Manufacturing method of canine distemper virus trace detection sensor, the sensor and method for detecting trace of canine distemper virus

Abstract

The present invention discloses a method for fabricating a sensor for trace detection of canine distemper virus. A single-mode optical fiber is obtained, first hydroxylated on its surface, then silanized, and immersed in a canine distemper virus antibody solution to obtain a canine distemper virus antibody-modified optical fiber. Then, single-mode optical fibers are butt-welded at both ends with a dislocation to obtain the sensor. The present invention also discloses a sensor prepared by the above method and a method for detecting the trace amount of canine distemper virus using the sensor. Two sensors are connected through a single-mode optical fiber. One is used as a sensing arm and immersed in a canine distemper virus antigen solution, and the other is used as a reference arm and immersed in a PBS buffer solution. Then, interference spectra at different concentrations of the canine distemper virus antigen solution are obtained respectively. The troughs in the same wavelength range are selected to obtain the corresponding spectral intensities. A linear fitting is used to obtain a formula for trace detection of canine distemper virus. Then, according to this formula, the concentration of an unknown concentration of canine distemper virus antigen solution can be detected.

Description

Technical Field

[0001] The present invention relates to the technical field of sensors, and particularly relates to a manufacturing method of a canine distemper virus trace detection sensor, the sensor, and a method for detecting canine distemper virus traces. Background Art

[0002] Canine distemper virus (CDV) is a highly lethal infectious virus pathogen, mainly existing in domestic canids, wild canids, and cruciferous animals. It can be transmitted through air, feces, and urine, with extremely strong infectivity, high incidence in the puppy stage, and a mortality rate that can be as high as over 80%. Relevant reports indicate that CDV can infect non-human primates, which also indicates the possibility of its development into a zoonosis. Currently, there is no effective treatment method for canine distemper virus, and prevention can only be carried out through early diagnosis and vaccination. Therefore, developing a CDV sensor with high sensitivity and fast response is of great significance for the early detection and targeted treatment of canine distemper virus.

[0003] Currently, there have been many related studies on the detection of canine distemper virus. The main detection methods include real-time reverse transcription polymerase chain reaction detection method, immunohistochemistry method, reverse transcription recombinase polymerase amplification method, double monoclonal antibody sandwich enzyme-linked immunosorbent assay, electrochemical detection method, and paper-based microfluidic immunoassay, etc. Although these methods can achieve highly sensitive and specific detection of canine distemper virus, there are still problems such as poor portability and the need for professional personnel to operate. Compared with the above detection methods, fiber optic sensors have the advantages of high sensitivity, small size, easy operation, and low cost. Over the years, biosensors have made rapid progress, such as the detection of antigens through fiber optic surface plasmon resonance (SPR).

[0004] Currently, some people use surface plasmon resonance (SPR) technology to adsorb PMMA polymer on a gold sensor to detect CDV virus, and its detection limit reaches 10 6 pg / ml, and the detection range is 0.004 - 0.2 ng / ml. In addition, some people have also used an SPR sensor labeled with gold nanoparticles to achieve the detection of CDV virus, and its detection limit reaches 700 pg / ml, and the detection range is 0 - 0.0015 ng / ml. However, these two surface plasmon resonance technologies require the modification of precious metals such as gold nanoparticles, which is relatively costly. In addition, the detection limits of such technologies are relatively high, which is not conducive to the trace detection of CDV virus. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned prior art, the technical problem to be solved by the present invention is: how to provide a manufacturing method of a canine distemper virus trace detection sensor that is easy to manufacture and has a low manufacturing cost.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for fabricating a trace detection sensor for canine distemper virus, comprising the following steps:

[0008] (1) Obtain a single-mode optical fiber and clean it thoroughly. Immerse the single-mode optical fiber in a sodium hydroxide solution for hydroxylation treatment to hydroxylate the surface of the single-mode optical fiber, obtaining a hydroxylated single-mode optical fiber. Then take it out, rinse it with deionized water, and dry it to a constant weight.

[0009] (2) Immerse the hydroxylated single-mode optical fiber in an APTES solution for silanization treatment to silanize the surface of the hydroxylated single-mode optical fiber, obtaining a silanized single-mode optical fiber. Then take it out, rinse it with deionized water, and dry it to a constant weight.

