CN115389469A - Surface plasma resonance sensing device and antibiotic concentration detection method - Google Patents

Surface plasma resonance sensing device and antibiotic concentration detection method Download PDF

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CN115389469A
CN115389469A CN202211076821.4A CN202211076821A CN115389469A CN 115389469 A CN115389469 A CN 115389469A CN 202211076821 A CN202211076821 A CN 202211076821A CN 115389469 A CN115389469 A CN 115389469A
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antibiotic
spr
resonance
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prism
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罗云瀚
施伟成
胡诗琦
袁锦明
陈耀飞
刘贵师
陈雷
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Jinan University
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    • G01N21/553—Attenuated total reflection and using surface plasmons
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Abstract

The invention discloses a surface plasma resonance sensing device, which comprises a bridge wedge-shaped prism, a light source, a collimating lens group, a spectrometer, an infrared polarizer and a coupling lens, wherein the bridge wedge-shaped prism is arranged on the front end of the light source; the front surface and the rear surface of the bridge wedge prism are parallel to each other, and the upper surface, the left side surface and the right side surface are vertical to the front surface; the left side surface and the right side surface of the bridge wedge-shaped prism form acute angles with the upper surface; the bottom of the bridge wedge prism is provided with a groove-shaped opening; the top of the groove-shaped opening is a plane, and forms an included angle beta with the upper surface of the bridge wedge-shaped prism, and the included angle beta is larger than 0 degree; the upper surface of the bridge wedge prism is plated with an SPR metal film; the top of the groove-shaped opening is plated with a reflecting film. The invention also discloses an antibiotic concentration detection method based on the surface plasma resonance sensing device. The surface plasma resonance sensing device has high sensitivity and temperature self-compensation capability, and can reduce the detection limit when used for detecting the concentration of antibiotics.

Description

Surface plasma resonance sensing device and antibiotic concentration detection method
Technical Field
The invention relates to the field of optical detection and optical sensing, in particular to a surface plasma resonance sensing device and an antibiotic concentration detection method.
Background
Surface Plasmon Resonance (SPR) sensing is an optical sensing method that uses the property of the SPR effect that it is very sensitive to changes in the refractive index of the surrounding environment. Compared with the traditional detection sensing method using biochemical markers such as radioactivity or fluorescence, the SPR sensor judges the biochemical reaction on the surface of the sensor through the change of the refractive index value of the sensing surface, has the advantages of high speed, high sensitivity, no marker, real-time monitoring and the like, and is widely applied to a plurality of fields such as environmental monitoring, food safety, medicine detection and the like. Sensitivity is one of the most important performance indicators for SPR sensors and determines the minimum change in analyte that the sensor can detect. However, the increased sensitivity also means that the sensor is more sensitive to changes in ambient temperature, which will seriously affect the accuracy of the sensor for the detection of low concentrations of antibiotics.
In order to compensate for temperature variations, a series of research works have been proposed. However, only a few SPR temperature compensation sensors based on prism coupling have been reported and are essentially simulation efforts. For example, lin et al simulated an SPR sensor based on prism coupling for sensitivity dependence on temperature, but did not compensate for the effect on temperature. Xiao et al theoretically propose a method of combining angle and intensity modulation to achieve the purpose of temperature compensation, but two lasers are needed to provide light sources at the same time, and the system is complicated. Luo et al proposed a temperature compensation method in simulation that combines angle modulation and wavelength modulation. The method needs to adjust the angle of incident light for many times, and is inconvenient for practical test.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks and deficiencies of the prior art, the present invention provides a surface plasmon resonance sensor device with ultra-high sensitivity, which can perform temperature self-compensation by using two SPR resonance valleys.
Another object of the present invention is to provide an antibiotic concentration detection method based on the surface plasmon resonance sensing apparatus, which improves and realizes the accuracy of antibiotic detection at low concentration.
The purpose of the invention is realized by the following technical scheme:
a surface plasma resonance sensing device comprises a bridge wedge prism, a light source, a collimating lens group, a spectrometer, an infrared polarizer and a coupling lens;
the bridge wedge prism has the following structure: the front surface and the rear surface of the bridge wedge prism are parallel to each other, and the upper surface, the left side surface and the right side surface are vertical to the front surface; the included angle between the left side surface of the bridge wedge-shaped prism and the upper surface is an acute angle; the right side surface of the bridge wedge-shaped prism forms an acute angle with the upper surface; the bottom of the bridge wedge prism is provided with a groove-shaped opening; the top of the groove-shaped opening is a plane and forms an included angle beta with the upper surface of the bridge wedge-shaped prism, and the beta is larger than 0 degree; the upper surface of the bridge wedge prism is plated with an SPR metal film; the top of the groove-shaped opening is plated with a reflecting film;
the light emitted from the light source is coupled to an optical fiber and collimated by a collimating lens group, the collimated light beam passes through an infrared polarizer to generate P-type polarized light, and the P-type polarized light is perpendicular to one side surface of the bridge wedge prism at a first incidence angle theta 1 The first resonance valley is formed after the first resonance valley is formed; after being reflected on the reflection film, the light beam is at a second incident angle theta due to the inclination angle beta of the reflection film 2 Then the light is incident on the SPR metal film again to form a second resonance valley, so that two sensing areas are formed, and the light is emitted out perpendicularly to the other side surface of the bridge wedge prism, and finally is coupled to a receiving optical fiber through a coupling lens to be received by a spectrometer.
