Biosensor based on carbohydrate binding domain and preparation method and application thereof

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a biosensor based on a carbohydrate binding domain, and a preparation method and application thereof. The biosensor is prepared by sequentially performing electrode surface activation, nanomaterial modification, CBM immobilization, and blocking liquid blocking on the electrode. The HA electrochemical biosensor prepared according to this technology realizes highly specific quantitative analysis of HA by detecting changes in electrochemical signals. It not only has a fast detection speed, which can be completed in 10 minutes, but also has low cost, a wide quantitative range, and does not rely on large instruments and professional operators. It is suitable for on-site detection scenarios and for use as an industrial automation sensor. The present invention can also be used for rapid quantitative detection of other carbohydrates by replacing different types of CBMs.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a biosensor based on a carbohydrate binding domain and a preparation method and application thereof. Background Art

[0002] HA is a linear, unbranched natural polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units connected by β-1,4 glycosidic bonds. It is widely distributed in the extracellular matrix, skin, and serum of animal and human connective tissue. It is an important component of the dermis of human skin and one of the main components of the extracellular matrix and intercellular matrix. It plays an important physiological function in maintaining the normal function of the human body. HA has a wide range of applications in food, medical care, cosmetics and other fields due to its unique physical and chemical properties (such as biocompatibility, moisturizing, viscoelasticity) and biological activities (such as anti-inflammation, tissue repair, and immune regulation).

[0003] At present, conventional HA analysis uses high performance liquid chromatography (HPLC), which has high accuracy, sensitivity and specificity, but requires expensive instruments and professional operators. Colorimetric methods such as the carbazole method and the phenol-sulfuric acid method indirectly estimate the HA in the sample by detecting the monosaccharide content produced after HA hydrolysis. These methods have long detection times and low specificity, and the presence of other sugars in the sample will seriously affect the accuracy of the method.

[0004] Therefore, there is a need for a method that can quickly, accurately and specifically detect the HA content in a sample, preferably a method that can detect in real time and on-site.

[0005] In recent years, the research and development of electrochemical sensors has been very rapid. Due to their characteristics of fast detection speed, low cost, and independence from large instruments and professional operators, they have been widely used in the field of rapid detection.

[0006] Electrochemical immunosensors are based on the specific binding between antigens and antibodies to detect and analyze the target. The basic principle is that when antigens and antibodies bind to form a complex through specific interactions, the sensor technology converts this event into a detectable electrical signal. Further measurement of the electrical signal can clarify the specific binding degree of the antibody to the corresponding antigen, and ultimately achieve quantitative research on the sample to be tested. Immunosensors have the advantage of high specificity. However, due to the complexity of the polysaccharide structure and low immunogenicity, it is difficult for the immune system to recognize it. Therefore, specific antibodies for polysaccharides are often difficult to obtain, limiting the application of polysaccharide immunosensors.

[0007] Carbohydrate-binding module (CBM) is a class of proteins with polysaccharide binding ability. It exists as an independent domain of carbohydrate-active enzymes. It has no catalytic activity, but can specifically bind to polysaccharide substrates. CBM is a component of carbohydrate-active enzymes, so it can reflect the microscopic heterogeneity of natural polysaccharides and has accurate polysaccharide recognition capabilities. Taking advantage of the specific binding characteristics of CBM with polysaccharides, CBM can be used instead of antibodies to construct polysaccharide electrochemical biosensors, circumventing the difficulty of obtaining polysaccharide antibodies. However, it is difficult for ordinary CBM to meet the requirements of specificity and stability at the same time. For example, some proteins can bind to multiple proteins with poor specificity; some proteins have poor stability, and the test results will be very different after being used as sensors for a few days. This limits the application of CBM in biosensors. Summary of the invention

[0008] The technical problem to be solved by the present invention is that it is difficult for ordinary CBM to meet the requirements of specificity and stability at the same time. For example, some proteins can bind to multiple proteins and have poor specificity; some proteins have poor stability and the detection results will be very different after being used as sensors for a few days, which limits the application of CBM in biosensors.

