Biosensor for detecting endotoxin as well as method and application thereof

Abstract

The invention belongs to the field of electrochemical detection, and particularly relates to a biosensor for detecting endotoxin as well as a method and application thereof. The invention provides a bioelectrode and a biosensor, and detection of endotoxin based on the bioelectrode and the biosensor. According to the present invention, the polypeptide is adopted as the recognition receptor to specifically recognize and capture the endotoxin molecule, and the signal probe is labeled on the carbohydrate chain of the endotoxin through the borate cross-linking reaction so as to achieve the rapid electrochemical detection of the endotoxin; the lowest detection lower limit of the endotoxin biosensor obtained by the invention is 2.17 pM, and the endotoxin biosensor can accurately detect endotoxin in a range of 5.0 pM to 5.0 nM. The electrochemical rapid detection method for the endotoxin has the advantages of simplicity, convenience, rapidness, low cost, high sensitivity, good selectivity and the like, and is suitable for instant detection of the endotoxin.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical detection, and in particular relates to a biosensor for detecting endotoxin and a method and application thereof. Background Art

[0002] Endotoxin, as the main component of the outer membrane of Gram-negative bacteria, can cause symptoms such as fever, diarrhea, sepsis, organ failure, etc., resulting in millions of deaths worldwide each year.

[0003] At present, the gold standard method for endotoxin detection is the horseshoe crab test, the basic principle of which is that endotoxin can activate the C-factor in the horseshoe crab reagent, and the activated C-factor further activates the B-factor or (1,3)-β-D-glucan activates the G-factor, which can activate procoagulant to coagulase, causing the soluble coagulogen to be converted into coagulant protein, resulting in a gelation reaction. However, the horseshoe crab test has shortcomings such as long detection time and easy to produce false positive results. Summary of the invention

[0004] In order to solve the problems of long endotoxin detection time and high false positive rate in the prior art, the present invention provides a biosensor for detecting endotoxin. Based on the sensor, polypeptides are used as recognition receptors to specifically recognize and capture endotoxin molecules, and signal probes are labeled on the sugar chains of endotoxins through a borate cross-linking reaction, thereby realizing rapid electrochemical detection thereof.

[0005] The first aspect of the present invention aims to provide a bioelectrode.

[0006] The purpose of the second aspect of the present invention is to provide a method for preparing the bioelectrode of the first aspect of the present invention.

[0007] The third aspect of the present invention aims to provide a biosensor.

[0008] The fourth aspect of the present invention aims to provide the use of the bioelectrode of the first aspect of the present invention and / or the biosensor of the third aspect of the present invention in the preparation of endotoxin detection.

[0009] The fifth aspect of the present invention aims to provide a kit.

[0010] The sixth aspect of the present invention aims to provide a method for detecting endotoxin.

[0011] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0012] A first aspect of the present invention provides a bioelectrode, wherein a recognition polypeptide is bound to the surface of the bioelectrode.

[0013] Preferably, the amino acid sequence of the recognition polypeptide is KKNYSSSISSIHGGGC (SEQ ID NO: 1).

[0014] Preferably, the active sites on the electrode surface that are not bound to the recognition polypeptide are blocked by a blocking agent.

[0015] In some embodiments of the present invention, the electrode is a gold electrode.

[0016] In some embodiments of the present invention, the blocking agent is mercaptohexanol.

[0017] In the first aspect of the present invention, the recognition polypeptide KKNYSSSISSIHGGGC is combined with the electrode in a covalently bound manner, and can specifically recognize and capture endotoxin. In order to improve the accuracy of the detection, the active sites on the electrode surface that are not bound to the recognition polypeptide are further blocked using a blocking agent to prevent the foreign proteins in the sample from binding to the electrode and affecting the detection effect. Those skilled in the art should know that substances that specifically recognize and bind to endotoxins include polypeptides, nucleic acid aptamers, etc. Therefore, other polypeptide sequences and nucleic acid aptamer sequences that can specifically bind to endotoxins can also become alternatives to the present invention.

[0018] The second aspect of the present invention provides a method for preparing the bioelectrode of the first aspect of the present invention, comprising the following steps:

[0019] The recognition polypeptide is incubated with the electrode, and a blocking agent is added to block the electrode to obtain a bioelectrode.