Specifically, the temperature compensation is performed according to the following calculation formula:
Figure BDA0003830324670000021
wherein, Δ λ 1 And Δ λ 2 Respectively, a first incident angle theta 1 And a second incidence theta 2 The generated wavelength drift, wherein delta n and delta T are respectively the variation of the external refractive index and the variation of the temperature; m is a sensitivity matrix, where M ni And m Ti Refractive index and temperature sensitivity of the SPR sensor, respectively; i =1,2.
Preferably, the thickness of the reflective film is 150 to 250nm.
Preferably, the reflective film is a silver thin film.
Preferably, the SPR metal film is a gold film, a silver film or a copper film.
Preferably, the slot-like opening is square in cross-section.
Preferably, the first incident angle θ 1 Is 65 deg., and theta 1 -θ 2 ＝2β。
An antibiotic concentration detection method based on the surface plasmon resonance sensing device comprises the following steps:
chemically modifying an antibiotic antibody on the SPR metal film;
preparing antibiotic solutions with different concentrations;
introducing antibiotic solutions with different concentrations into the surface plasma resonance sensing device, and testing the resonance wavelength drift amount corresponding to the antibiotic solution with each concentration to obtain the corresponding relation between the total drift amount of the resonance wavelength and the concentration of the antibiotic solution;
and introducing the antibiotic solution to be tested into the surface plasma resonance sensing device, testing the resonance wavelength drift amount corresponding to the antibiotic solution to be tested, and obtaining the concentration test result of the antibiotic solution to be tested according to the corresponding relation between the total drift amount of the resonance wavelength and the concentration of the antibiotic solution.
Preferably, the antibiotic antibody is chemically modified on the SPR metal film, specifically:
introducing a PBS buffer solution until the surface plasma resonance sensing device system runs stably, and processing resonance valley generated by the PBS buffer solution to obtain resonance wavelength of the PBS buffer solution to form a PBS baseline; after the PBS baseline is stable, introducing MPA solution; then introducing PBS buffer solution, and washing off residual MPA solution; after the baseline is stabilized, introducing EDC/NHS solution to activate carboxyl on the surface of the SPR metal film, and then introducing PBS buffer solution to wash away residual EDC/NHS mixed solution; and then introducing an antibiotic antibody solution to be modified to ensure that activated carboxyl on the surface of the SPR metal film is fully combined with the antibody, and then introducing a PBS buffer solution to wash away residual antibiotic antibody.
Preferably, the measuring of the resonance wavelength shift amount corresponding to each concentration of the antibiotic solution specifically comprises:
and (3) introducing PBS buffer solution until the SPR resonance wavelength is stable, taking the resonance wavelength spectral line at the moment as a reference baseline for the specific binding of the antigen and the antibody, introducing antibiotic solutions with different concentrations after the reference baseline is stable, and calculating the resonance wavelength drift amount corresponding to each concentration by comparing the PBS baseline before and after the antibiotic solutions are introduced.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The surface plasma resonance sensing device has high sensitivity and temperature self-compensation capability. By adjusting the incidence angle and the inclination angle of the wedge-shaped bridge prism, two cascaded sensing areas with low crosstalk and high sensitivity are formed in one sensor, and the highest refractive index sensitivity reaches 43166.7nm/RIU. By detecting the two sensing areas simultaneously, combining the refractive index sensitivity and the temperature sensitivity, the self-compensation of the temperature is realized by using the sensitivity matrix, and the measurement error after compensation can be reduced by two orders of magnitude at most from 0.19% to 0.007%.
(2) The surface plasma resonance sensing device is characterized in that a gold film, a silver film or a copper film is plated on the upper surface of the bridge wedge prism, and high sensitivity can be realized by changing the incident angle.
(3) The antibiotic concentration detection method of the invention realizes temperature self-compensation by utilizing the complementary action of two sensing areas with different sensitivities, and improves and realizes the accuracy of antibiotic detection with low concentration.
(4) The antibiotic concentration detection method has the advantages of real-time monitoring and no marking.