[0009] In order to solve the above problems, the present invention provides an electrochemical biosensor based on CBM and a preparation method thereof, discovers and screens a CBM with both high specificity and high stability, and for the first time realizes the fixation of a CBM with specific binding ability to polysaccharides on an electrode, replacing the antibodies used in traditional immunosensors, and preparing an electrochemical biosensor capable of quantitatively detecting polysaccharides, thereby avoiding the difficulty in obtaining polysaccharide antibodies. The sensor detects polysaccharides by detecting changes in electrochemical signals. The present invention also uses the above sensor to realize the rapid sensing detection of HA in a sample for the first time. The CBM-based HA sensor has the advantages of strong specificity, fast detection speed, and wide quantitative range. At the same time, it does not rely on large instruments and professional operators, and provides a solution for the rapid identification and analysis of HA.

[0010] To achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing an electrochemical biosensor based on CBM, comprising the following steps:

[0011] (1) Immersing the electrode in an activation solution for activation to increase the active sites on the electrode surface and improve the electrochemical activity of the electrode;

[0012] (2) adding nanomaterials to the surface of the electrode after activation in step (1), depositing the nanomaterials on the surface of the electrode until the response current tends to be stable, and then washing and drying; the nanomaterials have a large specific surface area and also have good catalytic activity and biocompatibility, thereby optimizing the catalytic performance of the electrode and the fixation efficiency of the CBM;

[0013] (3) dropping 5 to 200 μL of CBM on the surface of the electrode prepared in step (2), incubating for 0.1 to 2 h under constant temperature and humidity conditions for deposition, and then washing and drying; by precisely controlling the incubation conditions, the consistency and stability of the sensor can be improved;

[0014] (4) Add a blocking solution to the surface of the electrode prepared in step (3), wash and dry after the reaction, and the preparation of the electrochemical biosensor is now complete.

[0015] The currently confirmed binding function of CBMs covers almost all known natural polysaccharides, which means that the CBM of various polysaccharides can be screened out as needed, and the corresponding polysaccharide electrochemical biosensor can be prepared using the method provided by the present invention, providing a powerful tool for the accurate analysis of polysaccharides. In addition, since the coding gene sequence of CBM is clear, it is easier to achieve heterologous expression and batch preparation compared to antibodies that require animal immunization, and the cost is lower. When carbohydrates (usually polysaccharides) bind to CBM, the sensor can quantify the polysaccharide by detecting changes in electrochemical signals.

[0016] Furthermore, the activation solution in step (1) is a mixed solution of 0.1-10 mM potassium ferrocyanide and 0.1-10 mM potassium ferrocyanide; activation is performed by cyclic voltammetry (CV) scanning, and the CV scanning conditions are scanning potential range: -2-2 V, scanning speed: 10-500 mV / s, and scanning 1-50 cycles. A wider scanning potential range ensures that the electrode surface can undergo a wide range of redox reactions, which helps to expose more active sites on the electrode surface. Multiple cycles can ensure that the electrode surface repeatedly undergoes the redox process, which helps to uniformly and thoroughly activate the electrode surface.

[0017] Furthermore, in step (2), nanomaterials are deposited on the electrode surface by CV scanning; the nanomaterials are nanogold AuNPs and / or nanoplatinum PtNPs, with a particle size of 10 to 50 nm. The smaller the particle size of the nanomaterial, the larger its surface area per unit volume. This means that smaller nanoparticles can provide more surface active sites, which helps to improve the sensitivity of the sensor. Nanomaterials with larger particle sizes are less likely to agglomerate due to their lower surface energy. The choice of particle size should be based on the target object, working environment and expected performance indicators of the sensor.

[0018] Furthermore, in step (3), the incubation temperature is 1-50°C and the humidity is 20%-80%. A lower incubation temperature helps maintain the stability of the protein and reduce denaturation and nonspecific adsorption; while a higher temperature can accelerate the fixation process and shorten the incubation time, which is beneficial for the rapid preparation of the sensor.

[0019] Furthermore, in step (3), the CBM or variant of the sequence shown in SEQ ID NO: 1 is used as a recognition tool. The CBM can be produced in a variety of ways, including the collection and purification of natural proteins, the collection and purification of recombinant proteins, and chemical synthesis of proteins.