[0020] In some embodiments of the present invention, the electrode is a gold electrode.

[0021] In some embodiments of the present invention, the blocking agent is mercaptohexanol.

[0022] In some embodiments of the present invention, the concentration of the recognition polypeptide is 0.1-10 μM.

[0023] In some embodiments of the present invention, the incubation time is 1 to 120 minutes.

[0024] In some embodiments of the present invention, the concentration of the mercaptohexanol is 0.1-10 mM.

[0025] In some embodiments of the present invention, the sealing time is 1 to 120 minutes.

[0026] In some embodiments of the present invention, the incubation and sealing temperature is 4-37°C.

[0027] In the second aspect of the present invention, there is no strict standard for the amount of the recognition polypeptide and the blocking agent used, as long as they can completely cover the electrode surface. After the incubation and blocking steps, a cleaning step can also be included, wherein the cleaning includes using a buffer solution such as PBS to clean the residual solution on the electrode surface.

[0028] The third aspect of the present invention provides a biosensor comprising the bioelectrode and the signal probe according to the first aspect of the present invention.

[0029] Preferably, the signal probe includes at least one of ferrocene, methylene blue, thionine, and quantum dots.

[0030] Preferably, the signal probe has a boronic acid group.

[0031] In some embodiments of the present invention, the signal probe is ferrocene modified with boric acid, specifically ferrocene boric acid (FcBA).

[0032] In the third aspect of the present invention, the endotoxin molecule contains a long sugar chain, and there are cis-diol sites on the majority of monosaccharide residues that make up the sugar chain. The present invention finds that the cis-diol site can undergo a covalent cross-linking reaction with a boronic acid group, so a large number of signal probes can be labeled on the sugar chain of endotoxin by means of a borate cross-linking reaction, thereby achieving its simple, rapid, low-cost, highly sensitive and highly selective electrochemical detection. Those skilled in the art should know that the covalent cross-linking characteristics of the boronic acid group and the cis-diol site can be widely used in the detection of other substances. The method of modifying the signal probe using a boronic acid group in the present invention will bring new technical inspiration to other detection technologies.

[0033] The fourth aspect of the present invention provides use of the bioelectrode of the first aspect of the present invention and / or the biosensor of the second aspect of the present invention in the preparation of a product for detecting endotoxin.

[0034] Preferably, the product comprises at least one of a detection reagent and a detection kit.

[0035] The fifth aspect of the present invention provides an endotoxin detection kit, comprising the bioelectrode of the first aspect of the present invention and / or the biosensor of the third aspect of the present invention.

[0036] Preferably, the endotoxin detection kit has an endotoxin standard solution.

[0037] Preferably, the endotoxin detection kit has a negative control and a positive control.

[0038] Preferably, the endotoxin detection kit also has instructions.

[0039] In the fifth aspect of the present invention, the endotoxin standard solution refers to a group of endotoxin solutions with a concentration gradient determined by concentration. For example, 0, 1pM, 10pM, 100pM, 1000pM 10-fold gradient dilution of endotoxin standard solution, or 0, 100pM, 200pM, 400pM, 800pM, 1600pM 2-fold gradient dilution of endotoxin standard solution, or 0, 100pM, 250pM, 500pM, 750pM, 1000pM irregular gradient dilution of endotoxin standard solution. The purpose of the endotoxin standard solution is to draw a standard curve for determining the concentration of the sample. Those skilled in the art should know that the specific concentration and specific dilution method of the standard solution should not be a limitation of the present invention.

[0040] The negative control refers to a reference for judging whether the test sample does not contain endotoxin. When the test result of the sample changes less than that of the negative control, it is judged that the sample does not contain endotoxin; conversely, if the test result of the sample changes more than that of the negative control, it is judged that the sample contains endotoxin.

[0041] The positive control is a reference for determining whether the test sample contains endotoxins and is used to determine system errors. If the positive control cannot obtain the expected test results, it is determined that there is a problem with the test system.

[0042] The instructions record the method of use of the endotoxin detection kit, specific information of various reagents, and other information that can be used and referred to by users.

[0043] A sixth aspect of the present invention provides a method for detecting endotoxin, comprising the following steps:

[0044] 1) incubating a sample to be tested with the bioelectrode according to any one of claims 1 or 2;

[0045] 2) Add signal probe solution and incubate;

[0046] 3) Detection.