Drawings
Fig. 1 is a schematic perspective view of a bridge wedge prism according to an embodiment of the present invention.
Fig. 2 is a front view of a bridge wedge prism of an embodiment of the present invention.
Fig. 3 is a bottom view of a bridge wedge prism of an embodiment of the present invention.
Fig. 4 is a schematic view of a conventional wedge prism.
Fig. 5 is an optical path diagram of a surface plasmon resonance sensing apparatus according to an embodiment of the present invention.
FIG. 6 is a graph of simulated reflectance spectra at an incident angle of 62 to 66 with an external refractive index of 1.331.
FIG. 7 is a diagram showing the electric field intensity distribution in the x direction and a two-dimensional electric field intensity distribution in the Au-SPR model at an incident angle of 65 °.
FIG. 8 is a diagram showing the distribution of the electric field intensity in the x direction of the Au-SPR model and a two-dimensional electric field intensity diagram of the bridge wedge prism according to the embodiment of the present invention under the condition that the incident angle is 63 °.
Fig. 9 is a diagram illustrating simulated changes of the reflection spectrum with the external refractive index when the bridge wedge prism of the embodiment of the present invention has the incident angles of 65 °,63 °,65 ° +63 °, respectively.
FIG. 10 is a piecewise linear fit graph of the resonant wavelength as a function of the external index of refraction for a bridge wedge prism according to an embodiment of the present invention at an angle of incidence of 65 + 63.
FIG. 11 is a reflection spectrum generated by increasing the external RI of two sensing regions (SPR-1 and SPR-2) of a bridge wedge prism from 1.331 to 1.343RIU according to an embodiment of the present invention.
FIG. 12 is a piecewise linear fit plot of the resonant wavelength as a function of the external RI as the external RI of the two sensing regions (SPR-1 and SPR-2) of a bridge wedge prism in accordance with an embodiment of the present invention increases from 1.331 to 1.343 RIU.
FIG. 13 is a reflection spectrum of SPR-2 increasing from 1.331 to 1.343RIU while maintaining 1.331RIU at the outer RI of SPR-1 for a bridge wedge prism according to an embodiment of the present invention.
FIG. 14 is a piecewise linear fit plot of the resonant wavelength of SPR-2 increasing from 1.331 to 1.343RIU versus the external RI for bridge wedge prism of embodiments of the present invention maintaining a 1.331RIU at the external RI of SPR-1.
FIG. 15 is a reflection spectrum of SPR-1 increasing from 1.331 to 1.343RIU with the bridge wedge prism of an embodiment of the present invention maintaining 1.331RIU at the outer RI of SPR-2.
FIG. 16 is a piecewise linear fit graph of resonant wavelength versus external RI for SPR-1 increasing from 1.331 to 1.343RIU while maintaining a 1.331RIU at external RI for SPR-2 for a bridge wedge prism of an embodiment of the present invention.
FIG. 17 is a graph of resonance wavelength versus temperature and a linear fit of SPR-1 generated by the bridge wedge prism of the embodiment of the present invention under different external RI conditions.
FIG. 18 is a graph of resonance wavelength versus temperature for SPR-2 generated by the bridge wedge prism of the embodiment of the present invention under different external RI and a linear fitting graph.
FIG. 19 is a refractive index comparison before and after temperature compensation of a bridge wedge prism sensor according to an embodiment of the present invention.
Fig. 20 is a comparison of measurement errors before and after temperature compensation of the bridge wedge prism sensor according to the embodiment of the present invention.
FIG. 21 is a graph showing the resonance wavelength of the binding process of a streptomycin antibody with time during antibiotic detection in accordance with an embodiment of the present invention.
FIG. 22 is a plot of the resonant wavelength shift over time during antibiotic detection in accordance with an embodiment of the present invention.
FIG. 23 is a graph showing the relationship between the shift amount of the resonance wavelength and the streptomycin concentration in the antibiotic detection process according to the embodiment of the present invention and the result of exponential fitting.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
The embodiment provides a surface plasma resonance sensing device based on a small incident angle, and the reliability of the sensitivity improvement of the surface plasma resonance sensing device is proved through simulation and experiments. The bridge wedge prism of example 1 of the present invention is obtained by providing a slot-like opening in the bottom of a conventional wedge prism, schematically shown in fig. 1 to 3, having the following structure: the front surface and the rear surface of the bridge wedge prism are parallel to each other, and the upper surface, the left side surface and the right side surface are vertical to the front surface; the included angle between the left side surface of the bridge wedge-shaped prism and the upper surface is an acute angle; the included angle between the right side surface of the bridge wedge-shaped prism and the upper surface is an acute angle; the bottom of the bridge wedge prism is provided with a groove-shaped opening; the top 11 of the groove-shaped opening is a plane and forms an included angle beta with the upper surface 12 of the bridge wedge-shaped prism, and the beta is larger than 0 degree; the upper surface of the bridge wedge prism is plated with an SPR metal film; the top of the groove-shaped opening is plated with a reflecting film.
