A multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding and its supporting equipment

Abstract

The present invention discloses a multi-index, linear range-adjustable optical fiber chemiluminescence biosensor and supporting equipment based on the embedding of a polymer sleeve. By embedding a polymer sleeve on an optical fiber, the biorecognition molecule is fixed on the sleeve by physical adsorption, thereby preparing an optical fiber probe modified with a specific biorecognition molecule. By modifying different biorecognition molecules on sleeves of different sizes and assembling them into a string, it is used as a carrier for multi-index detection, thereby realizing the simultaneous detection of multiple targets in the same sample. The present invention simplifies the production process of the optical fiber chemiluminescence probe, avoids the use of chemical reagents when coupling biorecognition molecules to the optical fiber surface, and can achieve adjustable linear range and simultaneous detection of multiple targets through controllable assembly and series combination of sleeves, thereby reducing the detection cost, and can realize on-site rapid detection in combination with portable detection equipment.

Description

Technical Field

[0001] The invention belongs to the field of biosensing, and relates to a multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding and its supporting equipment. Background Art

[0002] With the rapid economic development and the improvement of people's living standards, the demand for accurate, sensitive and rapid detection of hazardous factors in food and environment and biomarkers in in vitro diagnosis has increased. Therefore, it is of great practical significance to develop low-cost, excellent performance and easy-to-operate on-site rapid detection equipment.

[0003] At present, the main detection methods for hazard factors and biomarkers are instrumental analysis, enzyme-linked immunosorbent assay (ELISA) and biosensor. Traditional instrumental analysis has high detection sensitivity, but requires expensive large-scale precision instruments, complex pre-treatment steps and professional operators. ELISA has high sensitivity and low cost, but the linear range is narrow, and the clapping step required in the washing process will cause experimental errors due to different operations of the experimenters. Biosensor is a new detection method with the advantages of fast detection speed, high sensitivity and simple operation. It has been widely used in environmental monitoring, in vitro diagnosis, rapid food safety detection and other fields.

[0004] Biosensors are devices that convert biological signals into other quantitatively detectable optical, electromagnetic and other signals, including biorecognition systems and signal conversion systems. When biorecognition molecules (antibodies, DNA molecules or enzymes, etc.) interact with the target, the signal conversion system can convert changes in biological information into measurable changes in detection signals, such as charge, heat, mass and pH changes, and then present them in intuitive ways such as numbers or pictures. Chemiluminescent sensors are analytical devices that determine the concentration of a target based on the intensity of chemiluminescence. They have both the high sensitivity of chemiluminescence and the high specificity of biorecognition reactions. They are particularly suitable for quantitative analysis of trace targets in complex samples. Compared with other optical biosensors, they do not require components such as excitation light sources and spectrometers, effectively reducing the complexity and cost of the instrument.

[0005] Optical fiber can conduct light through the principle of total reflection. The optical fiber chemiluminescence sensor that combines optical fiber with chemiluminescence not only has biological recognition, separation and enrichment, and signal transmission in one, but also has a simpler washing process, which can simplify the operation and make the equipment portable. In addition, the anti-interference ability of optical fiber is extremely strong, which is particularly suitable for field operation. The current optical fiber biosensor production optical fiber probe must first be processed by physical stripping and chemical corrosion, and then the biorecognition molecules are fixed on the optical fiber surface by chemical bonds using silicon chemistry methods. The process is cumbersome and time-consuming and requires chemical reagents. In addition, the surface area of ​​the optical fiber core is small (the diameter is usually several hundred microns), resulting in a small amount of biorecognition molecules such as antigens / antibodies or enzymes fixed on its surface, which limits the improvement of sensitivity and the expansion of the linear range. In the early stage, scholars have developed a fiber optic immunosensor (CN 109596604A) that regulates signal intensity and detection linear range by changing the length of the optical fiber sensing response core and the number of optical fibers, which can be used for the detection of veterinary drug residues. However, the method is cumbersome and time-consuming to modify the complete antigen step, and multiple optical fibers are required to adjust the linear range. Therefore, how to simplify the production process of optical fiber probes while ensuring the adjustable linear range is of great significance for rapid on-site detection. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of cumbersome and time-consuming probe preparation process of optical fiber biosensors, the need to use chemical reagents, and high cost, and to provide an automated detection method and portable detection equipment with low cost, simple operation, adjustable linear range, multiplex detection, high sensitivity and automation, using an optical fiber embedded in a polymer sleeve as a solid phase carrier and chemiluminescence as a detection substrate.

[0007] The technical solution of the present invention is as follows:

[0008] Polystyrene (PS) refers to a polymer synthesized from styrene monomers by free radical polymerization. It is a colorless and transparent thermoplastic. PS can be used as a solid phase carrier to adsorb biorecognition molecules, such as antibodies and other protein molecules. This is because protein molecules can bind to the PS surface through the interaction of hydrophobic groups, avoiding the disadvantages of chemical coupling methods such as the need for chemical reagents and multi-step operations.