[0010] (3) Immerse the silanized single-mode optical fiber in a canine distemper virus antibody solution to modify the canine distemper virus antibody on the surface of the silanized single-mode optical fiber. Then take it out, rinse off the residual canine distemper virus antibody on the surface with a PBS buffer solution with a pH value of 7.2 - 7.4, and then immerse it in a skim milk powder blocking solution. Take it out and dry it to a constant weight. Cut the two ends of the optical fiber flat respectively to obtain a canine distemper virus antibody-modified optical fiber.

[0011] (4) Obtain two single-mode optical fibers, respectively perform misaligned fusion splicing with the two ends of the canine distemper virus antibody-modified optical fiber, so that the cores of the canine distemper virus antibody-modified optical fiber are respectively docked with the cladding and core of the single-mode optical fiber, thereby fabricating the sensor.

[0012] As an optimization, in step (1), prepare a sodium hydroxide solution with a concentration of 10 - 15% using absolute ethanol, immerse the single-mode optical fiber in the sodium hydroxide solution for 2 - 5 hours, and then dry it at a constant temperature of 40 - 60°C in a vacuum drying oven.

[0013] As an optimization, in step (2), prepare an APTES solution with a concentration of 10 - 15% using absolute ethanol, immerse the hydroxylated single-mode optical fiber in the APTES solution for 1 - 2 hours, and then dry it at a constant temperature of 40 - 60°C in a vacuum drying oven.

[0014] As an optimization, in step (3), dilute to obtain a canine distemper virus antibody solution with a concentration of 0.1 - 10000 pg / ml using a PBS buffer solution with a pH value of 7.2 - 7.4. Immerse the silanized single-mode optical fiber in the canine distemper virus antibody solution at a temperature of 30 - 37°C for 1 - 2 hours, and then immerse it in a skim milk powder blocking solution with a mass fraction of 10 - 15%.

[0015] As an optimization, in step (3), the length of the canine distemper virus antibody-modified optical fiber is 4.7 - 5.7 cm.

[0016] The present invention discloses a trace detection sensor for canine distemper virus, which is prepared by the manufacturing method of the trace detection sensor for canine distemper virus described above.

[0017] The present invention also discloses a trace detection method for canine distemper virus, comprising the following steps:

[0018] (1) Obtain the trace detection sensor for canine distemper virus described above. One end of the trace detection sensor for canine distemper virus is connected to a light source device, and the other end is connected to a spectrometer.

[0019] (2) Prepare multiple portions of canine distemper virus antigen solutions with different concentrations.

[0020] (3) Immerse the trace detection sensor for canine distemper virus into multiple portions of canine distemper virus antigen solutions with different concentrations respectively. Turn on the light source device to obtain the transmission spectra of the canine distemper virus antigen solutions with corresponding concentrations.

[0021] (4) According to the transmission spectra of the canine distemper virus antigen solutions with each concentration, select the same section of interference troughs among them, obtain the spectral intensity corresponding to this trough, and through linear fitting based on the spectral intensity, obtain y = a - bx, that is, x = (a - y) / b, where y is the spectral intensity corresponding to this interference trough, a is the correction coefficient, b is the gain coefficient, and x is the concentration of canine distemper virus antigen.

[0022] (5) Immerse the trace detection sensor for canine distemper virus into the canine distemper virus antigen solution to be detected. Turn on the light source device and obtain the transmission spectrum of this solution. Select the central wavelength of the corresponding trough to obtain the spectral intensity corresponding to this trough, and substitute it into the formula x = (a - y) / b to calculate the concentration of canine distemper virus antigen in this solution.

[0023] The present invention also discloses a trace detection method for canine distemper virus, comprising the following steps:

[0024] (1) Obtain two trace detection sensors for canine distemper virus described above. Connect the two trace detection sensors for canine distemper virus through a single-mode optical fiber. The other ends of the two trace detection sensors for canine distemper virus are respectively connected to a light source device and a spectrometer.

[0025] (2) Prepare multiple portions of canine distemper virus antigen solutions with different concentrations, and mix them with a PBS buffer solution with a pH value of 7.2 - 7.4.