Specifically, the bridge wedge prism of this embodiment is made of K9 glass, and a silver film layer with a thickness of 200 nm is plated on the top of the slot-like opening to serve as a reflector. The SPR metal film is a gold thin film (may be a silver thin film or a copper thin film).
To achieve temperature compensation, two angles of incidence need to be chosen, two resonance valleys with high sensitivity are generated on the SPR sensor, and they can be clearly distinguished. Considering that the range of the crossflow spectrometer is (650-1700 nm) and the 62 ° angle of incidence produces a trough out of the range of the spectrometer, we chose SPR-2 as the minimum 63 ° angle of incidence produced trough. According to the Fresnel reflection law, the first incident angle is assumed to be theta 1 The second incident angle is theta 2 The wedge prism has an inclination angle beta, then theta 1 -θ 2 =2 β, i.e. the difference between the two angles of incidence is twice the angle of inclination. In consideration of the precision of the prism fabrication in the present embodiment, the inclination angle of the wedge prism is set to 1 °, and thus the bridge wedge prism is designed to have incident angles of 65 ° and 63 °. The prism thickness of the sensing area was set to 10mm and the distance between the two sensing areas was 14.58 mm. As shown in fig. 4, the conventional wedge prism has upper and lower surfaces parallel to each other; front and rear surfaces parallel to each other; the front surface and the back surface are vertical to the upper surface and the lower surface, the left side surface and the right side surface are inclined planes, and the included angles between the inclined planes and the upper surface are acute angles. In the same goldThe distance between the two sensing zones of a conventional wedge prism is 38.97 mm, for thickness and angle of incidence. It can be seen that the bridge wedge prism of this embodiment is a 62.6% reduction in size over conventional wedge prisms, which is advantageous in reducing the amount of sample required for biological testing. In addition, the design of two piers facilitates the installation of the prism and protects the reflective silver mirror.
Fig. 5 is an optical path diagram of the surface plasmon resonance sensing apparatus according to embodiment 1, which includes a light source 1, a collimating lens group 2, an infrared polarizer 3, a bridge wedge prism 4 with an SPR metal film plated on the upper surface, a coupling lens 5, and a spectrometer 6; the arrows in the figure indicate the direction of movement of the light beam. The specific optical path transmission process is as follows:
l1: the light emitted from the light source is coupled by the optical fiber and then emitted out, and is collimated by the collimating lens group;
l2: the collimated light beam passes through an infrared polarizer to generate P-type polarized light;
l3: the light beam is perpendicular to one side face side of the bridge wedge prism at a first incident angle theta 1 The light is incident on the SPR metal film to form a first resonance valley SPR-1;
l4: after being reflected on the silver mirror, the light beam is at a second incident angle theta due to the inclination angle beta of the mirror 2 To form a second resonance valley SPR-2, thereby forming two sensing areas;
l5: the light beam exits perpendicular to the other side surface of the bridge wedge prism and couples the exiting light through a coupling lens onto a receiving fiber and is received by a spectrometer.
Further, in example 1, the following theory and simulation analysis are performed on the bridge wedge prism by using a finite element method, and the specific simulation process is as follows:
s1: the simulated wavelength range is set to 700-1700 nm with a step size of 1 nm, depending on the spectrometer measurable range;
s2: selecting a gold film with the SPR metal material of 50 nanometers according to an experiment;
s3: setting different incidence angle values and refractive index bases by using a prism type Kretschmann structure to obtain corresponding transmission spectral lines;
s4: because the bottom of the wedge prism has an inclination angle, and the incident angles are different, the reflectivity of the emergent light can be expressed as follows:
R＝R 1 R 2 (4)
wherein R1 and R2 refer to the reflectivity of P-polarized light at the two sensing regions, respectively. According to the simulation process, the reflection spectra of a series of wedge-shaped SPR sensors are calculated, wherein the refractive index of the liquid to be measured is 1.331-1.343 RIU, the step length is 0.03RIU, and the simulation result is shown in figures 7-8. As can be seen from the graph, at an ambient refractive index of 1.331RIU, the SPR-1 resonance wavelength generated at a 65 ℃ incident angle is 890nm, and the SPR-2 resonance wavelength generated at a 63 ℃ incident angle is 1213nm, with little cross talk between the two valleys. The spectrum of the SPR is gradually red-shifted with the increase of external RI, the resonance wavelength generated by SPR-1 is shifted from 890nm to 1022nm, and the refractive index sensitivity is 8683.3nm/RIU (1.331-1.337 RIU) and 13266.7nm/RIU (1.337-1.343 RIU); while SPR-2 produced a shift in resonance wavelength from 1213nm to 1632nm with index sensitivities of 26666.7nm/RIU (1.331-1.337 RIU) and 43166.7nm/RIU (1.337-1.343 RIU).