[0009] A multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding, wherein the optical fiber is embedded with one or more polymer sleeves of different sizes, and biorecognition molecules are modified on the sleeves to prepare an optical fiber probe modified with biorecognition molecules.

[0010] Preferably, the optical fiber is a quartz optical fiber.

[0011] The polymer sleeve has a spherical, conical, cylindrical or other shape.

[0012] The polymer sleeve is made of polystyrene (PS), polyacrylamide (PAM), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET) or others;

[0013] The biorecognition molecule is a capture antibody or a detection antibody or an antibody or a complete antigen that can generate a competitive immune reaction or a double antibody sandwich immune reaction with the target to be detected.

[0014] The method for preparing the optical fiber chemiluminescent biosensor with multiple indexes and adjustable linear range based on polymer sleeve embedding comprises the following steps:

[0015] 1) Insert the optical fiber into the polystyrene sleeve for assembly;

[0016] 2) placing the optical fiber probe assembled in step 1) incubated in the biorecognition molecule and washing with PBST solution;

[0017] 3) The optical fiber probe treated in step 2) is placed in bovine serum albumin for sealing operation to obtain an optical fiber probe assembled with a polymer cladding.

[0018] Preferably, in step 1), the fitting method is interference fit;

[0019] Preferably, in step 2), the incubation is carried out at 37° C. for 1.5-2.5 hours, and in the PBST solution, the molar concentration of PBS is 0.005-0.015 M, and the mass fraction of Tween is 0.03-0.08%;

[0020] Preferably, in step 3), the mass fraction of bovine serum albumin is 3-7%.

[0021] An automated detection device based on a multi-index optical fiber chemiluminescent biosensor with adjustable linear range embedded in a polymer sleeve. The structure of the automated detection device is as follows:

[0022] The optical fiber probe is embedded in the polymer sleeve, and the optical fiber probe is connected to the optical fiber bracket. The optical fiber bracket, photon counter and control system components are also arranged on the bracket. A motor bracket and a movable guide rail are installed under the bracket. The motor is installed on the motor bracket, and the motor shaft is connected to the ball screw. One end of the mobile platform is fixedly connected to the sealing box, and the other end is connected to the ball screw.

[0023] Preferably, two guide wheels are respectively installed at the left and right ends of the movable platform, and the guide wheels are embedded in the movable grooves on both sides of the guide rail.

[0024] Preferably, the sealing box is made of black polyethylene, polyvinyl chloride, polystyrene or others.

[0025] When using the automated testing equipment for testing, the control system component controls the sealing box to move forward to open the sealing box, loads the optical fiber probe embedded in the polymer sleeve into the optical fiber holder, and then loads the centrifuge tube containing the reaction reagent into the test tube holder in the sealing box. When the measurement starts, the sealing box moves backward along the guide rail to close to form a closed space, and the optical fiber probe is extended into the reaction reagent for measurement. After the measurement is completed, the measurement result is processed by the control system and sent to the mobile phone.

[0026] The multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding or the preparation method of the multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding or the automated detection equipment of the multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding is used for detecting target objects by chemiluminescence method or fluorescent immunoassay method or enzyme-linked immunosorbent assay, and the target objects include fungal toxins, pathogenic microorganisms, antibiotics, pesticides and veterinary drugs, disease markers or others.

[0027] Preferably, when used for direct chemiluminescence method, the following steps are specifically included:

[0028] SB1: embedding optical fibers into polymer sleeves of different sizes, coupling different biorecognition molecules a on the surfaces of polymer sleeves of different sizes to obtain polymer sleeve-embedded optical fiber probes;

[0029] SB2: Insert the optical fiber probe into a mixed solution of the object to be tested and the biorecognition molecule b for biological reaction. After the reaction, wash the optical fiber probe to remove the non-specifically adsorbed biorecognition molecule b, and match the biorecognition molecule a with the biorecognition molecule b. The biorecognition molecule a and the biorecognition molecule b are capture antibody and detection antibody labeled with chemiluminescent substrate, detection antibody and capture antibody labeled with chemiluminescent substrate, antibody and antigen labeled with chemiluminescent substrate, or antigen and antibody labeled with chemiluminescent substrate.

[0030] SB3: Place the washed fiber optic probe into the fiber optic holder, and then place the centrifuge tube containing the chemiluminescent substrate into the test tube holder in the sealed box. When the measurement starts, the sealed box moves backward along the guide rail to close to form a closed space. The fiber optic probe is inserted into the chemiluminescent substrate for measurement. After the measurement is completed, the measurement result is processed by the control system and sent to the mobile phone. The measurement is completed.

[0031] Further preferably, in step SB2, the chemiluminescent substrate is acridinium ester or isoluminol, but is not limited to the above types.

[0032] Further preferably, in step SB3, the inducer is hydrogen peroxide or sodium hydroxide, but is not limited to the above types.