[0026] (3) Take the canine distemper virus trace detection sensor near one end of the light source device as the reference arm, and the canine distemper virus trace detection sensor near one end of the spectrometer as the sensing arm. Immerse the reference arm in the PBS buffer solution, and immerse the sensing arm in one of the canine distemper virus antigen solutions. After turning on the light source device, record the transmission spectrum data on the spectrometer;

[0027] (4) Repeat step (3) to detect all the canine distemper virus antigen solutions and obtain their transmission spectra. Select the same interference wave valley segment among them, obtain the spectral intensity corresponding to this wave valley, and obtain y = a + bx through linear fitting according to the spectral intensity, that is, x = (y - a) / b, where y is the spectral intensity corresponding to this interference wave valley, a is the correction coefficient, b is the gain coefficient, and x is the canine distemper virus antigen concentration;

[0028] (5) Immerse the reference arm in the PBS buffer solution, and immerse the sensing arm in the canine distemper virus antigen solution to be detected. Turn on the light source device and obtain the transmission spectrum of this solution. Select the central wavelength of the corresponding wave valley, obtain the spectral intensity corresponding to this wave valley, and substitute it into the formula x = (y - a) / b to calculate the concentration of the canine distemper virus antigen in this solution.

[0029] In the present invention, the surface of the single-mode optical fiber is hydroxylated to form silanol groups on its surface; then silanization treatment is carried out so that the surface of the optical fiber carries amino groups of APTES; finally, the optical fiber probe is immersed in the canine distemper virus antibody, and the carboxyl group of the antibody and the amino group on the surface of the optical fiber undergo a dehydration condensation reaction, so that the canine distemper virus antibody is modified onto the surface of the optical fiber. Soaking the optical fiber with the skim milk blocking solution can block the redundant sites on the optical fiber and avoid non-specific binding.

[0030] The light emitted by the light source device undergoes core mismatch at the misalignment between the single-mode optical fiber and the canine distemper virus antibody-modified optical fiber. Part of the light enters the cladding of the misaligned canine distemper virus antibody-modified optical fiber, and the other part of the light continues to be transmitted in the core of the canine distemper virus antibody-modified optical fiber, and finally enters the spectrometer through the misaligned single-mode optical fiber. When the canine distemper virus antibody-modified optical fiber is immersed in the canine distemper virus antigen solution, the canine distemper virus antigen specifically binds to the antibody on the surface of the optical fiber, and the effective refractive index on the surface of the optical fiber changes, but the refractive index of the core remains unchanged. Therefore, with the change of the surface effective refractive index, the m-order interference wave valley will change, and the change amount can be expressed as:

[0031]

[0032] where Δn effis the difference between the effective refractive index of the optical fiber core and the effective refractive index of the cladding, Δn is the change in the refractive index difference, and L is the length of the optical fiber modified with canine distemper virus antibody. It can be seen from Equation (1) that when the effective refractive index of the fiber surface changes, the interference trough will shift. Therefore, the shift information of the interference trough can be observed by a spectrometer, and then the trace amount of canine distemper virus can be detected.

[0033] Compared with the prior art, the present invention has the following beneficial effects: The method for fabricating the sensor of the present invention is simple and easy, does not require the consumption of precious metals, has a low cost, and the fabricated sensor has a good effect on the detection of canine distemper virus antigen ears. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the transmission spectrum diagram of the corresponding troughs of the sensor in Example 1 of the present invention in canine distemper virus antigen solutions with concentrations of 0.1 pg / ml, 1 pg / ml, and 10 pg / ml;

[0035] Figure 2 is the transmission spectrum diagram of the corresponding troughs of the sensor in Example 1 of the present invention in canine distemper virus antigen solutions with concentrations of 100 pg / ml, 1 ng / ml, and 10 ng / ml;

[0036] Figure 3 is the linear fitting diagram of the interference trough near the trough of 1558 nm in the spectrum diagrams of the sensor in Example 1 of the present invention in canine distemper virus antigen solutions with different concentrations;