In order to compare the SPR sensitivity generated by two different incident angles with the electric field, the electric field distribution with the incident angles of 65 and 63 and the ambient refractive index of 1.331 was simulated by FEM, as shown in FIGS. 9 to 10. An obvious evanescent field exists on the interface of the gold film and the surrounding refractive index liquid, and when the incident angle of SPR is 63 degrees, the penetration distance of the evanescent wave is 1100nm, which is 2 times of that of 65 degrees. The improvement in SPR sensitivity is related to an increase in the overlap integral of the electromagnetic field inside the liquid to be measured, and therefore, when the incident angle is 63 °, the SPR sensor has a stronger electric field at the metal interface, resulting in higher sensitivity.
Refractive index sensitivity detection for the bridge wedge prism of an embodiment of the invention:
this example performed three sets of refractive index sensitivity experiments to test the performance of the bridge wedge prism sensor. A first group: the spectra of the refractive index sensitivity test are shown in FIG. 11, with the sequential introduction of aqueous ethylene glycol solutions having refractive indices of 1.331, 1.334, 1.337, 1.340, and 1.343RIU into the SPR-1 and SPR-2 sensing regions. From the test results, it can be seen that the resonance wavelength is red-shifted with an increase in the external refractive index. The relationship of the resonance wavelength of SPR-1 and SPR-2 to the refractive index and the piecewise linear fit of the experimental results are shown in FIG. 12. The resonance wavelength generated by SPR-1 is shifted from 893.9nm to 1018nm, and the refractive index sensitivity is 7866.7nm/RIU (1.331-1.337 RIU) and 12816nm/RIU (1.337-1.343 RIU); while SPR-2 produced a shift in resonance wavelength from 1213nm to 1618nm with index sensitivities of 24333.3nm/RIU (1.331-1.337 RIU) and 43166.7nm/RIU (1.337-1.343 RIU).
Second group: the SPR-1 sensing region was kept in water (n = 1.331) and ethylene glycol aqueous solutions of the same refractive index range were sequentially introduced into the SPR-2 sensing region for testing, the obtained spectrum is shown in fig. 13, and the corresponding relationship between resonance wavelength and refractive index and piecewise fitting are shown in fig. 14; the resonance wavelength of SPR-1 is constant (maintained at 894 nm), while the resonance wavelength of SPR-2 shifts from 1216 to 1627nm; the refractive index sensitivities were 24166.7nm/RIU (1.331-1.337 RIU) and 44333.3nm/RIU (1.337-1.343 RIU).
Third group: the SPR-2 sensing area was kept in water (n =1.331 RIU) and an aqueous solution of ethylene glycol in the same refractive index range was added to the SPR-1 sensing area for testing, and the resulting spectrum is shown in fig. 15, and the corresponding relationship between resonance wavelength and refractive index and the piecewise fit is shown in fig. 16. The resonance wavelength of SPR-2 is constant (kept at 1214 nm), while the resonance wavelength of SPR-1 drifts from 894.1 to 1036nm; the refractive index sensitivities were 8383.3nm/RIU (1.331-1.337 RIU) and 15266.7nm/RIU (1.337-1.343 RIU).
The bridge wedge prism of embodiment 1 of the invention realizes higher sensitivity with a simple gold film structure by changing the incident angle, and is superior to other sensitivity-enhanced SPR sensors, such as MoS 2 Bimetallic layer, graphene oxide nanosheet, near infrared SPR @65 DEG and SPR sensor based on hyperbolic metamaterial (Table 1).
TABLE 1 comparison of SPR sensors of different sensitization modes
Figure BDA0003830324670000081
Example 2
In this embodiment 2, a temperature self-compensation method for a surface plasmon resonance sensing apparatus is provided. For example, the surface plasmon resonance sensing apparatus in embodiment 1 of the present invention is self-compensated for temperature.
The relationship between the resonance wavelength and the ambient temperature at different refractive indices in example 2 is shown in fig. 17 to 18. When the temperature was reduced from 55 ℃ to 25 ℃, a refractive index of 1.331RIU produced a red shift in the resonance wavelength from 871.3nm to 894.6nm. The relationship between resonant wavelength and temperature is nearly linear, while the calculated sensitivity is approximately-0.6757 nm/deg.C (SPR-1@1.331RIU) and-1.9647 nm/deg.C (SPR-2@1.331RIU). The temperature sensitivity of the sensor increases with the increase of the refractive index, and reaches the maximum of-1.1786 nm/DEG C (SPR-1@1.343RIU) and-3.6214 nm/DEG C (SPR-2@1.343RIU), which is caused by the higher refractive index sensitivity of the SPR sensor in the long wave band.