[0047] Preferably, the incubation time is 1 to 120 min.

[0048] Preferably, the incubation temperature is 4-37°C.

[0049] Preferably, the signal probe is ferrocenoboric acid.

[0050] In some embodiments of the present invention, a washing step is further included after the incubation, and the washing includes washing the residual solution on the surface of the electrode with a buffer solution such as PBS.

[0051] In some embodiments of the present invention, the concentration of the signal probe is 0.01-10 mM.

[0052] In some embodiments of the present invention, the detection is the detection of electrical signals (redox current signals), specifically the detection is performed using a reference electrode and a counter electrode; the reference electrode includes but is not limited to at least one of a hydrogen electrode, a calomel electrode, and a silver / silver chloride electrode; the counter electrode includes but is not limited to at least one of a platinum electrode and a carbon electrode.

[0053] In the sixth aspect of the present invention, there is no strict standard for the amount of the detection sample and signal probe used, as long as it can completely cover the electrode surface.

[0054] The beneficial effects of the present invention are:

[0055] The present invention provides a bioelectrode and a biosensor, and performs endotoxin detection based on the bioelectrode and the biosensor. The present invention uses polypeptides as recognition receptors to specifically recognize and capture endotoxin molecules, and labels the signal probes to the sugar chains of endotoxins through borate cross-linking reactions, thereby achieving rapid electrochemical detection thereof. The endotoxin biosensor obtained by the present invention has a minimum detection limit of 2.17pM, and can accurately detect endotoxins within the range of 5.0pM to 5.0nM. The endotoxin electrochemical rapid detection method of the present invention has the advantages of being simple, rapid, low cost, high sensitivity, and good selectivity, and is suitable for instant detection of endotoxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a schematic diagram of the detection principle of the present invention.

[0057] Figure 2 These are the feasibility study results of the biosensor of the present invention.

[0058] Figure 3 The distribution of P, B and Fe elements in the biosensor of the present invention.

[0059] Figure 4 This is a sensitivity test of the biosensor of the present invention.

[0060] Figure 5 This is a specificity test of the biosensor of the present invention. DETAILED DESCRIPTION

[0061] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0062] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0063] Figure 1 The core principle of the present invention is as follows:

[0064] Endotoxin molecules contain a long sugar chain, and most of the monosaccharide residues that make up the sugar chain have cis-diol sites. Since cis-diol sites can undergo covalent cross-linking reactions with boric acid groups, a large number of signal probes can be labeled on the sugar chain of endotoxin by means of borate cross-linking reactions, achieving simple, rapid, low-cost, highly sensitive, and highly selective electrochemical detection.

[0065] Example 1 Preparation of bioelectrode

[0066] A 0.5 μM (7.5 μL) solution of a polypeptide terminally modified with a cysteine ​​residue (whose sequence is KKNYSSSISSIHGGGC, SEQ ID NO: 1) was dropped onto the surface of the gold electrode and incubated for 30 min; then, the remaining active sites on the electrode surface were blocked with a 1.0 mM mercaptohexanol (MCH) solution for 15 min to obtain a bioelectrode.

[0067] Example 2 Feasibility Study of Biosensor

[0068] The surface of the bioelectrode prepared in Example 1 was incubated with a 5.0 nM endotoxin solution for 30 min, and then immersed in a 0.15 mM ferrocene boric acid (FcBA) solution for 15 min (the pH of the solution was 8.5). The obtained electrode was inserted into a 50 mM lithium perchlorate solution, and a saturated calomel electrode (SCE) and a platinum wire were used as the reference electrode and the counter electrode, respectively, and detection was performed using square wave voltammetry (SWV) (initial potential: 0.8 V; potential increment: 4 mV; termination potential: 0 V).

[0069] Test results such as Figure 2 As shown in the figure, for the fully modified gold electrode, a strong reduction peak can be observed at 0.4 V; if no peptide, endotoxin or FcBA is added during the modification of the electrode surface, no obvious reduction peak can be observed. It can be seen that the obtained electrochemical signal can be used for the detection of endotoxin.