[0020] SEQ ID NO.1:

[0021] EETTTNLVKNGDFTTTTKTSGAWSGQAAKDWETWVPGNVKKENGKVTVDNGRLHISSTDTYRVAVHQTVDVDPSKKYLLGYDVETKDLKGSGVRVRLHGLTAEGKDLEPKEFIHTPFKNGSKKERVEQVLTVSPQTRKLKVELFFENSIGQAWLDNVSLVEHIEKVPETPEQKPEPAQPES

[0022] The CBM variant has at least 30% homology with the amino acid sequence of SEQ ID NO.1. The amino acid sequence of SEQ ID NO:1 has been screened to be able to specifically recognize and bind to HA, and this specific binding ability is the basis for the high sensitivity and high specificity of the sensor. Variants with at least 30% homology usually retain key structural domains and functional sites. These regions determine the binding ability of CBM to HA, so the variant can still effectively recognize and bind to HA.

[0023] Furthermore, in step (4), 5 to 200 μL of blocking solution is added to the electrode surface and reacted under constant temperature and humidity conditions for 0.1 to 2 hours; the blocking solution is bovine serum albumin BSA and / or skimmed milk powder SMP, the concentration of the blocking solution is 0.5 to 10%, the reaction temperature is 1 to 50°C, and the humidity is 20% to 80%.

[0024] Both BSA and SMP have excellent blocking effects, which can effectively prevent the adsorption of nonspecific proteins or other biomolecules on the electrode surface. This property helps to improve the detection specificity of the sensor and reduce background noise. A moderate concentration of blocking solution can maximize the specificity of detection while maintaining the sensitivity of the sensor, reducing errors and noise. Moderate blocking conditions provide a more balanced environment, which is conducive to the uniform distribution and effective blocking of the blocking solution.

[0025] Furthermore, step (2) is washed with ultrapure water and dried with nitrogen.

[0026] Furthermore, in step (3) and step (4), the mixture is washed with phosphate buffer PB and dried with nitrogen.

[0027] Furthermore, the electrode system is composed of a working electrode, a counter electrode and a reference electrode.

[0028] Furthermore, the pH of the PB solution is 5 to 11, and the concentration is 0.01 to 0.5M.

[0029] A CBM-based electrochemical biosensor prepared by the above method can complete HA content detection within 10 minutes by DPV scanning. In the wide range of HA concentrations of 10 to 10000 μg / mL, there is a good regression relationship between HA content and response signal (R2>0.99), and the detection limit of the method is 1.5 μg / mL.

[0030] An application of the above-mentioned CBM-based electrochemical biosensor in detecting HA.

[0031] A method for detecting HA using the electrochemical biosensor comprises the following steps:

[0032] (1) Prepare HA solution standard and perform gradient dilution to obtain a series of HA solutions with known concentrations;

[0033] (2) adding the HA solution of known concentration to the sensor electrode, reacting under constant temperature and humidity conditions, then recording the intensity of the electrochemical signal response, and drawing a standard curve with the HA concentration as the horizontal axis and the electrochemical signal change as the vertical axis;

[0034] (3) Add the test solution to the working electrode of the sensor, perform detection using the same operation as step (2), record the electrochemical signal response intensity, and substitute it into the standard curve drawn in step (2) to calculate the HA concentration in the test solution.

[0035] Furthermore, the electrochemical signal changes include changes in the current, voltage, resistance, and capacitance of the sensor, and the electrochemical methods used include CV scanning, DPV scanning, and electrochemical impedance spectroscopy.

[0036] Furthermore, the CV scanning parameters are scanning potential range: -2 to 2 V, scanning speed: 10 to 500 mV / s, and scanning for 1 to 50 cycles.

[0037] Furthermore, the scanning parameters of the differential pulse voltammetry (DPV) are scanning range: -2 to 2 V, potential increment 1 to 10 mV, pulse amplitude 10 to 100 mV, pulse width 10 to 100 ms, sampling width 10 to 50 ms, and pulse period 100 to 1000 ms.

[0038] Furthermore, the electrochemical impedance spectroscopy (EIS) parameters are a frequency range of 10-2 to 106 Hz, an amplitude of 1 to 100 mV, and an initial potential of 0.1 to 1V.