The higher the sensitivity of the sensor, the more affected by ambient temperature variations, and the bridge wedge prism can compensate for temperature by two resonance valleys. As can be seen from fig. 2, 4 and fig. 17 to 18, since the sensitivity of the SPR resonance wavelength to temperature and refractive index depends on the incident angle, by measuring the SPR wavelength response at two different incident angles, the temperature and refractive index changes can be determined. The total wavelength change resulting from the simultaneous changes in temperature and refractive index at two selected angles of incidence is shown below.
Figure BDA0003830324670000091
Wherein, Δ λ 1 And Δ λ 2 Respectively corresponding to the incident angle theta 1 And theta 2 The generated wavelength shifts, Δ n and Δ T, correspond to the variations of the external refractive index and temperature, respectively. M is a full vector matrix (sensitivity matrix) of cross-sensitivity that takes into account RI variations and temperature variations, where M ni And m Ti (i =1,2) are the refractive index and temperature sensitivity, respectively, of the SPR sensor. From equation (5), the variation of RI and temperature with wavelength drift can be obtained by inverting the matrix. It is worth noting that this sensitivity matrix is always reversible, since the SPR resonance wavelength has different sensitivities to different angles of RI and temperature changes. Thus, taking 1.331RIU as an example, the change in refractive index and temperature can be calculated by wavelength shift at two angles of incidence:
Figure BDA0003830324670000092
the amount of change in refractive index and temperature can be calculated by inverse matrix as long as the SPR wavelength shifts at the two angles of incidence are calculated.
The temperature self-compensation process of the surface plasmon resonance sensing device of the embodiment under different refractive indexes is as follows:
n1: a reaction tank with the height of 2 cm is manufactured on a bridge wedge prism plated with an SPR metal film by using UV glue and a glass sheet.
N2: a bridge wedge prism with a reaction cell was placed in the optical path of the surface plasmon resonance sensing apparatus of example 1.
N3: the solution to be measured is heated to 60 ℃ on a hot bench, and then 5mL of the refractive index solution is added into the reaction tank, and the temperature is reduced from 55 ℃ to 25 ℃.
N4: a thermometer was placed in the test solution to monitor the real-time temperature and the spectral data was recorded every 5 ℃.
N5: as the temperature is lowered, the refractive index of the test solution increases and the resonant wavelength is also red-shifted. And (4) calculating the SPR wavelength drift of two incident angles, and calculating the variation of the refractive index and the temperature through the formula (6) to complete the self-compensation of the temperature.
Fig. 19 shows the temperature-compensated refractive index of water calculated by the equation (6) as a function of temperature. The results show that the RI of deionized water decreases with increasing temperature, which is consistent with the temperature dependence of the refractive index of water. For further comparison, theoretical curves of the refractive index of water as a function of temperature were calculated according to the reference. As can be seen from fig. 19, with the proposed temperature compensation method, the measurement error is reduced. Is composed ofIn more specific comparison, we define the error as the absolute value of the difference from the theoretical value. As can be seen from the calculation results of fig. 20, the measurement error of the refractive index is significantly reduced after the temperature compensation using the two SPR resonances. For example, when the temperature is 30 ℃, the measurement error is calculated to be from 1.19 × 10 -5 Reduced to 1.00X 10 -7 RIU, two orders of magnitude reduction.
Example 3
In embodiment 3 of the present invention, the surface plasmon resonance sensing apparatus is applied to detect the concentration of antibiotics, and the specific steps are as follows:
(1) Chemical modification of antibiotic antibodies on SPR metal membranes
Soaking the prepared bridge wedge prism of 5nmCr-50nm Au in acetone for 10 minutes, washing with deionized water, soaking in absolute ethyl alcohol for 10 minutes, washing with deionized water, and finally drying with nitrogen; the processed bridge wedge prism is installed into the SPR sensing system. And after successful installation, chemical modification can be carried out, firstly, a PBS buffer solution is introduced by using a syringe pump until the SPR sensing system runs stably, and resonance valley generated by the PBS buffer solution can obtain resonance wavelength after data processing to form a base line. After the PBS baseline stabilized, a 10mM MPA solution was passed into the microfluidic channel for 10 minutes at a flow rate of 50. Mu.L/min. Subsequently, the MPA solution remaining in the microfluidic channel was washed away by passing PBS buffer at a flow rate of 50. Mu.L/min for 10 minutes. And after the baseline is stabilized, introducing EDC/NHS solution at the flow rate of 50 mu L/min for 10 minutes, wherein the solution is prepared by mixing 0.4MEDC and 0.1M NHS in equal volume and has the function of activating carboxyl on the surface of the SPR metal membrane, and then introducing PBS buffer solution to wash away residual EDC/NHS mixed solution. Then, a solution of rabbit anti-streptomycin antibody or streptomycin solution to be modified was introduced at a flow rate of 50. Mu.L/min for 20 minutes to allow the activated carboxyl group of the SPR metal membrane to sufficiently bind to the antibody, and then PBS buffer was introduced to wash off the remaining streptomycin antibody. The active sites not bound to the antibody were blocked by passing 1M ethanolamine solution for 20 minutes, and the residual ethanolamine solution was washed off by passing PBS buffer to stabilize the baseline.