[0033] When used for enzymatic chemiluminescence method, the method specifically comprises the following steps:

[0034] SC1: embedding optical fibers into polymer sleeves of different sizes, coupling different biorecognition molecules a on the surfaces of polymer sleeves of different sizes to obtain polymer sleeve-embedded optical fiber probes;

[0035] SC2: inserting the optical fiber probe into the object to be tested to perform a biological reaction, and washing the optical fiber probe after the reaction;

[0036] SC3: The washed optical fiber probe is inserted into the biorecognition molecule b for biological reaction, and the optical fiber probe is washed after the reaction to remove the non-specifically adsorbed biorecognition molecule b, and the biorecognition molecule a is matched with the biorecognition molecule b; the biorecognition molecule a and the biorecognition molecule b are capture antibody and detection antibody, detection antibody and capture antibody, antibody and antigen, or antigen and antibody;

[0037] SC4: Place the washed fiber optic probe into the fiber optic holder, and then place the centrifuge tube containing the chemiluminescent substrate into the test tube holder in the sealed box. When the measurement starts, the sealed box moves backward along the guide rail to close to form a closed space. The fiber optic probe is inserted into the chemiluminescent substrate for measurement. After the measurement is completed, the measurement result is processed by the control system and sent to the mobile phone. The measurement is completed.

[0038] Further preferably, in step SC2, the biological reaction time is 30 minutes.

[0039] Further preferably, in step SC3, the biorecognition molecule b is an HRP enzyme-labeled antibody, the biological reaction time is 30 minutes, and the chemiluminescent substrate is luminol or acridinium ester, but is not limited to the above types.

[0040] Further preferably, in step SC3, the biorecognition molecule b may also be an ALP enzyme-labeled antibody, and the chemiluminescent substrate may be AMPPD or APS-5, but is not limited to the above types.

[0041] The optical fiber chemiluminescence biosensor with multiple indices and adjustable linear range based on polymer sleeve embedding can be used for multiplex detection. Different biorecognition molecules are modified by polymer sleeves of different sizes and connected in series. After the sleeve bundle is inserted into the sample, different biorecognition molecules specifically bind to the corresponding targets. According to the different sizes of the polymer sleeves, different targets to be detected can be easily distinguished by the naked eye, thereby realizing multi-target detection.

[0042] In order to achieve the purpose of rapid on-site detection, the present invention provides a multi-index, linear range-adjustable optical fiber chemiluminescence biosensor portable detection device based on polymer sleeve embedding. During the detection process, it is only necessary to prepare the relevant corresponding reagents and consumables for pre-packaging. After the reaction is completed, the optical fiber probe and the chemiluminescence substrate are placed on the corresponding detection device carrier platform. The device can read and analyze the chemiluminescence signal and send the result to the mobile phone, which can meet the on-site detection of different objects.

[0043] Compared with traditional optical fiber biosensors, the present invention can simplify the preparation process of optical fiber probes by embedding polymer sleeves, shorten the preparation time, avoid the use of chemical reagents, and only need to replace the polymer sleeves after detection, without replacing the optical fiber, thereby realizing ultra-low-cost detection; secondly, polymer sleeves of different sizes can not only adsorb different numbers of biorecognition molecules, thereby detecting targets in different detection concentration ranges and realizing adjustable linear range, but also can distinguish different sizes of sleeves by naked eyes to identify different analytes, thereby realizing simultaneous detection of multiple targets; thirdly, the chemiluminescence induced by the target during the detection process can be efficiently collected and transmitted by the optical fiber through the refraction and total reflection of the polymer sleeve; finally, the optical fiber as a solid phase carrier of the reaction can avoid the step of approving in the traditional ELISA washing process, making the operation simple and the degree of automation high. The method has good analytical performance and has good application prospects in the fields of food safety, environmental monitoring, in vitro diagnosis, etc.

[0044] The beneficial effects of the present invention are as follows:

[0045] 1) Low cost, easy operation, and good stability: The present invention avoids the use of chemical reagents in the process of making optical fiber probes, is easy to operate, has good stability, and has low cost for polymer sleeves, which is suitable for mass production;

[0046] 2) Adjustable linear range: Polymer sleeves of different sizes can adsorb different numbers of biorecognition molecules, thereby detecting targets in different detection concentration ranges and achieving adjustable linear range;

[0047] 3) Simultaneous detection of multiple targets: polymer sleeves of different sizes are assembled into strings after adsorbing different biorecognition molecules. After the immune reaction is over, the size of the sleeve can be identified by the naked eye to determine the target to be detected, thus achieving easy-to-operate simultaneous detection of multiple targets;

[0048] 4) High sensitivity: The polymer sleeve can increase the surface area of ​​the optical fiber probe and bind more biorecognition molecules. The sleeve and the antibody are combined through physical adsorption, which can better maintain the activity of the antibody, thereby more effectively reacting with the analyte. Finally, the refraction and total reflection of the light by the sleeve can achieve efficient collection of chemiluminescent signals.