[0039] The beneficial effects of the present invention are:

[0040] 1. This invention is the first to use CBM as the core recognition element to prepare polysaccharide electrochemical biosensors, replacing the traditional antibodies. This innovative design avoids the problem of difficulty in obtaining antibodies due to the complex structure and low immunogenicity of polysaccharides, and solves the development difficulties of polysaccharide electrochemical sensors.

[0041] 2. The present invention firstly constructs a novel HA electrochemical biosensor based on the CBM or variants of the sequence shown in SEQ ID NO: 1 as a recognition tool, and realizes rapid sensing detection of HA by detecting changes in electrochemical signals. The metal nanomaterials not only enhance the conductivity but also increase the electrode surface area, thereby improving the fixation efficiency of CBM. Using CBM as a recognition element improves the specificity of the detection method.

[0042] 3. Due to CBM's accurate recognition ability of HA and its own excellent characteristics, the sensor has the advantages of strong specificity, fast detection speed and wide quantitative range when detecting HA. HA content detection can be completed within 10 minutes through DPV scanning. In the wide range of HA concentration of 10 to 10000 μg / mL, there is a good regression relationship between HA content and response signal (R2>0.99), and the detection limit of the method is 1.5 μg / mL.

[0043] 4. The HA biosensor of the present invention has a simple preparation process. In addition to using traditional solid electrodes, it can also be prepared based on screen-printed carbon electrodes (SPCE), which do not require pre-treatment operations such as polishing, and are highly portable, low-cost, and can be used and thrown away, which is more suitable for on-site rapid detection scenarios.

[0044] 5. The detection process can be carried out by a portable electrochemical analyzer, which does not rely on large instruments and professional operators. It is easy to operate and can be applied to rapid on-site detection of HA. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 :CV curves of the sensor at different modification stages

[0046] Figure 2 :DPV curves of different concentrations of HA detected by the sensor

[0047] Figure 3 : Linear relationship between the natural logarithm of HA concentration and the change in DPV response current

[0048] Figure 4 : Sensor stability test results

[0049] Figure 5 :Linear relationship between the natural logarithm of HA concentration and the change in DPV response current measured by the miniaturized sensor

[0050] Figure 6 : Schematic diagram of portable HA sensor rapid detection, the red circle is the sensor body (left: computer interface and sensor, right: smartphone interface) DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased or obtained by existing methods. Steps S1, S2, S3, etc. of the present invention do not represent a strict sequential relationship, and the order can be appropriately adjusted as needed.

[0053] Example 1: A biosensor based on SPCE and CBM, the preparation method of which is as follows:

[0054] S1. The original unmodified SPCE was characterized by CV, DPV and EIS in a characterization solution containing potassium ferrocyanide and potassium ferrocyanide, and the basic performance data of the electrode was recorded; then after each modification step, the electrode was re-characterized by the same CV, DPV and EIS to track the changes in electrode performance.

[0055] In S1, the activation solution is prepared by dissolving 0.5-5 mM potassium ferrocyanide, potassium ferrocyanide and 0.1-0.5 M potassium chloride in ultrapure water. The parameters of the CV scan are potential scanning range: -2-2 V, scanning speed: 10-500 mV / s, and scanning for 1-50 cycles. The parameters of the DPV scan are potential scanning range: -2-2 V, potential increment 1-10 mV, pulse amplitude 10-100 mV, pulse width 10-100 ms, sampling width 10-50 ms, and pulse period 100-1000 ms. The EIS parameters are frequency range 10 -2 ~10 6 Hz, amplitude 1~100mV, initial potential 0.1~1V.

[0056] S2. Immerse the SPCE in the activation solution and activate it through CV cycle scanning to increase the active sites on the electrode surface and improve the electrochemical reaction activity of the electrode.

[0057] In S2, the activation solution is prepared by dissolving 0.5-5 mM potassium ferrocyanide and potassium ferrocyanide in 0.2 mM PB at pH 7-8. The CV scanning conditions are scanning potential range: -2-2 V, scanning speed: 10-500 mV / s, and scanning 1-50 cycles.

[0058] S3. Add AuNPs to the surface of the electrode prepared in S2, and perform CV scanning on the electrode until the electrochemical response current curve of the electrode reaches stability. Then discard the remaining liquid on the surface of the electrode, wash it with ultrapure water for multiple times and blow it dry with nitrogen to obtain AuNPs / SPCE.