FIG. 21 shows the resonance wavelength curve with time of the binding process of streptomycin antibody. It can be seen that when the streptomycin antibody enters the SPR metal film surface, the resonance wavelength rapidly red-shifts, which is caused by the difference in refractive index of the streptomycin antibody and PBS. The resonance wavelength slowly red-shifts with time until reaching a stable state because the streptomycin antibody and the activated carboxyl group aggregate exist on the SPR metal film, and the streptomycin antibody tends to be stable about 15 minutes after the streptomycin antibody is introduced, and reaches a saturated state. The residual antibody solution was then washed clean by passing through PBS buffer, resulting in a blue shift of the resonance wavelength, followed by ethanolamine blocking of unbound activation sites.
(2) Testing the amount of shift of resonance wavelength corresponding to each concentration of antibiotic solution
Streptomycin solutions with different concentrations were prepared, and streptomycin powder was dissolved in PBS buffer and diluted to streptomycin solutions of 0.2, 0.5, 1, 5, 20, 100, 200, 500 μ g/mL.
Firstly, PBS buffer solution is introduced until SPR resonance wavelength is stable, the resonance wavelength spectral line at the time is used as a reference baseline for specific binding of the antigen and the antibody, streptomycin solutions with different concentrations are introduced for 10min from low to high at the flow rate of 50 mu L/min after the reference baseline is stable, and then the residual streptomycin solution is washed away by PBS. The curve of the shift of the resonance wavelength with time during the detection is shown in fig. 22.
(3) And introducing the antibiotic solution to be tested into the surface plasma resonance sensing device, testing the resonance wavelength drift amount corresponding to the antibiotic solution to be tested, and obtaining the concentration test result of the antibiotic solution to be tested according to the relation between the total drift amount of the resonance wavelength and the concentration of the antibiotic solution.
In the above detection process, as can be seen from the time-resonance wavelength curve in fig. 22, after the streptomycin solution is introduced, the refractive index of the surface of the SPR metal film increases due to the fact that the streptomycin is bound to the antibody bound to the SPR metal film, and the resonance wavelength is red-shifted. However, it can be seen from the figure that when the streptomycin concentration is lower than 1. Mu.g/mL, the resonance wavelength is only slightly red-shifted because the molecular weight of streptomycin is small, only 581Da. When the concentration of streptomycin is lower, the refractive index change generated by the streptomycin combined on the SPR metal film is less, so that the shift amount of the resonance wavelength is smaller; the resonance wavelength started to drift significantly when the concentration of streptomycin was greater than 5. Mu.g/mL. After being washed by PBS, the resonance wavelength generates blue shift phenomenon, which is because PBS washes away streptomycin physically adsorbed on the surface of SPR metal film, but the resonance wavelength does not completely fall back to the state before being washed by streptomycin, which indicates that the antigen and the antibody have been specifically combined. The shift amount of the resonance wavelength corresponding to each concentration can be calculated by comparing the PBS baselines before and after streptomycin introduction, as shown in 0, and a relation graph of the total shift amount of the resonance wavelength and the streptomycin concentration is made, as shown in FIG. 23. It can be seen that the drift amount of the resonance wavelength is increased along with the increase of the concentration of streptomycin, and the detection Limit (LOD) of streptomycin detected by a sensing system at an incident angle of 65 degrees can be calculated to be 1.291 mug/mL through exponential fitting according to the minimum resolution of 0.03nm of a spectrometer.