[0037] Figure 4 is the transmission spectrum diagram of the corresponding troughs of the sensing arm in Example 2 of the present invention in canine distemper virus antigen solutions with concentrations of 0.1 pg / ml, 1 pg / ml, and 10 pg / ml;

[0038] Figure 5 is the transmission spectrum diagram of the corresponding troughs of the sensing arm in Example 2 of the present invention in canine distemper virus antigen solutions with concentrations of 100 pg / ml, 1 ng / ml, and 10 ng / ml;

[0039] Figure 6 is the linear fitting diagram of the interference trough near the trough of 1542 nm in the spectrum diagrams of the sensing arm in Example 2 of the present invention in canine distemper virus antigen solutions with different concentrations;

[0040] Figure 7 is the transmission spectrum diagram of a single reference arm in phosphate buffer solution, a single sensor in canine distemper virus antigen solution, and the reference arm and the sensing arm after cascading, when the canine distemper virus antibody-modified optical fiber in Example 2 of the present invention is 2.7 cm;

[0041] Figure 8Transmission spectra of the canine distemper virus antibody - modified optical fiber in Example 2 of the present invention at 3.7 cm, for a single reference arm in phosphate - buffered solution, a single sensor in canine distemper virus antigen solution, and after cascading, the transmission spectra of the reference arm and the sensing arm;

[0042] Figure 9 Transmission spectra of the canine distemper virus antibody - modified optical fiber in Example 2 of the present invention at 4.7 cm, for a single reference arm in phosphate - buffered solution, a single sensor in canine distemper virus antigen solution, and after cascading, the transmission spectra of the reference arm and the sensing arm;

[0043] Figure 10 Transmission spectra of the canine distemper virus antibody - modified optical fiber in Example 2 of the present invention at 5.7 cm, for a single reference arm in phosphate - buffered solution, a single sensor in canine distemper virus antigen solution, and after cascading, the transmission spectra of the reference arm and the sensing arm;

[0044] Figure 11 Response - time test chart of the sensing arm in canine distemper virus antigen solution with a concentration of 1 pg / ml in Example 2 of the present invention;

[0045] Figure 12 Selectivity test chart of the sensing arm in different antigens in Example 2 of the present invention;

[0046] Figure 13 Chart of the change in the valley light intensity near 1550 nm of the sensing arm between 20 - 55 °C in Example 2 of the present invention;

[0047] Figure 14 Chart of the stability test of the sensing arm within 140 minutes in Example 2 of the present invention;

[0048] Figure 15 Chart of the change in light intensity caused by the sensing arm in positive and negative samples of canine distemper virus activity in Example 2 of the present invention. Specific Embodiment 1

[0050] The manufacturing method of the canine distemper virus trace - detection sensor in this specific embodiment includes the following steps:

[0051] (1) Obtain a single - mode optical fiber and clean it thoroughly. Immerse the single - mode optical fiber in a sodium hydroxide solution for hydroxylation treatment to make the surface of the single - mode optical fiber hydroxylated, obtaining a hydroxylated single - mode optical fiber. Then take it out, rinse it with deionized water, and dry it to a constant weight;

[0052] (2) Immerse the hydroxylated single - mode optical fiber in an APTES solution for silanization treatment to make the surface of the hydroxylated single - mode optical fiber silanized, obtaining a silanized single - mode optical fiber. Then take it out, rinse it with deionized water, and dry it to a constant weight;

[0053] (3) Immerse the silanized single-mode optical fiber in the canine distemper virus antibody solution to modify the canine distemper virus antibody on the surface of the silanized single-mode optical fiber. Then take it out and rinse off the residual canine distemper virus antibody on the surface with PBS buffer solution with a pH value of 7.4. Then immerse it in the skim milk powder blocking solution and take it out and dry it to a constant weight. Cut the two ends of the optical fiber flat respectively to obtain the canine distemper virus antibody-modified optical fiber;

[0054] (4) Obtain two single-mode optical fibers and perform misaligned fusion splicing with the two ends of the canine distemper virus antibody-modified optical fiber respectively, so that the cores of the canine distemper virus antibody-modified optical fiber are respectively butt-jointed with the cladding and core of the single-mode optical fiber, thereby manufacturing the sensor.