[0059] In the S3, the particle size of AuNPs is 15 to 50 nm, the dosage is 50 to 500 μL, and the CV scanning conditions are: scanning potential range: -2 to 2 V, scanning speed 10 to 200 mV / s.

[0060] S4. CBM solution was added dropwise to the surface of the electrode prepared in S3, incubated at a constant temperature and humidity, washed with PB for several times and dried with nitrogen to obtain CBM / AuNPs / SPCE.

[0061] The CBM sequence is shown in SEQ ID NO: 1:

[0062] EETTTNLVKNGDFTTTTKTSGAWSGQAAKDWETWVPGNVKKENGKVTVDNGRLHISSTDTYRVAVHQTVDVDPSKKYLLGYDVETKDLKGSGVRVRLHGLTAEGKDLEPKEFIHTPFKNGSKKERVEQVLTVSPQTRKLKVELFFENSIGQAWLDNVSLVEHIEKVPETPEQKPEPAQPES

[0063] The gene encoding the above CBM has a nucleotide sequence of SEQ ID NO.2 or any gene that can be translated into SEQ ID NO.1.

[0064] SEQ ID NO.2:

[0065] GAAGAGACTACGACTAATTTGGTAAAAAATGGGGATTTTACAACCACAACAAAGACTTCAGGGGCCTGGTCAGGACAGGCAGCTAAAGACTGGGAAACTTGGGTGCCGGGTAATGTGAAAAAGGAAAATGGAAAG GTAACCGTTGATAACGGGCGACTCCATATCTCTTCAACCGATACATACCGAGTAGCGGTTCACCAAACTGTGGATGTTGATCCTAGTAAAAAATATCTCTTAGGCTATGATGTGGAAAACTAAGGATCTGAAAGGTA GCGGAGTGCGCGTTCGGTTACACGGTTTGACAGCAGAAGGGAAGGATTTGGAGCCGAAAGAATTTATCCATACCCCTTTTAAAAATGGCAGCAAGAAAGAGCGTGTAGAGCAAGTCTTAACTGTCTCACCACAAAC TCGCAAACTGAAGGTCGAACTATTTTTTGAAAATAGTATCGGTCAGGCTTGGCTTGACAACGTTTCGTTGGTTGAACATATTGAAAAAGTGCCAGAAACACCGGAACAAAAACCAGAACCTGCTCAGCCAGAATCG

[0066] In the S4, the amount of CBM used is 10 to 200 μL, the incubation temperature is 4 to 50° C., and the incubation time is 0.5 to 2 h.

[0067] S5. Add BSA solution to the electrode surface prepared in S4, incubate at constant temperature and humidity to block nonspecific binding sites, then wash with PB several times and blow dry with nitrogen to obtain BSA / CBM / AuNPs / SPCE. At this point, the HA biosensor is ready.

[0068] In the S5, the BSA concentration is 1-5%, the dosage is 10-200 μL, the incubation temperature is 4-50° C., and the incubation time is 0.5-2 h.

[0069] Combination Figure 1 After AuNPs were electrodeposited onto the SPCE surface, the peak current increased significantly because AuNPs accelerated the electron transfer rate on the electrode surface. With the gradual modification of CBM, BSA and HA, the peak current also decreased accordingly, which was mainly due to the fact that proteins and HA hindered the electron transfer process, indicating that the sensor was successfully modified at each stage.

[0070] Example 2: A biosensor based on a glassy carbon electrode (GCE) and a CBM, the preparation method of which is as follows:

[0071] S1. The original unmodified GCE was characterized by CV, DPV and EIS in a characterization solution containing potassium ferrocyanide and potassium ferrocyanide, and the basic performance data of the electrode was recorded; then after each modification step, the electrode was re-characterized by the same CV, DPV and EIS to track the changes in electrode performance.

[0072] In S1, the activation solution is prepared by dissolving 0.5-5 mM potassium ferrocyanide, potassium ferrocyanide and 0.1-0.5 M potassium chloride in ultrapure water. The parameters of the CV scan are potential scanning range: -2-2 V, scanning speed: 10-500 mV / s, and scanning for 1-50 cycles. The parameters of the DPV scan are potential scanning range: -2-2 V, potential increment 1-10 mV, pulse amplitude 10-100 mV, pulse width 10-100 ms, sampling width 10-50 ms, and pulse period 100-1000 ms. The EIS parameters are frequency range 10 -2 ~10 6 Hz, amplitude 1~100mV, initial potential 0.1~1V.

