic detection range, so that The reflected light intensity returns to the corresponding linear region, and the reflected light intensity and modulation voltage at this time are recorded. During data processing, the ordinate of the measurement result is first normalized, that is, the measured reflected light intensity is converted into a normalized reflected light intensity according to the formula in step 202, and then according to the normalized light intensity obtained in step 206, The linear coefficient v ₁ of the normalized reflected light intensity changing with the modulation voltage of the external field is used to calculate the actual size of the detection signal and obtain the final result.

The method of this embodiment can not only increase the dynamic range, but also avoid the adverse effects on the detection sensitivity and linearity caused by the movement and deformation of the SPR reflectivity curve during the external field modulation process, thereby improving the performance of the tunable SPR biosensor when using external field modulation. The quality of the data obtained during testing, that is, improving accuracy and reducing errors.

Table 4 shows the detection ranges corresponding to different external field modulation voltages after normalization and dynamic linear region processing.

Table 4

Voltage sensitivity Linearity Refractive index detection range 0V 25782.6 0.99978 1.3310-1.3324=0.0014 -10V 25156.25 0.99981 1.3317-1.3331=0.0014 -20V 24433.37 0.99981 1.3325-1.3339=0.0014 -30V 23558.65 0.9998 1.3332-1.3347=0.0015 -40V 22676.89 0.99982 1.3341-1.3356=0.0015 -50V 21631.43 0.9998 1.335-1.3366=0.0016 -60V 20505.04 0.99977 1.336-1.3377=0.0017 -70V 19273.29 0.99974 1.337-1.3389=0.0019

It can be seen from Table 4 that after normalization processing, the detection sensitivity under a larger modulation voltage has also been greatly improved, so that under the regulation of the external field modulation voltage, the detection range of the refractive index is from the initial 1.3310-1.3324 when the electric field voltage increases to 1.3310-1.3389 when the external field modulation voltage is 0-70V. Therefore, the detection range of the refractive index is increased to 5.9 times of the initial value, while the linearity is always above 0.9997, which ensures the accuracy of the detection data.

In the actual test, pure water and substances with known refractive indices such as glucose solutions with different concentrations were selected. Using the scheme of the second embodiment above, the measured data reached the basis of keeping the detection sensitivity and linearity basically unchanged. The effect of expanding the detection range.

In the above-mentioned embodiment, the SPR detection system may also use other types of prisms, gratings or other optical couplers capable of coupling the light beam into the tunable SPR biochip to replace the high refractive index prism 3 . The light detector 4 can be a CCD, a photodiode, or other sensors sensitive to light intensity.

It is worth noting that the electric field tunable WCSPR biochip 5 in the above embodiment can also be replaced by other tunable SPR biochips, such as Coupled Plasmon-Waveguide Resonance (CPWR for short), waveguide-coupled surface plasmon resonance (Waveguide-Coupled SPR, referred to as WCSPR) or other structures that can tune the detection signal by modulating the physical properties of the medium layer (such as refractive index and thickness) through electrical, magnetic, thermal, and acoustic methods. Specifically, the tunable medium layer can be made of electro-optic materials, magneto-optic materials, thermo-optic materials, acousto-optic materials, or other materials whose physical properties (such as refractive index and thickness) change under the action of an external field. Materials that can be used for the metal layer include pure metals, alloys, metal compounds, or other materials that can serve as SPW carriers. Wherein, said pure metal includes gold, silver, chromium, copper and aluminum. The detection layer is a substance to be detected, a modified substance, a label substance or a combination thereof. Similarly, the voltage source 8 in this embodiment can also be replaced by other field generators. The field generator is a controllable device of various electric fields, magnetic fields, temperature, and sound waves, and the physical properties of the tunable medium layer of the tunable SPR biochip are tuned by the field generator.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit them. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications or equivalent replacements to the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and all of them should be included in the scope of the present invention. within the scope of the claims.

Claims (6)

1., based on a detection method for the SPR detection system of outfield modulation, comprise the following steps:

1) with refractive index or known first, second sample solution of concentration, drawn the sensitivity of 0 outer described SPR detection system after the match by angle scanning, and demarcate according to the range of linearity of first, second sample solution SPR angle scanning curve the dynamic range detected; Calculate the sensitivity of 0 outer described SPR detection system after the match, and demarcate according to the range of linearity of first, second sample solution SPR angle scanning curve the dynamic range detected;

2) a certain angle in selected described dynamic range, as incident angle, is carried out outer field scan, is obtained the linear coefficient that reflective light intensity changes with outfield strength;

3) solution to be measured is passed in the sample cell on the tunable SPR biochip of described SPR detection system, selected and step 2) identical incident angle, when reflective light intensity exceeds or be about to exceed described dynamic range, keep same incident angle, regulate the outfield strength applied, make detected intensity of reflected light remain in described dynamic range, read outfield strength now and intensity of reflected light;

4) based on step 2) intensity of reflected light under current outfield strength is converted to 0 outer equivalent reflective light intensity after the match by the linear coefficient that draws, the sensitivity of the 0 outer described SPR detection system after the match obtained based on step 1) again, utilizes this 0 outer equivalent reflective light intensity meter after the match to calculate refractive index or the concentration of solution to be measured.

2. detection method according to claim 1, it is characterized in that, in described step 1), angle scanning is carried out respectively after the match at the outer of varying strength, obtain the SPR angle scanning curve of the first sample solution and the second sample solution, demarcate the dynamic range corresponding to the outfield corresponding to each intensity according to the range of linearity of first, second sample solution SPR angle scanning curve;

In described step 3), when the reflective light intensity of solution to be measured is more than 0 during outer dynamic range after the match, by regulating outfield strength, in the dynamic range under making detection signal intensity of reflected light get back to corresponding outfield strength, and then read outfield strength and intensity of reflected light.

3. detection method according to claim 1, is characterized in that, the described range of linearity is the intensity of reflected light region making the linearity be greater than 0.9995.

4. detection method according to claim 2, it is characterized in that, in described step 1), based on the range of linearity of the SPR angle scanning curve under each outfield strength, using the maximum reflection light intensity of the range of linearity and minimal reflection light intensity as 1 and 0, normalized is done to SPR angle scanning curve; Further, with the SPR angle scanning curve after the normalization of the first sample solution and the second sample solution, the normalization sensitivity of 0 outer described SPR detection system is after the match calculated;

Described step 2) in, using the maximum reflection light intensity of the range of linearity and minimal reflection light intensity as 1 and 0, the curve that the reflective light intensity that external field scan obtains changes with outfield strength does normalized;

In described step 3), according to read-out outfield strength, the normalization SPR angle scanning curve of the corresponding outfield strength drawn according to step 1), is normalized read-out intensity of reflected light;

In described step 4), based on step 2) linear coefficient of curve that changes with outfield strength of reflective light intensity after the normalization that draws, intensity of reflected light after normalization under current outfield strength is converted to 0 outer normalization equivalent reflective light intensity after the match, the normalization sensitivity of the 0 outer described SPR detection system after the match obtained based on step 1) again, utilizes this normalization equivalent reflective light intensity meter to calculate refractive index or the concentration of solution to be measured.

5. detection method according to claim 1, is characterized in that, first, second sample solution described is 1 × PBS, 2 × PBS, deionized water, 1:200 phosphate buffer or glycerite.

6. detection method according to claim 1, is characterized in that, described outfield is electric field, thermal field, magnetic field or sound field, and described outfield strength is voltage, temperature, magnetic field intensity or the sound intensity.