Electrochemical biosensor for detecting staphylococcus aureus

Abstract

The invention discloses an electrochemical biosensor for detecting staphylococcus aureus, and belongs to the technical field of biosensors. The method comprises preparation of a biosensor electrode and modification of the biosensor electrode. The sensor is arranged in the wound negative pressure foam dressing, the biological agent in the sensor reacts and feeds information change back to the electrochemical workstation, wound infection signals are captured in time, targeted treatment is performed, the wound healing speed is greatly increased, the workload of manual observation, sampling and inspection is relieved, the detection time is shortened, and the detection efficiency is improved. The sensor is light, small and exquisite in shape, simple and convenient in preparation process and low in raw material cost, the used biological agent is easy to extract, the clinical dosage requirement is conveniently met, and the economic burden of a patient is light.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, in particular to an electrochemical biosensor for detecting Staphylococcus aureus. Background Art

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It is also the main pathogen causing hospital infection, which seriously affects the healing speed of patients' wounds and endangers their health. At present, there are two main methods for routine detection of Staphylococcus aureus in clinical medicine: 1. Smear staining microscopy and bacterial culture: This is a traditional detection method with low cost and easy to use. It is detected by staining or isolating and purifying clinical specimens. However, this method is very time-consuming. It usually takes 1-2 days to obtain a single colony, and then the bacteria are identified by biochemical reactions after isolation and culture. It takes a long time and can no longer meet the requirements of modern clinical rapid detection; 2. Rapid detection method: immunological method (latex agglutination method, enzyme-linked immunosorbent assay, chemiluminescent enzyme-linked immunosorbent assay), molecular biology method (multiplex PCR technology, real-time fluorescence quantitative PCR, loop-mediated isothermal amplification technology LAMP, gene chip technology detection) and biosensor technology. Although immunological and molecular biological methods greatly shorten the detection time, they require expensive instruments, complex preparation processes and high professional skills of operators. At present, biosensors have poor clinical practicality, the sensor manufacturing process is complex and costly, and the sensor response signal is unstable due to interference from other biological factors in the blood sample. Therefore, it is of great practical significance to prepare a biosensor for Staphylococcus aureus detection that is fast, simple, specific, has stable signals, and has low manufacturing cost.

[0003] In recent years, the methods for detecting Staphylococcus aureus have been constantly innovated, but the traditional staining microscopy, bacterial culture method and rapid test method are still the ones that are truly suitable for hospital inspection needs and are often used. Traditional methods take too long and are only suitable for small hospitals with small scale and few pathogens. Although rapid test methods greatly shorten the time, some require expensive instruments, which are too expensive. Some tests are affected by many factors and require high professional skills of operators, which are not easy to master. Biosensor detection is a new technology that has emerged in recent years, but the current situation is that it is not very clinically applicable, has high production costs, and has complicated preparation processes. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies in the above-mentioned background technology and to provide an electrochemical biosensor for detecting Staphylococcus aureus using a biosensor.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention is an electrochemical biosensor for detecting Staphylococcus aureus, comprising the following steps:

[0007] Preparation of biosensor electrodes;

[0008] Modify the sensor electrodes.

[0009] Furthermore, the preparation of the biosensor electrode: using organic polymer material polyimide as raw material, cutting it according to the required size, and using a laser engraving system to carbonize the polyimide material at high temperature to generate a graphene film according to the sensor electrode pattern to form an electrode substrate. The sensor is a three-electrode system, including a working electrode, a counter electrode, and a reference electrode.

[0010] Further, the sensor electrode is modified: the chitosan particles are dissolved with 1M hydrochloric acid HCL solution, and then the pH value is adjusted to 5-6 with 1M sodium hydroxide NaOH solution. After stirring and filtering the undissolved particles in the chitosan solution, the electrode is immersed in the chitosan solution and deposited by electrochemical deposition for 300-600 seconds to obtain a chitosan-modified graphene electrode; a layer of silver chloride Agcl is electroplated on the reference electrode and dried.

[0011] The electrochemical method, fixation of sortase A: dilute sortase A dry powder particles with HEPES buffer, drop the diluted enzyme solution onto the working electrode of the sensor, add glutaraldehyde solution after drying, and let it stand and dry for 12 hours before using for measurement.

[0012] The prepared sensor is placed in a negative pressure foam dressing. When there is a Staphylococcus aureus infection, the sortase A in the sensor electrode will specifically bind to the surface protein of Staphylococcus aureus. The sensor electrode will collect the signal and send it to the electrochemical workstation. The workstation uses differential pulse voltammetry to detect changes in the electrical signal, thereby sensing Staphylococcus aureus.