[0073] S2. Soak the GCE in ultrapure water to remove surface impurities, polish the electrode surface with alumina, and ultrasonically clean it with ethanol and ultrapure water for 10 minutes respectively. Soak the cleaned electrode in the activation solution and perform CV cycle scanning until the electrochemical response current curve reaches stability to ensure that the electrode surface is fully activated. The activated GCE is ultrasonically cleaned again with ethanol and ultrapure water for 10 minutes respectively, and then dried for use.

[0074] In S2, the activation solution is a 0.1-1 M dilute sulfuric acid solution. The CV scanning conditions are: scanning potential range: -2-2 V, scanning speed: 10-500 mV / s, scanning 1-50 cycles.

[0075] S3. PtNPs were added dropwise to the electrode surface prepared in S2, and the electrode was subjected to CV scanning until the electrochemical response current curve of the electrode reached stability. The remaining liquid on the electrode surface was then discarded, the electrode surface was washed with ultrapure water for multiple times and dried with nitrogen to obtain PtNPs / GCE.

[0076] In the S3, the particle size of PtNPs is 15 to 50 nm, the dosage is 100 to 1000 μL, and the CV scanning conditions are scanning potential range: -2 to 2 V, and scanning speed 10 to 200 mV / s.

[0077] S4. CBM solution was added dropwise to the surface of the electrode prepared in S3, incubated at constant temperature and humidity, washed with PB for multiple times and dried with nitrogen to obtain CBM / PtNPs / GCE.

[0078] In the S4, the amount of CBM is 50-500 μL, the incubation temperature is 4-50° C., and the incubation time is 0.5-2 h.

[0079] S5. Add SMP solution to the surface of the electrode prepared in S4, incubate at constant temperature and humidity to block nonspecific binding sites, then wash with PB several times and blow dry with nitrogen to obtain SMP / CBM / PtNPs / GCE. At this point, the preparation of the GCE-based HA sensor is completed.

[0080] In the S5, the SMP concentration is 1-10%, the dosage is 50-500 μL, the incubation temperature is 4-50° C., and the incubation time is 0.5-2 h.

[0081] The rest are the same as in Example 1.

[0082] Embodiment 3:

[0083] The CBM sequence used in step 4 is shown in SEQ ID NO: 3:

[0084] SEQ ID NO.3

[0085] NLVANGEFISTTAPSGNWKELAATNWETWIPANVKKENGQVRIDEGRLHISSTASYRVAVHQTVDVDPNKRYLFSYDVETKDLKGSGVRVRLRSLTAEGKDLSPQEFAYTPYKNGSQAEHIEQILTVSPETRKLKVELFFENSVGQAWLDNISLVEYV

[0086] The rest are the same as in Example 1 or 2.

[0087] Embodiment 4:

[0088] The CBM sequence used in step 4 is shown in SEQ ID NO: 4:

[0089] SEQ ID NO.4

[0090] NLVKNGDFTTTTKTSGAWSGQAAKDWETWVPGNVKKENGKVTVDNGRLHISSTDTYRVAVHQTVDVDPSKKYLLGYDVETKDLKGSGVRVRLHGLTAEGKDLEPKEFIHTPFKNGSKKERVEQVLTVSPQTRKLKVELFFENSIGQAWLDNVSLVE

[0091] The rest are the same as in Example 1 or 2.

[0092] Embodiment 5:

[0093] The CBM sequence used in step 4 is shown in SEQ ID NO: 5:

[0094] SEQ ID NO.5

[0095] NLVANGEFASTTAASGNWADPAATNWETWIPANVKKENGQVRIDEGRLHISSTASYRVAVHQTVDVDPNKRYLFSYDVETKDLKGSGVRVRLRSLTAEGKDLSPQEFAYTPYKNGSQAEHIEQILTVSPETRKLKVELFFENSVGQAWLDNISLVEYV

[0096] The rest are the same as in Example 1 or 2.