[0055] In this specific embodiment, in step (1), prepare a 10% sodium hydroxide solution with absolute ethanol and immerse the single-mode optical fiber in the sodium hydroxide solution for 2 hours, and then dry it at a constant temperature of 60 °C in a vacuum drying oven.

[0056] In this specific embodiment, in step (2), prepare a 10% APTES solution with absolute ethanol and immerse the hydroxylated single-mode optical fiber in the APTES solution for 1 hour, and then dry it at a constant temperature of 60 °C in a vacuum drying oven.

[0057] In this specific embodiment, in step (3), dilute with PBS buffer solution with a pH value of 7.4 to obtain a canine distemper virus antibody solution with a concentration of 100 ng / ml. Immerse the silanized single-mode optical fiber in the canine distemper virus antibody solution at a temperature of 37 °C for 1 hour, and then immerse it in the skim milk powder blocking solution with a mass fraction of 10%.

[0058] In this specific embodiment, the length of the canine distemper virus antibody-modified optical fiber in step (3) is 4.7 cm.

[0059] A canine distemper virus trace detection sensor is prepared by the manufacturing method of the canine distemper virus trace detection sensor described in any one of the above.

[0060] A method for trace detection of canine distemper virus includes the following steps:

[0061] (1) Obtain the canine distemper virus trace detection sensor described above. One end of the canine distemper virus trace detection sensor is connected to the light source device, and the other end is connected to the spectrometer;

[0062] (2) Prepare canine distemper virus antigen solutions with concentrations of 0.1 pg / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 1 ng / ml and 10 ng / ml;

[0063] (3) Immerse the trace detection sensor for canine distemper virus into the above-mentioned canine distemper virus antigen solutions with different concentrations respectively. Turn on the light source device to obtain the transmission spectra of the canine distemper virus antigen solutions with corresponding concentrations, as Figure 1 and Figure 2 shown;

[0064] (4) According to the transmission spectra of the canine distemper virus antigen solutions with various concentrations, select the interference trough with a trough wavelength near 1558 nm, obtain the spectral intensity corresponding to this trough, and through linear fitting based on the spectral intensity, get y = -33.23159 - 0.04283x, as Figure 3 shown, with the fitting coefficient R 2 = 0.98072, that is, x = (-33.23159 - y) / 0.04283;

[0065] (5) Immerse the trace detection sensor for canine distemper virus into the canine distemper virus antigen solution I to be detected. Turn on the light source device and obtain the transmission spectrum of this solution. Select the light intensity corresponding to the trough with a central wavelength near 1558 nm to be -33.294. Calculate the concentration of the canine distemper virus antigen in this solution to be 1.4586 pg / ml according to the formula;

[0066] Immerse the trace detection sensor for canine distemper virus into the canine distemper virus antigen solution II to be detected. Turn on the light source device and obtain the transmission spectrum of this solution. Select the light intensity corresponding to the trough with a central wavelength near 1558 nm to be -33.536. Calculate the concentration of the canine distemper virus antigen in this solution to be 7.123 pg / ml according to the formula; Specific Embodiment 2

[0068] A trace detection method for canine distemper virus, comprising the following steps:

[0069] (1) Obtain two of the above-mentioned trace detection sensors for canine distemper virus. Connect the two trace detection sensors for canine distemper virus through a single-mode optical fiber, and connect the other ends of the two trace detection sensors for canine distemper virus to a light source device and a spectrometer respectively;

[0070] (2) Canine distemper virus antigen solutions with concentrations of 0.1 pg / ml, 1 pg / ml, 10 pg / ml, 100 pg / ml, 1 ng / ml, and 10 ng / ml, in combination with a PBS buffer solution with a pH value of 7.4;

[0071] (3) Take the canine distemper virus trace detection sensor near one end of the light source device as the reference arm, and the canine distemper virus trace detection sensor near one end of the spectrometer as the sensing arm. Immerse the reference arm in the PBS buffer solution, and immerse the sensing arm in one of the canine distemper virus antigen solutions. After turning on the light source device, record the transmission spectrum data on the spectrometer;