[0049] 5) Portable detection equipment: The detection equipment is lightweight and portable, integrating chemiluminescence signal collection and result analysis, and can realize rapid on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : Overall structure diagram of portable detection equipment, where Figure 1 a is a stereogram of the overall structure of the portable testing equipment; Figure 1 b is a top view of the overall structure of the portable detection equipment; Figure 1 c is a detailed diagram of the fiber optic probe in the portable detection device, including: polymer sleeve 01, fiber optic probe 02, fiber optic bracket 03, photon counter 04, control system component 05, bracket 06, sealing box 07, moving platform 08, moving guide rail 09, ball screw 10, guide wheel 11, moving groove 12, motor bracket 13, motor 14.

[0051] Figure 2 : Overall structural diagram of the portable multi-channel detection equipment.

[0052] Figure 3 : Schematic diagram of the assembly of the polystyrene sleeve and the optical fiber on the positioning base.

[0053] Figure 4 : Schematic diagram of the detection of vomitoxin based on a multi-index fiber-optic chemiluminescent biosensor with adjustable linear range embedded in a polymer sleeve.

[0054] Figure 5 : Optimization of DON complete antigen concentration.

[0055] Figure 6 : Optimization of isoluminol-labeled DON antibody concentration.

[0056] Figure 7 : Standard curve of detection of vomitoxin based on a multi-index fiber optic chemiluminescence biosensor with adjustable linear range embedded in a polymer sleeve.

[0057] Figure 8 : Schematic diagram of the multi-index, linear range-adjustable fiber optic chemiluminescent biosensor based on polymer sleeve embedding for the detection of procalcitonin.

[0058] Fig. 9 : Comparison of standard curves for the detection of procalcitonin based on a multi-index fiber optic chemiluminescence biosensor with adjustable linear range embedded in a polymer sleeve and a chemically modified fiber optic chemiluminescence immunoassay.

[0059] Fig.10 : Schematic diagram of the multi-index fiber optic chemiluminescence biosensor with adjustable linear range embedded in a polymer sleeve for simultaneous detection of three inflammatory markers.

[0060] Fig.11 : Standard curves for simultaneous detection of three inflammatory markers based on a multi-index fiber optic chemiluminescence biosensor with adjustable linear range embedded in a polymer sleeve.

[0061] Fig.12 : Comparison of the stability of the multi-index, linear range-adjustable fiber optic chemiluminescence biosensor embedded in a polymer sleeve and the chemically modified fiber optic chemiluminescence immunoassay for detecting procalcitonin. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below by specific examples. It should be understood by those skilled in the art that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0063] Test materials

[0064] 3-Aminopropylmethyldiethoxysilane (3-ADMS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich.

[0065] Deoxynivalenol (DON) antibody (3 mg / mL), deoxynivalenol complete antigen (DON-BSA) (10 mg / mL) were purchased from Shandong Landu Biotechnology Co., Ltd.

[0066] DON standard: purchased from J&K Technology Co., Ltd.

[0067] C-reactive protein (CRP), CRP capture antibody (CRP-Ab1), CRP enzyme-labeled antibody, procalcitonin (PCT), PCT capture antibody (PCT-Ab1), PCT enzyme-labeled antibody, interleukin 6 (IL6), IL6 capture antibody (IL6-Ab1), IL6 enzyme-labeled antibody: purchased from Abcam.

[0068] Enzyme-labeled goat anti-mouse IgG: purchased from Shanghai Biotime Biotechnology Co., Ltd.

[0069] Photon counter and photon counting unit: purchased from Beijing Hamamatsu Photon Counting Co., Ltd.

[0070] Quartz optical fiber (SFS400 / 440 / 700T Superguide UV-Vis): purchased from Shanghai Wenyi Optoelectronics Technology Co., Ltd.

[0071] Example 1 Construction of a multi-index, linear range-adjustable optical fiber chemiluminescence biosensor based on polymer sleeve embedding

[0072] Depending on the target to be detected, the optical fiber was assembled with spherical polystyrene sleeves of different radii (vomitoxin: sleeve radius 1.5 mm, C-reactive protein: sleeve radius 1.5 mm, procalcitonin: sleeve radius 2.5 mm, interleukin 6: sleeve radius 3.5 mm), and the sleeve was immersed in the corresponding biorecognition molecules (10 μg / mL DON-BSA, 10 μg / mL CRP-Ab1, 20 μg / mL PCT-Ab1, 40 μg / mL IL6-Ab1) at 4°C overnight, and the unbound biorecognition molecules were washed away with PBS; the remaining active sites were blocked with 5% bovine serum albumin solution for 2 hours and then stored at 4°C for later use.

[0073] Example 2 Construction of portable automated detection device

[0074] The invention provides a portable automatic detection device.

[0075] See also Figure 1 As shown, an automated detection device based on a multi-index optical fiber chemiluminescent biosensor with adjustable linear range embedded in a polymer sleeve, the structure of the automated detection device is:

[0076] The optical fiber probe 02 is embedded in the polymer sleeve 01, the biorecognition molecule is modified on the polymer sleeve 01, the optical fiber probe 02 is connected to the optical fiber bracket 03, the optical fiber bracket 03, the photon counter 04 and the control system component 05 are also arranged on the bracket 06, and the motor bracket 13 and the movable guide rail 09 are installed under the bracket 06. The motor 14 is installed on the motor bracket 13, and the motor shaft is connected to the ball screw 10. One end of the mobile platform 08 is fixedly connected to the sealing box 07, and the other end is connected to the ball screw 10.