[0097] Example 6: Application of the biosensor of Example 1 above in quantitative detection of HA, the specific method is as follows:

[0098] (1) HA was dissolved in 0.02 M PB and gradiently diluted to obtain a series of HA solutions with known concentrations: 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, and 10000 μg / mL.

[0099] (2) 50 μL of the above HA solution was added dropwise to the sensor electrode prepared in Example 1. After incubation at room temperature for 10 min, the remaining liquid on the electrode surface was discarded, the electrode surface was cleaned with PB and blown dry with nitrogen. The biosensor was connected to an electrochemical workstation, and the sensor incubated with different concentrations of HA was characterized by the DPV method, with a scanning potential range of -0.2 to 0.6 V, an amplitude of 0.05 V, and a sensitivity of 10 -5 A / V, and obtain the DPV curve at different HA concentrations ( Figure 2 ), record the response current data; draw a standard curve with the natural logarithm of HA concentration as the horizontal axis and the DPV peak current change ΔI as the vertical axis ( Figure 3 ). As can be seen from the figure, in the wide range of 20 to 10000 μg / mL, there is a good regression relationship between HA content and ΔI, and the correlation coefficient R 2 >0.99, the linear fitting equation was y=9.019x+4.051, and the detection limit of the method was 2.5μg / mL, showing a wider detection range and a lower detection limit.

[0100] (3) 50 μL of the test solution with a HA concentration of 200 μg / mL was dripped onto the prepared sensor electrode, and the DPV curve of the test solution was obtained using the steps shown in (2). The detection was performed three times in parallel, and the average response current change was 24.76 μA. The HA concentration in the solution was calculated to be 197.77 μg / mL by substituting it into the standard curve, indicating that the method has good accuracy.

[0101] Example 7: Determination of spike recovery of HA biosensor

[0102] The biosensor prepared in Example 1 was used to measure HA solutions with concentrations of 50, 200, and 2000 μg / mL as low, medium, and high samples, which were spiked (50 μg / mL) and mixed thoroughly. The detection was performed using the steps shown in Example 6 (2). Each concentration group was tested three times in parallel, the response current was recorded, and the HA concentration and spike recovery rate were calculated, as shown in Table 1.

[0103] Table 1 Results of spike recovery at low, medium and high concentrations

[0104]

[0105] The spiked recovery of the quantitative detection method of HA biosensor ranged from 97.12 to 102.49%, indicating that the method had good accuracy. The relative standard deviation gradually decreased with the increase of sample concentration, which means that the precision of the sensor was better under medium and high concentration conditions, and the relative standard deviation at each concentration was within an acceptable range, indicating that the biosensor had good reproducibility.

[0106] Example 8: Determination of specificity of HA biosensor

[0107] To a 200 μg / mL HA solution, interfering substances (chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, alginate, pectin) were added at a concentration of 200 μg / mL. The HA concentration in the above solution was determined using the biosensor prepared in Example 1 using the steps shown in Example 6 (2). The response current was recorded and the recovery rate was calculated, as shown in Table 2.

[0108] Table 2 Recovery rate of biosensor in the presence of different interferents

[0109]

[0110]

[0111] Chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin and HA belong to the same glycosaminoglycans, and alginate and pectin contain uronic acid as HA. Combined with Table 2, even in the presence of a large number of interfering substances with similar structures or properties, the sensor can still effectively identify and quantify the target HA, showing the application value of the sensor for HA detection in complex samples, which is due to the good binding specificity of CBM to HA.

[0112] Example 9: Storage stability determination of HA biosensor

[0113] The biosensor prepared in Example 1 was stored at 4°C for 30 days. On the 1st, 2nd, 5th, 10th, 15th and 30th days, the HA standard with a concentration of 200 μg / mL was measured using the steps shown in Example 6 (2). The response current was recorded in three parallel replicates. Figure 4 After being stored for 30 days, the response current of the sensor of the present invention still remains above 90% of the initial response current, showing good stability.

[0114] Example 10: Miniaturization of HA Biosensor

[0115] (1) HA was dissolved in 0.02 M PB and gradiently diluted to obtain a series of HA solutions with known concentrations, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000 μg / mL. 50 μL of the above solution was added dropwise to the biosensor electrode prepared in Example 1 and incubated at room temperature for 10 min.