[0070] Furthermore, the surface of the electrode was subjected to energy spectrum analysis, and the results were as follows: Figure 3 As shown in the energy spectrum analysis results, it can be seen that there are a large number of P, B and Fe elements on the electrode surface, which is consistent with the presence of endotoxin and FcBA on the electrode surface.

[0071] Example 3 Sensitivity Detection of Biosensor

[0072] Endotoxin standard solutions (concentrations of 0, 5, 10, 50, 100, 500, 1000, and 5000 pM, respectively) were prepared, and incubated with the bioelectrode prepared in Example 1 for 30 min, respectively, and immersed in a 0.15 mM ferrocene boric acid (FcBA) solution for 15 min (the pH of the solution was 8.5); the obtained electrode was inserted into a 50 mM lithium perchlorate solution, and a saturated calomel electrode (SCE) and a platinum wire were used as the reference electrode and the counter electrode, respectively, and detection was performed by square wave voltammetry (SWV) (initial potential: 0.8 V; potential increment: 4 mV; termination potential: 0 V).

[0073] Test results such as Figure 4 As shown in the figure, the reduction current increases with the increase of endotoxin concentration. Moreover, in the range of 5.0pM to 5.0nM, there is a good linear relationship between the reduction peak current and the endotoxin concentration. The linear regression equation can be expressed as y = -6.423-2.301lgx (where y is the reduction peak current, in μA; x is the endotoxin concentration, in nM), and the correlation coefficient R 2 =0.99, and the lower limit of detection is calculated to be 2.17 pM. Therefore, the method of the present invention can be used for high-sensitivity detection of endotoxin.

[0074] Example 4 Specific detection of biosensor

[0075] Different types of standard solutions to be tested were prepared, including: 5.0 nM endotoxin, 50 nM human chorionic gonadotropin (HCG), 50 nM mucin 1 (MUC1), 50 nM carcinoembryonic antigen (CEA), 50 nM cyclodextrin, 50 nM dextran and 50 nM glucose.

[0076] The bioelectrode in Example 1 and the detection method in Example 2 were used to detect the corresponding reduction peak currents of different solutions. The results are shown in Figure 5 shown.

[0077] from Figure 5 It can be seen that only endotoxin can produce a strong reduction peak current, while the reduction peak currents produced by other interfering components are close to that of the blank control without endotoxin. It can be seen that the method of the present invention can be used for high-selective detection of endotoxin.

Claims

1. A bioelectrode, characterized in that: The surface of the bioelectrode is bound to a recognition polypeptide; The amino acid sequence of the recognition polypeptide is KKNYSSSISSIHGGGC (SEQ ID NO: 1).

2. The bioelectrode according to claim 1, characterized in that: The active sites on the surface of the bioelectrode that are not bound to the recognition polypeptide are blocked by a blocking agent; Preferably, the blocking agent comprises mercaptohexanol.

3. The method for preparing the bioelectrode according to claim 1, comprising the following steps: The recognition polypeptide is incubated with the electrode, and a blocking agent is added to block the electrode to obtain a bioelectrode.

4. A biosensor, characterized in that: The biosensor comprises the biosensor according to claim 1 or 2, and a signal probe.

5. The biosensor according to claim 4, characterized in that: The signal probe includes at least one of ferrocene, methylene blue, thionine, and quantum dots; Preferably, the signal probe has a boronic acid group.

6. Use of the bioelectrode according to any one of claims 1 to 2 and / or the biosensor according to any one of claims 3 to 5 in the preparation of a product for detecting endotoxin.

7. An endotoxin detection kit, characterized in that: The bioelectrode comprises the bioelectrode according to any one of claims 1 to 2, and / or the biosensor according to any one of claims 4 to 5.

8. The endotoxin detection kit according to claim 7, characterized in that: The endotoxin detection kit comprises an endotoxin standard solution; Preferably, the endotoxin detection kit has a negative control and a positive control; Preferably, the endotoxin detection kit also has instructions.

9. A method for detecting endotoxin, comprising the following steps: 1) incubating a sample to be tested with the bioelectrode according to any one of claims 1 or 2; 2) Add signal probe solution and incubate; 3) Detection.

10. The method according to claim 9, characterized in that: The incubation time is 5 to 120 minutes; The incubation temperature is 4-37°C.

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