[0077] Preferably, two guide wheels 11 are respectively installed at the left and right ends of the movable platform 08 , and the guide wheels 11 are embedded in the movable grooves 12 on both sides of the guide rail 09 .

[0078] Preferably, the sealing box is made of black polyethylene, polyvinyl chloride, polystyrene or others.

[0079] When using the automated detection equipment for detection, the control system component 05 controls the sealing box 07 to move forward to open the sealing box 07, installs the optical fiber probe 02 embedded in the above-mentioned polymer sleeve 01 into the optical fiber bracket 03, and then installs the centrifuge tube containing the reaction reagent into the test tube bracket in the sealing box. When the measurement starts, the sealing box 07 moves backward along the guide rail 09 to close to form a closed space, and the optical fiber probe 01 extends into the reaction reagent for measurement. After the measurement is completed, the measurement result is processed by the control system and sent to the mobile phone.

[0080] The control part mainly includes a signal processing module, a signal sending module, and a control module. The signal processing module processes the photoelectric signal collected by the photon counter, and sends the processed detection result to the mobile end through the signal sending module. The control module controls the forward and reverse rotation of the motor through the internal relay, thereby controlling the sealed box to move along the track.

[0081] See also Figure 2 As shown, when the automated detection device is multi-channel, it is only necessary to couple the corresponding biorecognition molecules to the polymer sleeve on each optical fiber, and each group of biorecognition molecules corresponds to the target to be detected, so that multiple groups of targets can be measured simultaneously.

[0082] Example 3 Direct chemiluminescence detection of vomitoxin

[0083] like Figure 3 As shown, a spherical polystyrene sleeve with a radius of 1.5 mm and a through hole with a diameter of 395 μm is assembled with an optical fiber on a base. The diameter of the optical fiber core is 400 μm. The biorecognition molecules can be fixed on the polystyrene sleeve through the interaction of hydrophobic groups.

[0084] Taking the fiber optic biosensor fixed biorecognition molecule a as DON-BSA complete antigen and biorecognition molecule b as isoluminol labeled DON antibody (ABEI-DON-Ab) as an example, the detection principle is explained. Figure 4 As shown, first, a polystyrene sleeve is assembled with an optical fiber, and the complete antigen of vomitoxin and BSA are adsorbed on the sleeve respectively to prepare a polystyrene sleeve embedded optical fiber probe for detecting vomitoxin. Then the optical fiber probe is reacted with a mixed solution of the sample to be tested and ABEI-DON-Ab, and the DON-BSA on the probe competes with the DON in the sample to be tested for the ABEI-DON-Ab in the solution, and the ABEI-DON-Ab bound to the optical fiber probe is related to the concentration of DON in the sample to be tested. Using the device of Example 2, the optical fiber probe is inserted into hydrogen peroxide, and the hydrogen peroxide reacts with isoluminol to generate a chemiluminescent signal, and the signal intensity is related to the concentration of DON in the sample to be tested.

[0085] The specific process is as follows:

[0086] 1. Preparation of fiber optic probe

[0087] 1) Assemble a spherical polystyrene sleeve with a radius of 1.5 mm and a through hole with a diameter of 395 μm with an optical fiber, the core diameter of the optical fiber is 400 μm, and then insert the optical fiber into 10 μg / mL DON-BSA, incubate at 37°C for 2 hours, and then wash with PBST;

[0088] 2) The optical fiber was inserted into a BSA solution (5%, w / v) at 37°C to block nonspecific sites for 1 hour, and then washed with PBST and stored at 4°C in the dark.

[0089] At this point, the fiber optic probe embedded in the polystyrene sleeve has been prepared.

[0090] 2. Optimization of DON-BSA concentration

[0091] The concentrations of DON-BSA used in preparing the sensor were 1, 2, 5, 10, and 20 μg / mL, the concentration of ABEI-DON-Ab was 5 μg / mL, the concentration of DON was 0, 1, 10, 100, 1000, and 10000 ng / mL, and the competitive immune reaction time was 30 minutes. Figure 5 It can be seen that there are obvious differences in the sensitivity of sensors prepared with complete antigens of different concentrations to target detection, among which the best effect is achieved when the DON-BSA concentration is 10 μg / mL.

[0092] 3. Optimization of ABEI-DON-Ab concentration

[0093] The concentrations of DON-BSA used in preparing the sensor were 10 μg / mL, the concentrations of ABEI-DON-Ab were 0.5, 1, 2, 5, and 10 μg / mL, the concentrations of DON were 0, 1, 10, 100, 1000, and 10000 ng / mL, and the competitive immune reaction time was 30 minutes. Figure 6 It can be seen that there are obvious differences in the sensitivity of target detection with different concentrations of labeled antibodies, and the best effect is achieved when the ABEI-DON-Ab concentration is 5 μg / mL.