[0072] (4) Repeat step (3) to detect all the canine distemper virus antigen solutions and obtain their transmission spectra. As Figure 4 and Figure 5 shown, select the interference trough near 1542 nm as the trough, obtain the spectral intensity corresponding to this trough, and obtain y = -47.15307 + 0.4786 through linear fitting according to the spectral intensity. As Figure 6 shown, the fitting coefficient R 2 = 0.98439, that is, x = (y + 47.15307) / 0.4786;

[0073] (5) Immerse the reference arm in the PBS buffer solution, and immerse the sensing arm in the canine distemper virus antigen solution III to be detected. Turn on the light source device and obtain the transmission spectrum of this solution. Select the light intensity corresponding to the trough near the central wavelength of 1542 nm as -46.54. According to the formula, calculate that the concentration of canine distemper virus antigen in this solution is 1.2789 pg / ml;

[0074] Immerse the reference arm in the PBS buffer solution, and immerse the sensing arm in the canine distemper virus antigen solution IV to be detected. Turn on the light source device and obtain the transmission spectrum of this solution. Select the light intensity corresponding to the trough near the central wavelength of 1542 nm as -40.127. According to the formula, calculate that the concentration of canine distemper virus antigen in this solution is 14.68 pg / ml.

[0075] The structure of the cascaded MZI sensor consists of two parts: the reference arm and the sensing arm. Both arms are of the Mach-Zehnder interference structure of single-mode - misaligned single-mode - single-mode. Among them, the reference arm is immersed in the phosphate buffer solution (PBS, pH = 7.4), and the sensing arm is immersed in the canine distemper virus antigen solutions with different concentrations. The free spectral ranges (FSRs) of these two arms are slightly different. The transmission spectra of a single reference arm in the phosphate buffer solution, a single sensor in the canine distemper virus antigen solution, and the cascaded reference arm and sensing arm are as Figures 7 to 10 shown. When the lengths of the optical fibers modified with canine distemper virus antibodies are 4.7 cm and 5.7 cm, there are more interference troughs, and the cascading effect is better, and the length is shorter, which is beneficial to the miniaturization of the optical fiber.

[0076] The vernier scale is a method to improve measurement accuracy based on the vernier effect. It consists of two scales with different precisions, where one slides along the other. The overlap between the lines on the two scales is used for measurement. The working principle of traditional sensors is mainly based on the spectral difference. The FSR difference between the sensing interferometer (FSR S ) and the reference interferometer (FSR R ) must be very small to adopt the vernier effect. As Figure 9 shown, the highest transmission peak appears at the position where the resonance peak of the sensing arm aligns with the resonance peak of the reference arm. The envelope function of the transmission peak is periodic and is called the FSR of the double-arm sensor, which is given by the following formula:

[0077]

[0078] where FSR R is the free spectral range of the reference arm, FSR S is the free spectral range of the sensing arm, and FSR C is the free spectral range of the envelope. Compared with the wavelength shift of the sensing interferometer, the shift of the cascaded sensor is amplified by a factor, and the amplification factor is defined as:

[0079]

[0080] Combining the response spectra of a single canine distemper virus trace detection sensor to canine distemper virus antigen solutions with different concentrations and the response spectra of the cascaded structure of two canine distemper virus trace detection sensors to canine distemper virus antigen solutions with different concentrations, as the concentration of the canine distemper virus antigen solution increases, the monitored valley interference intensity continuously increases. The maximum change in the spectral intensity of the cascaded MZI structure is 2.412 dB. Linear fitting is performed on the valley, and the linearity is 0.98439. The maximum change in the spectral intensity of a single MZI structure is 0.207 dB. Linear fitting is performed on the valley, and the linearity is 0.98072. By comparison, the vernier effect of the cascaded MZI is amplified by nearly 12 times.

[0081] The detection limit (LOD) formula of the cascaded MZI structure is expressed as follows:

[0082]

[0083] where K is the linear fitting slope (0.4786), and σ is the standard deviation of the slope (0.02691). According to the above formula, the detection limit of the sensor with the cascaded MZI structure is 0.1687 pg / ml.