[0013] The electrochemical method has the following detection conditions for differential pulse voltammetry: the starting point is 0.2V, the highest point is 1.0V, the electrochemical biosensor is a biosensor, chitosan is used as a carrier on the working electrode, sortase A is dripped and fixed on the working electrode, and the drip concentration of sortase A is very important for the detection limit.

[0014] According to the electrochemical method, sortase A is suitable for storage under low temperature conditions. After testing, sortase A still maintains good activity after 6 months at -20°C.

[0015] The present invention has the following beneficial effects:

[0016] 1. The present invention prepares a biosensor, utilizing the characteristics of the specific binding of sortase A on the working electrode of the sensor to the surface protein of Staphylococcus aureus, transmits the captured signal to the electrochemical workstation, and senses the change of the electrical signal through the electrochemical differential pulse voltammetry (DPV method), thereby sensing Staphylococcus aureus;

[0017] 2. The sensor of the present invention is placed in the trauma negative pressure foam dressing, which can monitor the wound condition at any time without the need for medical staff to collect samples and send them for inspection. The sensor can collect samples from the wound exudate, react with the biological agent in the sensor and feed back the information changes to the electrochemical workstation, so as to timely detect the wound infection, reduce the workload of manual observation, sampling and inspection, and shorten the detection time. There are currently no similar products on the market, and the present invention fills the gap in bedside body surface monitoring.

[0018] 3. This biosensor uses polyimide, an organic polymer material with excellent comprehensive performance, as its raw material. It is resistant to high temperatures and has strong stability. It is carbonized into a graphene film at high temperatures. It has the characteristics of efficient conductivity, high strength and light weight. The sensor is light and compact.

[0019] 4. The preparation process of this biosensor is relatively simple, the cost of raw materials is low, and the biological agents used are easy to extract, which is convenient for meeting clinical dosage requirements. This sensor is fast, highly sensitive, and highly specific, with a detection limit of 104cfu / mL.

[0020] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the sensor electrode pattern;

[0022] Figure 2 The schematic diagram of the preparation of the biosensor and the detection process of Staphylococcus aureus;

[0023] Figure 3 This is the principle diagram of differential pulse voltammetry;

[0024] Figure 4 The activity characteristic curve of sortase A detected by cyclic voltammetry;

[0025] Figure 5 This is the characteristic curve obtained by differential pulse voltammetry measurement. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0027] See also Figure 1-5 The present invention provides a technical solution: an electrochemical biosensor for detecting Staphylococcus aureus. The biosensor of the present invention is used for real-time monitoring of wound infection in patients with clinical burns and trauma in hospitals. The biosensor is wrapped in a trauma negative pressure foam dressing through a special process, and the exudate in the dressing can be directly sampled, and the collected signal is transmitted to the electrochemical workstation at all times. Through electrochemical detection means, the infection can be quickly discovered for targeted treatment. At present, there is no surface biosensor on the market that can be fixed in a trauma dressing and can sensitively and quickly detect Staphylococcus aureus.

[0028] This sensor is made with advanced technology, which improves the traditional sensor preparation process, which is complicated (such as raw materials need to be polished, calcined at high temperature, chemically treated, etc.), long preparation time, high cost and other problems. This product uses polyimide as the sensor raw material, which is one of the organic polymer materials with the best comprehensive performance. It can withstand high temperatures of more than 400°C. As a special engineering material, it has been widely used in aerospace, microelectronics, nano, liquid crystal, laser and other fields. According to the sensor requirements, such as Figure 1 As shown, the sensor electrode pattern is drawn using computer CAD technology, and then engraved on the polyimide material through a laser system. When the laser is irradiated on the polyimide material, local heating causes it to be carbonized and forms a graphene film, i.e., the sensor electrode. Graphene has the characteristics of efficient conductivity, high strength and light weight. A layer of chitosan is modified on the graphene working electrode by electrodeposition. After biological reagents are added to the surface of the chitosan film, the biosensor is completed. The sensor is packaged and wrapped in a negative pressure foam dressing for use.

[0029] Wound dressings are widely used in wound care, but existing dressings only have care functions such as wound bandaging and absorbing exudate. The infection of the wound in the dressing can only be observed or sampled for inspection by changing the dressing or repeatedly tearing off the dressing, resulting in untimely treatment of wound infection and slow recovery of the wound. The present invention designs a wound dressing containing a biosensor, which not only has the isolation, antibacterial and exudate adsorption functions of existing dressings, but the biosensor contained therein can also sample from the wound exudate at any time and timely feedback the signal to the electrochemical workstation, which is convenient for medical personnel to detect infection in time and treat symptomatically, greatly improving the efficiency of wound healing, reducing the workload of medical staff for sampling and inspection, and reducing the pain of patients. It is very practical in wound care.