[0116] (2) The electrochemical workstation in Example 6 was replaced with a portable electrochemical detection device, which was connected to the biosensor. The above-mentioned biosensor incubated with different concentrations of HA was characterized by the DPV method, with a scanning potential range of -0.2 to 0.6 V, an amplitude of 0.05 V, and a sensitivity of 10-5 A / V, record the response current data, and draw a standard curve with the natural logarithm of HA concentration as the horizontal axis and the DPV peak current change ΔI as the vertical axis.

[0117] Combination Figure 5 The standard curve equation is y = 7.0027x + 10.75, and the correlation coefficient is R 2 >0.99, and its quantitative range is extended to 10-10000 mg / mL compared with Example 6. The Pearson correlation coefficient of the results of Example 6 and Example 10 is calculated to be 0.994, indicating that the measurement results of the two in the quantitative analysis of HA are highly consistent. The test results of Examples 2-5 are not significantly different from those of Example 1, indicating that the biosensor of the present invention can realize real-time and wireless detection of HA by combining with a portable electrochemical detection device, and can conveniently transmit the detection results wirelessly to a smart phone for real-time monitoring ( Figure 6 ). The miniaturized HA sensor can complete the detection task instantly on site, simplifying the pretreatment steps required for traditional laboratory testing and getting rid of the dependence on special environments and large equipment.

Claims

1. A method for preparing a CBM-based electrochemical biosensor, characterized in that The following steps are involved: (1) Immerse the electrode in an activation solution for activation; (2) adding nanomaterials to the surface of the electrode after activation in step (1) to deposit the nanomaterials on the surface of the electrode until the response current tends to be stable, and then washing and drying; (3) dropping 5 to 200 μL of CBM on the surface of the electrode prepared in step (2), incubating for 0.1 to 2 h under constant temperature and humidity conditions for deposition, and then washing and drying; using the CBM or a variant of the sequence shown in SEQ ID NO: 1 as an identification tool, wherein the CBM variant has at least 30% homology with the amino acid sequence of SEQ ID NO: 1, and the CBM variant sequence is any one of the sequences shown in SEQ ID NO: 3-5; (4) Add a blocking solution to the surface of the electrode prepared in step (3), wash and dry after the reaction, and the preparation of the electrochemical biosensor is now complete.

2. The preparation method according to claim 1, characterized in that: The activation solution described in step (1) is a mixed solution of 0.1-10 mM potassium ferrocyanide and 0.1-10 mM potassium ferrocyanide; activation is performed by CV scanning, and the CV scanning conditions are scanning potential range: -2-2 V, scanning speed: 10-500 mV / s, and scanning 1-50 cycles.

3. The preparation method according to claim 1, characterized in that: Step (2) depositing nanomaterials on the electrode surface by CV scanning; the nanomaterials are nano-gold AuNPs and / or nano-platinum PtNPs, with a particle size of 10 to 50 nm.

4. The preparation method according to claim 1, characterized in that: In step (3), the incubation temperature is 1 to 50° C. and the humidity is 20% to 80%.

5. The preparation method according to claim 1, characterized in that: Step (4) adding 5-200 μL of blocking solution to the electrode surface and reacting for 0.1-2 hours under constant temperature and humidity conditions; the blocking solution is bovine serum albumin BSA and / or skimmed milk powder SMP, the concentration of the blocking solution is 0.5-10%, the reaction temperature is 1-50° C., and the humidity is 20%-80%.

6. A CBM-based electrochemical biosensor prepared by the method of any one of claims 1-5.

7. Use of the CBM-based electrochemical biosensor according to claim 6 in detecting HA.

8. A method for detecting HA using the electrochemical biosensor according to claim 6, characterized in that The following steps are involved: (1) preparing HA solution standards and performing gradient dilution to obtain a series of HA solutions with known concentrations; (2) adding the HA solution of known concentration to the sensor electrode, reacting under constant temperature and humidity conditions, then recording the intensity of the electrochemical signal response, and drawing a standard curve with the HA concentration as the horizontal axis and the electrochemical signal change as the vertical axis; (3) Add the test solution to the working electrode of the sensor, perform detection using the same operation as step (2), record the electrochemical signal response intensity, and substitute it into the standard curve drawn in step (2) to calculate the HA concentration in the test solution.

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