TABLE 2 amount of shift in resonance wavelength due to different concentrations of streptomycin
Figure BDA0003830324670000121
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A surface plasma resonance sensing device is characterized by comprising a bridge wedge prism, a light source, a collimating lens group, a spectrometer, an infrared polarizer and a coupling lens;
the bridge wedge prism has the following structure: the front surface and the rear surface of the bridge wedge prism are parallel to each other, and the upper surface, the left side surface and the right side surface are vertical to the front surface; the included angle between the left side surface of the bridge wedge-shaped prism and the upper surface is an acute angle; the right side surface of the bridge wedge-shaped prism forms an acute angle with the upper surface; the bottom of the bridge wedge-shaped prism is provided with a groove-shaped opening; the top of the groove-shaped opening is a plane, and forms an included angle beta with the upper surface of the bridge wedge-shaped prism, and the included angle beta is larger than 0 degree; the upper surface of the bridge wedge prism is plated with an SPR metal film; the top of the groove-shaped opening is plated with a reflecting film;
the light emitted from the light source is coupled to an optical fiber and collimated by a collimating lens group, the collimated light beam passes through an infrared polarizer to generate P-type polarized light, and the P-type polarized light is perpendicular to one side surface of the bridge wedge prism at a first incidence angle theta 1 The resonance wave is incident on the SPR metal to form a first resonance valley; after being reflected on the reflecting film, the light beam is at a second incident angle theta due to the inclined angle beta of the reflecting film 2 Then the two sensing areas are formed by forming a second resonance valley on the SPR metal film, and the two sensing areas are emitted out perpendicularly to the other side face of the bridge wedge prism, and finally the two sensing areas are coupled to a receiving optical fiber through a coupling lens and received by a spectrometer.
2. A surface plasmon resonance sensing apparatus according to claim 1, wherein the temperature compensation is performed according to the following calculation:
Figure FDA0003830324660000011
wherein, Δ λ 1 And Δ λ 2 Respectively, a first incident angle theta 1 And a second incidence theta 2 The generated wavelength drift, wherein delta n and delta T are the variation of the external refractive index and the variation of the temperature respectively; m is a sensitivity matrix, where M ni And m Ti Refractive index and temperature sensitivity of the SPR sensor, respectively; i =1,2.
3. A surface plasmon resonance sensing apparatus according to claim 1, characterized in that the thickness of the reflective film is 150-250 nm.
4. A plasmon resonance sensing apparatus according to claim 1 or 3, characterized in that the reflective film is a silver thin film.
5. A surface plasmon resonance sensing apparatus according to claim 1, wherein the SPR metal film is a gold film or a silver film or a copper film.
6. A surface plasmon resonance sensing apparatus according to claim 1, wherein the slot-like opening is square in cross-section.
7. The surface plasmon resonance sensing apparatus of claim 1, wherein said first angle of incidence θ 1 Is 65 deg., and theta 1 -θ 2 ＝2β。
8. A method for detecting antibiotic concentration based on the surface plasmon resonance sensor apparatus according to any of claims 1 to 7, comprising the steps of:
chemically modifying an antibiotic antibody on the SPR metal film;
preparing antibiotic solutions with different concentrations;
introducing antibiotic solutions with different concentrations into the surface plasma resonance sensing device, and testing the resonance wavelength drift amount corresponding to the antibiotic solution with each concentration to obtain the corresponding relation between the total drift amount of the resonance wavelength and the concentration of the antibiotic solution;
and introducing the antibiotic solution to be tested into the surface plasma resonance sensing device, testing the resonance wavelength drift amount corresponding to the antibiotic solution to be tested, and obtaining the concentration test result of the antibiotic solution to be tested according to the corresponding relation between the total drift amount of the resonance wavelength and the concentration of the antibiotic solution.
9. The method for detecting antibiotic concentration according to claim 8, wherein the antibiotic antibody is chemically modified on the SPR metal film, and the method comprises the following steps:
introducing a PBS buffer solution until the surface plasma resonance sensing device system runs stably, and processing resonance valley generated by the PBS buffer solution to obtain resonance wavelength of the PBS buffer solution to form a PBS baseline; after the PBS baseline is stable, introducing MPA solution; then introducing PBS buffer solution, and washing away residual MPA solution; after the baseline is stabilized, introducing EDC/NHS solution to activate carboxyl on the surface of the SPR metal film, and then introducing PBS buffer solution to wash away residual EDC/NHS mixed solution; and then introducing an antibiotic antibody solution to be modified to ensure that activated carboxyl on the surface of the SPR metal film is fully combined with the antibody, and then introducing a PBS buffer solution to wash away residual antibiotic antibody.
10. The method for detecting antibiotic concentration according to claim 9, wherein the measuring of the resonance wavelength shift amount corresponding to each concentration of antibiotic solution is specifically:
and (3) introducing a PBS buffer solution until the SPR resonance wavelength is stable, taking the resonance wavelength spectral line at the moment as a reference baseline for the specific binding of the antigen and the antibody, introducing antibiotic solutions with different concentrations after the reference baseline is stable, and calculating the resonance wavelength drift amount corresponding to each concentration by comparing the PBS baseline before and after the antibiotic solutions are introduced.
CN202211076821.4A 2022-09-02 2022-09-02 Surface plasma resonance sensing device and antibiotic concentration detection method Pending CN115389469A (en)

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