[0030] The following steps are involved:

[0031] Preparation of biosensor electrodes;

[0032] Modify the sensor electrodes.

[0033] Furthermore, the preparation of the biosensor electrode: using organic polymer material polyimide as raw material, cutting it according to the required size, and using a laser engraving system to carbonize the polyimide material at high temperature to generate a graphene film according to the sensor electrode pattern to form an electrode substrate. The sensor is a three-electrode system, including a working electrode, a counter electrode, and a reference electrode.

[0034] Further, the sensor electrode is modified: the chitosan particles are dissolved with 1M hydrochloric acid HCL solution, and then the pH value is adjusted to 5-6 with 1M sodium hydroxide NaOH solution. After stirring and filtering the undissolved particles in the chitosan solution, the electrode is immersed in the chitosan solution and deposited by electrochemical deposition for 300-600 seconds to obtain a chitosan-modified graphene electrode; a layer of silver chloride Agcl is electroplated on the reference electrode and dried.

[0035] The electrochemical method, fixation of sortase A: dilute sortase A dry powder particles with HEPES buffer, drop the diluted enzyme solution onto the working electrode of the sensor, add glutaraldehyde solution after drying, and let it stand and dry for 12 hours before using for measurement.

[0036] The prepared sensor is placed in a negative pressure foam dressing. When there is a Staphylococcus aureus infection, the sortase A in the sensor electrode will specifically bind to the surface protein of Staphylococcus aureus. The sensor electrode will collect the signal and send it to the electrochemical workstation. The workstation uses differential pulse voltammetry to detect changes in the electrical signal, thereby sensing Staphylococcus aureus.

[0037] The detection conditions of differential pulse voltammetry in the electrochemical method are: the starting point is 0.2V, the highest point is 1.0V, the electrochemical biosensor is a biosensor, chitosan is used as a carrier on the working electrode, and the sorting enzyme A is added and fixed to the working electrode. The addition concentration of the sorting enzyme A is very important for the detection limit. The biosensor that collects Staphylococcus aureus signals fills the gap in bedside detection of Staphylococcus aureus, provides detection convenience, captures wound infection signals in time and makes targeted treatments, greatly improves the wound healing speed, has a simple preparation process, a reasonable cost price, and a light economic burden on patients.

[0038] In the electrochemical method, sortase A is suitable for storage under low temperature conditions. After testing, sortase A still maintains good activity after 6 months at -20°C.

[0039] like Figure 1 As shown, working electrode radius: 1.5mm; electrode strip width: 2.0mm; electrode spacing:

[0040] 2.0mm; the length, width and thickness of the whole PI piece: 2.9cm×1.5cm×0.125mm.

[0041] Tool: CO2 laser cutter; Laser: Power 15% Speed ​​8% Height -0.1mm.

[0042] like Figure 3 As shown, through cyclic voltammetry detection, the biosensor prepared by the present invention underwent a redox reaction at the working electrode during the test, with an obvious CV cycle peak. This was the result after the electrode was prepared and stored at room temperature for the third day, indicating that the enzyme activity was acceptable under room temperature storage.

[0043] like Figure 5 As shown, after the activity of sortase A was detected by cyclic voltammetry, the electrode was cleaned and characteristic curves were obtained by differential pulse voltammetry for Staphylococcus aureus solutions with different concentrations.

[0044] Embodiment 1:

[0045] 1. Using organic polymer material polyimide as raw material, cut it according to size, and use a laser engraving system to carbonize the polyimide material at high temperature to generate a graphene film according to the sensor electrode pattern to form an electrode substrate. The sensor is a three-electrode system, including a working electrode, a counter electrode, and a reference electrode.

[0046] 2. Dissolve the chitosan particles with 1M hydrochloric acid HCL solution, then adjust the pH value to 5-6 with 1M sodium hydroxide NaOH solution, stir and filter the undissolved particles in the chitosan solution. Immerse the electrode in the chitosan solution and deposit it by electrochemical deposition for 300-600 seconds to obtain a chitosan-modified graphene electrode; electroplate a layer of silver chloride Agcl on the reference electrode and dry it.

[0047] 3. Use HEPES buffer to dilute the sortase A dry powder particles, add the diluted enzyme solution dropwise to the working electrode of the sensor, add glutaraldehyde solution after drying, and let it stand and dry for 12 hours to complete the solidification of sortase A.