[0084] The response time test was carried out when the concentration of the canine distemper virus antigen solution was 1 pg, and the response time was recorded every 3 minutes. To ensure the reliability of the experiment, the experiment was repeated three times, and the sensor response time was obtained as Figure 11As shown, it can be seen that the binding of antigen and antibody reacts relatively fast within the first 15 minutes, the reaction rate gradually slows down around 15 - 20 minutes, and tends to be stable after 25 minutes, indicating that the sites on the fiber optic surface are basically saturated at this time.

[0085] Prepare hemoglobin (HGB), avian influenza antigen (H9N2), abnormal prothrombin (DCP), bovine serum albumin (BSA), alpha - fetoprotein antigen (AFP), toxoplasma antigen (SAG1), ovalbumin (OVA), rabies antigen (Rabies) with equal concentrations to explore the selectivity of the sensor. Figure 12 The selectivity test results of the sensor show that the light intensity changes caused by other antigens are all less than 20% of the light intensity change caused by the canine distemper antigen with the same concentration, indicating that the sensor has excellent selectivity for the canine distemper antigen. Place the sensor in a constant temperature and humidity chamber, record it every 5 °C between 20 - 55 °C, and the change in the light intensity at the trough near 1550 nm of the sensor is as Figure 13 shown. The maximum change amount is 0.06 dB, and the temperature sensitivity is 0.0017 dB / °C. This value is much lower than the sensitivity to the canine distemper antigen, indicating that the sensor has good temperature stability. Figure 14 The time stability test results of the sensor show that the maximum spectral change within 140 minutes is 0.188 dB, which is negligible compared to the change amount of the canine distemper antigen measured by the cascaded MZI, indicating that the sensor has good time stability.

[0086] To verify the practicability of the sensor, clinical samples were detected for the sensor, as Figure 15 shown. Among them, numbers 1 - 3 are positive samples of canine distemper activity, and numbers 4 - 7 are negative samples of activity. It can be seen that the maximum value of the light intensity change caused by the positive samples is 1.039 dB, while the maximum value of the light intensity change caused by the negative samples is 0.154 dB, less than 20% of the light intensity change caused by the positive samples, and the detection of clinical samples of canine distemper antigen can be achieved.

[0087] The cascaded fiber optic MZI structure in this specific embodiment realizes the specific detection of canine distemper antigen by modifying the canine distemper antibody on the misaligned structure, successfully proving the successful modification of the canine distemper antibody and the specific binding of antigen and antibody. The test results show that the detection result of the sensor for the canine distemper antigen is about 12 times amplified compared with that of a single MZI sensor, the sensitivity is 0.4786 dB / log, the response time is 25 minutes, and the detection limit can reach 0.1687 pg / ml. And the sensor has the advantages of high sensitivity, good selectivity, low detection limit, etc., and has great significance and application prospects in the field of CDV specific detection.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Those of ordinary skill in the art should understand that any modifications or equivalent replacements made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for manufacturing a canine distemper virus trace detection sensor, characterized in that: It includes the following steps: (1) Obtain a single-mode optical fiber and clean it thoroughly. Immerse the single-mode optical fiber in a sodium hydroxide solution for hydroxylation treatment. Prepare a sodium hydroxide solution with a concentration of 10-15% using absolute ethanol, and immerse the single-mode optical fiber in the sodium hydroxide solution for 2-5 hours to hydroxylate the surface of the single-mode optical fiber, obtaining a hydroxylated single-mode optical fiber. Then take it out, rinse it with deionized water, and dry it to a constant weight at a constant temperature of 40-60°C in a vacuum drying oven; (2) Immerse the hydroxylated single-mode optical fiber in an APTES solution for silanization treatment. Prepare an APTES solution with a concentration of 10-15% using absolute ethanol, and immerse the hydroxylated single-mode optical fiber in the APTES solution for 1-2 hours to silanize the surface of the hydroxylated single-mode optical fiber, obtaining a silanized single-mode optical fiber. Then take it out, rinse it with deionized water, and dry it to a constant weight at a constant temperature of 40-60°C in a vacuum drying oven; (3) Dilute to obtain a canine distemper virus antibody solution with a concentration of 0.1-10000 pg / ml using a PBS buffer solution with a pH value of 7.2-7.