[0094] 4. Establishment of DON standard curve

[0095] Under the above conditions, a standard curve was established:

[0096] 1) The prepared optical fiber probe was inserted into a mixed solution of 15 μL 5 μg / mL ABEI-DON-Ab and 15 μL DON of different concentrations, reacted at 37°C for 30 min, and washed 3 times with PBST;

[0097] 2) inserting the optical fiber probe into a portable detection device and placing it into a hydrogen peroxide solution to collect and record data of the chemiluminescent signal;

[0098] 3) A standard curve was drawn with the logarithm of DON concentration as the abscissa and the difference in chemiluminescence intensity between the blank sample and samples with different concentrations of DON as the ordinate.

[0099] like Figure 7As shown in the figure, the greater the DON concentration, the greater the difference in chemiluminescence intensity. 4 ng / mL range, there is a good linear relationship between the difference in chemiluminescence intensity between the blank sample and the samples with different concentrations of DON and the logarithm of the DON concentration. The regression equation is y=1682.76x+4042.1, and the correlation coefficient is R 2 is 0.99.

[0100] 3. Detection of DON in wheat

[0101] The standard addition method was used to detect DON in wheat samples. The results are shown in Table 1. The recoveries were all in the range of 80.74% to 102.36%, and the relative standard deviations were all lower than 10.34%, indicating that this method has extremely high accuracy and precision and can be used for the detection of DON in cereals.

[0102] Table 1 Detection results of DON in wheat

[0103]

[0104] Example 4 Enzymatic chemiluminescence detection of procalcitonin

[0105] When the enzymatic chemiluminescence method is used, the detection principle is explained by taking the biorecognition molecule a fixed on the optical fiber biosensor as PCT capture antibody (PCT-Ab1) and the biorecognition molecule b as PCT enzyme-labeled antibody as an example. Figure 8 As shown, first, a spherical polystyrene sleeve with a radius of 2.5 mm and a through hole with a diameter of 395 μm is assembled with an optical fiber, and the diameter of the optical fiber core is 400 μm. PCT-Ab1 and BSA are adsorbed on the sleeve respectively to prepare a polystyrene sleeve embedded optical fiber probe for detecting PCT. Then the optical fiber probe reacts with the sample to be tested, and the PCT-Ab1 on the probe reacts with the PCT in the sample to be tested. The PCT bound to the optical fiber probe is related to the concentration of PCT in the sample to be tested. Then the optical fiber probe reacts with the PCT enzyme-labeled antibody, and the PCT enzyme-labeled antibody forms an immune complex with the PCT captured on the probe. The enzyme in the complex can catalyze hydrogen peroxide to produce an oxidative product, which reacts with luminol to produce a chemiluminescent signal, and the signal intensity is related to the concentration of PCT in the sample to be tested. In this example, the preparation conditions and detection conditions of the probe were optimized according to the method of Example 3 before detection. The optimal concentration of PCT-Ab1 was 20 μg / mL, and the optimal concentration of PCT enzyme-labeled antibody was 10 μg / mL. The logarithm of PCT concentration was used as the abscissa and the chemiluminescence intensity was used as the ordinate to draw a standard curve, and compared with the chemically modified optical fiber chemiluminescence immunoassay, as shown in FIG. Fig. 9 shown.

[0106] The PCT residues in human serum samples were detected by the standard addition method. The test results are shown in Table 2. The recovery rates were between 88.79% and 108.36%, and the relative standard deviations were less than 10.51%. The above results show that this method has high accuracy and precision and can be used for the quantitative analysis of PCT in human serum samples.

[0107] Table 2 Detection results of PCT in human serum

[0108]

[0109] Example 5 Fiber-optic chemiluminescent biosensor with adjustable linear range for simultaneous detection of three inflammatory markers

[0110] In order to verify that this method can be applied to the detection concentration range requirements of different subjects, three inflammatory markers CRP, PCT and IL6 in human serum were detected simultaneously. Fig.10 As shown, first, capture antibodies of CRP, PCT, and IL6 are modified on spherical polystyrene sleeves with radii of 1.5 mm, 2.5 mm, and 3.5 mm and through holes with a diameter of 395 μm, respectively, and the fiber core diameter is 400 μm, and then they are connected in series. The capture antibody modified on the polystyrene sleeve reacts with the target in the sample to form an immune complex, and the enzyme-labeled antibody can bind to the captured target to form an immune complex. After the immune reaction is completed, the three sleeves of different sizes in series are disassembled, and each sleeve is placed on a positioning base and assembled with a single optical fiber. The HRP enzyme in the immune complex on the sleeve can catalyze the chemiluminescent substrate to produce a chemiluminescent signal. The more targets in the sample, the more immune complexes are formed, the more HRP enzymes are bound, and the stronger the chemiluminescent signal is generated. Therefore, the concentration of the target in the sample is positively correlated with the chemiluminescence intensity, thereby determining the target content in the sample.