[0048] 4. The biosensor electrode prepared by this method was immersed in Staphylococcus aureus liquid for detection. The detection method was differential pulse voltammetry, and the conditions were: the starting point was 0.2V and the highest point was 1.0V.

[0049] The results measured by the above method have obvious and regular peak current values.

[0050] Example 2

[0051] 1. Using organic polymer material polyimide as raw material, cut it according to size, and use a laser engraving system to carbonize the polyimide material at high temperature to generate a graphene film according to the sensor electrode pattern to form an electrode substrate. The sensor is a three-electrode system, including a working electrode, a counter electrode, and a reference electrode.

[0052] 2. Dissolve the chitosan particles with 1M hydrochloric acid HCL solution, then adjust the pH value to 5-6 with 1M sodium hydroxide NaOH solution, stir and filter the undissolved particles in the chitosan solution. Immerse the electrode in the chitosan solution and deposit it by electrochemical deposition for 300-600 seconds to obtain a chitosan-modified graphene electrode; electroplate a layer of silver chloride Agcl on the reference electrode and dry it.

[0053] 3. Use 1% chitosan solution to dilute the sortase A dry powder particles, add the diluted enzyme solution dropwise onto the working electrode of the sensor, and let it stand and dry for 12 hours to complete the solidification of the sortase A.

[0054] 4. The biosensor electrode prepared by this method was immersed in Staphylococcus aureus liquid for detection. The detection method was differential pulse voltammetry, and the conditions were: the starting point was 0.2V and the highest point was 1.0V.

[0055] The results measured by the above method do not have obvious and regular peak current values.

[0056] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing an electrochemical biosensor for detecting Staphylococcus aureus, characterized in that: The following steps are involved: Preparation of biosensor electrodes: Using organic polymer material polyimide as raw material, cutting it according to the required size, and using a laser engraving system to carbonize the polyimide material at high temperature to generate a graphene film according to the sensor electrode pattern to form an electrode substrate; Modify the sensor electrodes.

2. The electrochemical biosensor for detecting Staphylococcus aureus according to claim 1, characterized in that: Preparation of biosensor electrodes: Using organic polymer material polyimide as raw material, cut it according to the required size, and use a laser engraving system to carbonize the polyimide material at high temperature to generate a graphene film according to the sensor electrode pattern to form an electrode substrate. The sensor is a three-electrode system, including a working electrode, a counter electrode, and a reference electrode.

3. The electrochemical biosensor for detecting Staphylococcus aureus according to claim 1, characterized in that: Modify the sensor electrode: dissolve the chitosan particles with 1M hydrochloric acid HCL solution, then adjust the pH value to 5-6 with 1M sodium hydroxide NaOH solution, stir and filter the undissolved particles in the chitosan solution, immerse the electrode in the chitosan solution, and deposit it by electrochemical deposition for 300-600 seconds to obtain a chitosan-modified graphene electrode; electroplate a layer of silver chloride Agcl on the reference electrode and dry it.

4. The electrochemical biosensor obtained according to the method of claims 1-3.

5. Use of the electrochemical biosensor according to claim 4 in detecting characteristic sequences of Staphylococcus aureus.

6. The electrochemical method for detecting Staphylococcus aureus using the electrochemical biosensor according to claim 4, characterized in that: Fixation of sortase A: dilute sortase A powder particles with HEPES buffer, drop the diluted enzyme solution onto the working electrode of the sensor, add glutaraldehyde solution after drying, and let it stand and dry for 12 hours before use for measurement; The prepared sensor is placed in a negative pressure foam dressing. When there is a Staphylococcus aureus infection, the sortase A in the sensor electrode will specifically bind to the surface protein of Staphylococcus aureus. The sensor electrode will collect the signal and send it to the electrochemical workstation. The workstation uses differential pulse voltammetry to detect changes in the electrical signal, thereby sensing Staphylococcus aureus.

7. The electrochemical method for detecting Staphylococcus aureus according to claim 6, characterized in that: The detection conditions of differential pulse voltammetry are: the starting point is 0.2V, the highest point is 1.0V, the electrochemical biosensor is a biosensor, chitosan is used as a carrier on the working electrode, and sortase A is added and fixed to the working electrode. The addition concentration of sortase A is very important for the detection limit.

8. The electrochemical method according to claim 6, characterized in that: Sortase A is suitable for storage under low temperature conditions. After testing, Sortase A still maintains good activity after 6 months at -20℃.

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