4. Immerse the silanized single-mode optical fiber in the canine distemper virus antibody solution at a temperature of 30-37°C for 1-2 hours to modify the canine distemper virus antibody on the surface of the silanized single-mode optical fiber. Then take it out, rinse off the residual canine distemper virus antibody on the surface using a PBS buffer solution with a pH value of 7.2-7.4, and then immerse it in a blocking solution of skim milk powder with a mass fraction of 10-15%. Take it out and dry it to a constant weight. After cutting the two ends of the optical fiber flat, a canine distemper virus antibody-modified optical fiber is obtained; (4) Obtain two single-mode optical fibers and perform misaligned fusion splicing with the two ends of the canine distemper virus antibody-modified optical fiber respectively, so that the cores of the canine distemper virus antibody-modified optical fiber are respectively butt-jointed with the cladding and core of the single-mode optical fiber, thereby manufacturing a sensor.

2. The manufacturing method of the canine distemper virus trace detection sensor according to claim 1, wherein: In step (3), the length of the canine distemper virus antibody-modified optical fiber is 4.7-5.7 cm.

3. A canine distemper virus trace detection sensor, characterized in that: It is prepared by the method for manufacturing a canine distemper virus trace detection sensor according to any one of claims 1 or 2.

4. A trace detection method for canine distemper virus, characterized in that: It includes the following steps: (1) Obtain the canine distemper virus trace detection sensor described in claim 3. One end of the canine distemper virus trace detection sensor is connected to a light source device, and the other end is connected to a spectrometer; (2) Prepare multiple portions of canine distemper virus antigen solutions with different concentrations; (3) Immerse the canine distemper virus trace detection sensor into multiple portions of canine distemper virus antigen solutions with different concentrations respectively. Turn on the light source device to obtain the transmission spectra of the canine distemper virus antigen solutions with corresponding concentrations; (4) According to the transmission spectra of the canine distemper virus antigen solutions with each concentration, select the same interference trough section among them, obtain the spectral intensity corresponding to this trough, and obtain y = a - bx through linear fitting according to the spectral intensity, that is, x = (a - y) / b, where y is the spectral intensity corresponding to this interference trough, a is the correction coefficient, b is the gain coefficient, and x is the canine distemper virus antigen concentration; (5) Immerse the canine distemper virus trace detection sensor into the canine distemper virus antigen solution to be detected, turn on the light source device and obtain the transmission spectrum of the solution. Select the central wavelength of the corresponding wave valley, obtain the spectral intensity corresponding to the wave valley, and substitute it into the formula x = (a - y) / b to calculate the concentration of canine distemper virus antigen in the solution.

5. A trace detection method for canine distemper virus, characterized in that: The method includes the following steps: (1) Obtain two canine distemper virus trace detection sensors described in claim 3. Connect the two canine distemper virus trace detection sensors through a single-mode optical fiber, and connect the other ends of the two canine distemper virus trace detection sensors to the light source device and the spectrometer respectively; (2) Configure multiple canine distemper virus antigen solutions with different concentrations, and mix them with PBS buffer solution with a pH value of 7.2 - 7.4; (3) Take the canine distemper virus trace detection sensor near the light source device as the reference arm, and the canine distemper virus trace detection sensor near the spectrometer as the sensing arm. Immerse the reference arm into the PBS buffer solution, and immerse the sensing arm into one of the canine distemper virus antigen solutions. After turning on the light source device, record the transmission spectrum data on the spectrometer; (4) Repeat step (3) to detect all the canine distemper virus antigen solutions and obtain their transmission spectra. Select the same interference wave valley segment among them, obtain the spectral intensity corresponding to the wave valley, and obtain y = a + bx through linear fitting according to the spectral intensity, that is, x = (y - a) / b, where y is the spectral intensity corresponding to the interference wave valley, a is the correction coefficient, b is the gain coefficient, and x is the concentration of canine distemper virus antigen; (5) Immerse the reference arm into the PBS buffer solution, and immerse the sensing arm into the canine distemper virus antigen solution to be detected. Turn on the light source device and obtain the transmission spectrum of the solution. Select the central wavelength of the corresponding wave valley, obtain the spectral intensity corresponding to the wave valley, and substitute it into the formula x = (y - a) / b to calculate the concentration of canine distemper virus antigen in the solution.

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