[0111] The specific process is as follows:

[0112] 1) The prepared optical fiber probe was inserted into different concentrations of CRP, PCT, and IL6, reacted at 37°C for 30 minutes, and washed 3 times with PBST;

[0113] 2) Insert the fiber optic probe into the enzyme-labeled antibody, react at 37°C for 30 min, and wash three times with PBST;

[0114] 3) The three sleeves were disassembled and assembled on a single optical fiber on a positioning base, respectively, and then inserted into a portable detection device and a newly prepared chemiluminescent substrate solution (50 μL of luminol and hydrogen peroxide were taken and mixed immediately) was added to collect and record the data of the chemiluminescent signal;

[0115] A standard curve was drawn with the logarithm of the inflammatory marker concentration as the horizontal axis and the chemiluminescence intensity as the vertical axis. Fig.11 As shown;

[0116] The standard addition method was used to simultaneously detect the three inflammatory marker residues in serum samples with different detection concentration ranges. The process was the same as above. The results are shown in Table 3. The recovery rates were between 87.51% and 112.37%, and the relative standard deviations were less than 10.58%. The above results show that this method has high accuracy and precision and can be used for the quantitative analysis of IL6 in human serum samples.

[0117] Table 3 Detection results of three inflammatory markers in human serum

[0118]

[0119] Example 6 Storage stability verification

[0120] Traditional fiber optic chemiluminescence immunoassay requires the use of silicon chemistry to activate and change the groups on the surface of the optical fiber to couple biomolecules. This process uses chemical reagents such as hydrofluoric acid and sodium hydroxide, and the operation is complicated, resulting in poor stability of biomolecule coupling. The present invention uses the hydrophobic bonds and ionic bonds on the surface of the polystyrene sleeve to directly modify the biomolecules on the cladding surface, avoiding the use of chemical reagents, simplifying the biomolecule coupling steps, and at the same time, greatly improving the activity and stability of the biomolecules after coupling.

[0121] The PCT optical fiber probes made by chemical modification and polystyrene sleeve modification were stored for different time periods to compare their storage stability. The specific process is as follows:

[0122] Preparation of chemically modified fiber optic probes:

[0123] 1) Use a knife to gently scrape off 0.5 cm of the acrylic protective layer at the end of the optical fiber;

[0124] 2) The part where the acrylic protective layer was removed was etched with 24% hydrofluoric acid for 20 minutes to remove the silicon dioxide coating layer, and then washed with 0.1 mol / L NaOH and water for 3 times respectively, and dried with nitrogen;

[0125] 3) Soak the core part of the fiber with the coating removed in 0.1 mol / L NaOH for 30 min to activate the silanol groups on the surface of the optical fiber, wash it twice with water, wash it once with ethanol, and blow dry it with nitrogen;

[0126] 4) Soak the core part in a 1% (v / v) silanization agent 3-ADMS ethanol solution for 60 min to introduce amino groups to the surface of the optical fiber, wash it with ethanol three times, and blow dry it with nitrogen;

[0127] 5) The optical fiber was immersed in PBS buffer (0.1 mmol / L, pH=7.0) containing 0.015 mol / L EDC and 0.03 mol / L NHS for 1.5 h, and then immersed in 20 μg / mL PCT-Ab1 for coupling, and washed with PBS three times;

[0128] 6) Immerse the antibody-coupled optical fiber in 5% BSA solution (w / v) at 37°C for 1 hour, and wash with PBST three times;

[0129] 7) The prepared PCT fiber optic probe was stored at 4°C, and 1 ng / mL PCT was tested on days 0, 1, 3, 7, 14, and 30 to verify the antibody activity.

[0130] Preparation of optical fiber of the present invention:

[0131] 1) Place a spherical polystyrene sleeve with a radius of 1.5 mm and a through hole with a diameter of 395 μm on the positioning base and assemble it with the optical fiber, the core diameter of which is 400 μm;

[0132] 2) Insert the optical fiber with the sleeve into 20 μg / mL PCT-Ab1, incubate at 4°C overnight, and then wash three times with PBS;

[0133] 2) The optical fiber was inserted into a 5% BSA solution (w / v) at 37°C for 1 h, and then washed three times with PBST;

[0134] 3) The prepared PCT fiber optic probe was stored at 4°C, and 1 ng / mL PCT was detected on days 0, 60, 120, 180, 240, and 300 to verify the antibody activity.

[0135] Depend on Fig.12 It can be seen that the chemiluminescence intensity of PCT detected by the chemically modified antibody-fiber probe decreased significantly after 180 days of storage, and was almost completely inactivated after the 240th day; while the chemiluminescence intensity of PCT detected by the fiber probe assembled by the polystyrene sleeve remained almost unchanged in the first 180 days of storage, and the signal intensity remained 83% of the original signal value on the 240th day, and after 300 days of storage, its signal intensity was still more than 60% of the original intensity. The above results show that the antibody-fiber probe embedded in the polystyrene sleeve is less likely to be inactivated than the antibody coupled to the chemically modified fiber probe, and has better stability and storage capacity.

Claims

1. A method for preparing a multi-index optical fiber chemiluminescent biosensor with adjustable linear range based on polymer sleeve embedding, characterized in that: The optical fiber chemiluminescent biosensor is prepared by embedding an optical fiber into polymer sleeves of different sizes, modifying the biorecognition molecules on the sleeves, and preparing an optical fiber probe modified with the biorecognition molecules; The optical fiber is a quartz optical fiber; The biorecognition molecule is a capture antibody or a detection antibody or a complete antigen that can undergo a competitive immune reaction or a double antibody sandwich immune reaction with the target to be detected; The method comprises the following steps: 1) Insert the optical fiber into the polystyrene sleeve for assembly; 2) placing the fiber optic probe assembled in step 1) incubated in the biorecognition molecule and washing with PBST solution; 3) placing the optical fiber probe treated in step 2) in bovine serum albumin for sealing operation to obtain an optical fiber probe assembled with a polymer cladding; In the step 1), the fitting method is interference fit; In the step 2), the incubation is carried out at 37° C. for 1.5-2.5 hours, and in the PBST solution, the molar concentration of PBS is 0.005-0.015 M, and the mass fraction of Tween is 0.03-0.08%; In the step 3), the mass fraction of the bovine serum albumin is 3-7%.

2. The method for preparing a multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding according to claim 1, characterized in that: The structure of the automated detection device used in the method is: The optical fiber probe (02) is embedded in the polymer sleeve (01), and the optical fiber probe (02) is connected to the optical fiber bracket (03). The optical fiber bracket (03), the photon counter (04) and the control system component (05) are also arranged on the bracket (06). A motor bracket (13) and a movable guide rail (09) are arranged below the bracket (06). The motor (14) is installed on the motor bracket (13), and the motor shaft is connected to the ball screw (10). One end of the movable platform (08) is fixedly connected to the sealing box (07), and the other end is connected to the ball screw (10).

3. The method for preparing a multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding according to claim 2, characterized in that: Two guide wheels (11) are respectively mounted on the left and right ends of the mobile platform (08), and the guide wheels (11) are embedded in the moving grooves (12) on both sides of the guide rail (09).

4. The method for preparing a multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding according to claim 2, characterized in that: The sealing box is made of polyethylene, polyvinyl chloride or polystyrene.

5. The method for preparing the optical fiber chemiluminescence biosensor with multiple indexes and adjustable linear range based on polymer sleeve embedding according to any one of claims 1 to 4, characterized in that: The method is used for detecting target objects, which include mycotoxins, pathogenic microorganisms, antibiotics or pesticides and veterinary drugs.

6. The method for preparing a multi-index optical fiber chemiluminescence biosensor with adjustable linear range based on polymer sleeve embedding according to claim 5, characterized in that: When used for direct chemiluminescence method, the specific steps include: SB1: embedding optical fibers into polymer sleeves of different sizes, coupling different biorecognition molecules a on the surfaces of polymer sleeves of different sizes to obtain polymer sleeve-embedded optical fiber probes; SB2: inserting the optical fiber probe into a mixed solution of the object to be tested and the biorecognition molecule b to perform a biological reaction, washing the optical fiber probe after the reaction to remove the non-specifically adsorbed biorecognition molecule b, and matching the biorecognition molecule a with the biorecognition molecule b; the biorecognition molecule a and the biorecognition molecule b are a capture antibody and a detection antibody labeled with a chemiluminescent substrate, a detection antibody and a capture antibody labeled with a chemiluminescent substrate, an antibody and an antigen labeled with a chemiluminescent substrate, or an antigen and an antibody labeled with a chemiluminescent substrate; SB3: Place the washed fiber optic probe into the fiber optic holder, and then place the centrifuge tube containing the chemiluminescent substrate into the test tube holder in the sealed box. When the measurement starts, the sealed box moves backward along the guide rail to close to form a closed space. The fiber optic probe is inserted into the chemiluminescent substrate for measurement. After the measurement is completed, the measurement result is processed by the control system and sent to the mobile phone. The measurement is completed. When used for enzymatic chemiluminescence method, the method specifically comprises the following steps: SC1: embedding optical fibers into polymer sleeves of different sizes, coupling different biorecognition molecules a on the surfaces of polymer sleeves of different sizes to obtain polymer sleeve-embedded optical fiber probes; SC2: insert the fiber optic probe into the object to be tested for biological reaction, and wash the fiber optic probe after the reaction; SC3: The washed optical fiber probe is inserted into the biorecognition molecule b for biological reaction, and the optical fiber probe is washed after the reaction to remove the non-specifically adsorbed biorecognition molecule b, and the biorecognition molecule a is matched with the biorecognition molecule b; the biorecognition molecule a and the biorecognition molecule b are capture antibody and detection antibody, detection antibody and capture antibody, antibody and antigen, or antigen and antibody; SC4: Place the washed fiber optic probe into the fiber optic holder, and then place the centrifuge tube containing the chemiluminescent substrate into the test tube holder in the sealed box. When the measurement starts, the sealed box moves backward along the guide rail to close to form a closed space. The fiber optic probe is inserted into the chemiluminescent substrate for measurement. After the measurement is completed, the measurement result is processed by the control system and sent to the mobile phone. The measurement is completed.

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