CN105203756A - Method for preparing quick magnetic separation electrochemistry immunosensor and method for detecting staphylococcus aureus - Google Patents

Abstract

The invention discloses a method for preparing a quick magnetic separation electrochemistry immunosensor and a method for detecting staphylococcus aureus and belongs to the technical field of biosensing. Supernatant liquid is removed through separation and concentration under the effect of a magnetic field, sediment is dissolved through 0.1 M HNO3 and then subjected to ultrasounds, and the dissolved matter is then mixed with HAC/NaAC. A polished and cleaned electrode is steeped into the mixed solution, and staphylococcus aureus is detected through an SWV electrochemical method. Test results show that the concentration range of staphylococcus aureus detected by the sensor is 2.21*10<2>-2.21*10<7> CFU mL-1, and the detection limit is 83 CFU mL-1. In addition, the immunosensor successfully conducts quantitative detection on staphylococcus aureus in milk, compared with a traditional microorganism method, by means of the technology, the analyzing time is greatly shortened, operation is simple, and the requirement for quickly detecting staphylococcus aureus in the fields of food, environments and the like can be better met.

Description

Method for rapid magnetic separation electrochemical immunosensing and detection of Staphylococcus aureus

technical field

The invention provides a fast magnetic separation electrochemical immunosensing and a high-sensitivity detection method for Staphylococcus aureus, belonging to the technical field of biosensing.

Background technique

Staphylococcus aureus ( Staphylococcusaureus ) is a Gram-positive coccus that widely exists in nature. Staphylococcus aureus Enterotoxins ( Staphylococcus aureus Enterotoxins, SES) is an extracellular toxin secreted by Staphylococcus aureus, which can cause food poisoning, chest pain, vomiting, diarrhea, toxic shock and other symptoms, and its median lethal dose is 0.02 μg/kg. Staphylococcal enterotoxin B (SEB) is often detected in foods with high protein content, such as milk and dairy products. SEB is relatively stable, and after several minutes of high temperature heating, there are still residues, which should be given enough attention.

The current traditional methods for detecting Staphylococcus aureus include plate counting method, multi-tube fermentation method and membrane filtration method. These three methods all have some common disadvantages such as: the detection steps are relatively complicated, the detection cycle is long, and the labor intensity is relatively high. In order to meet the needs of the food industry, several methods for rapid detection of Staphylococcus aureus have been developed at home and abroad in recent years, such as: small-scale biochemical tests, immunological tests, methods based on nucleic acid probes, polymerase chain reaction, etc. These methods have advantages such as convenience and speed. But there are also some disadvantages such as: high detection cost, high dependence on automated instruments, etc. In recent years, in the fields of environmental monitoring and food safety, biosensors have played an increasingly important role in the detection of pathogens and organic matter. Given its high sensitivity and specificity, immunosensing has been recognized as one of the most effective detection tools.

CdTe quantum dots are generally stable, soluble in water, and can receive excitation light to generate fluorescence. Under the action of the cross-linking agent, the preparation of antibody-functionalized quantum dots can form a stable chemical bond between the antibody and the quantum dots, and the resulting connection product is firmly combined and relatively stable, and can specifically detect antigens. Magnetic nanospheres are nanoscale magnetic materials. In addition to the optical and surface properties of general nanoparticles, magnetic nanospheres also have catalytic activity, good magnetic guidance, biocompatibility, and biodegradability. They can be combined with many biomolecules binding to functionalize its surface. Fe ₃ O ₄ is the most used magnetic nanosphere. Fe ₃ O ₄ particles have small diameter, low toxicity, high sensitivity, stable performance, and easy access to raw materials. In addition, they have a large surface area and high surface activity. Therefore, a suitable cross-linking agent can be selected to bind specific antibodies to the magnetic nanospheres, so that the magnetic nanospheres have certain special functions. Under the action of crosslinking reagents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), functionalized magnetic nanospheres can Combined with a specific antigen, under the force of an external magnetic field, the magnetically functionalized biomolecules can move toward the direction of the magnetic field, thereby concentrating on a specific site.

Immunomagnetic bead separation technology uses antigen (or antibody)-coated magnetic beads to specifically bind to antibody (or antigen), and under the action of an external magnetic field, the sample can be separated from the complex matrix to achieve sample enrichment and concentration the goal of. Immunomagnetic bead separation technology is fast and specific, and has the advantages of solid-phase reagents. It does not need to enrich the bacteria to be detected, which helps to reduce the detection limit of the original detection method. Therefore, it has great application value in food, medicine and other aspects.

Contents of the invention

The purpose of the invention is to provide a biosensor with simple operation, short response time, low price and high sensitivity for detecting Staphylococcus aureus. The technical solution adopted by the present invention to solve the above technical problems is: an electrochemical biosensor for detecting Staphylococcus aureus, including an auxiliary electrode, a reference electrode and a working electrode. It is characterized in that: firstly, antibody-functionalized magnetic nanoparticles (Fe ₃ O ₄ -Ab) and antibody-functionalized quantum dots (QDs-Ab) are synthesized respectively. Two kinds of functionalized nanoprobes were combined to the surface of Staphylococcus aureus at the same time, and were rapidly separated by the action of a magnetic field. Combined with the electrochemical stripping voltammetry method, the rapid detection of Staphylococcus aureus in food could be realized.

A kind of rapid magnetic separation electrochemical immunosensing and its highly sensitive detection of Staphylococcus aureus, according to the following steps:

(1) Preparation of CdTe quantum dots:

6mM mercaptopropionic acid (MPA) was added to 2mMCdCl ₂ solution, the pH was adjusted to 9.0 with 1M NaOH solution, N ₂ was passed through and mixed for 30min, and then 0.0625M NaHTe solution was slowly added to form dark yellow CdTe quantum dots. The obtained quantum dot solution was refluxed for 10 hours, the attainable particle size was 2.6 nm, and the concentration was 4.7 μM. The prepared quantum dot solution was ultrafiltered at 15,000 g for 10 minutes at 4°C to remove excess mercaptopropionic acid, rinsed twice, and finally settled to volume with PBS at pH 7.4, and stored in a refrigerator at 4°C .

The reactants in the step (1) are added according to the mass ratio of Cd ²⁺ /MPA/HTe ^- 1:3:0.5.

(2) Functionalization of CdTe quantum dots:

Take the synthesized CdTe quantum dot solution (4.7μM), Staphylococcus aureus antibody at a concentration of 5mg/ml, and the cross-linking reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt Hydroxysuccinimide (EDC) and N-hydroxysuccinimide (NHS) solid particles were mixed and stirred at room temperature for 1 hour. After centrifugation, they were stored in a refrigerator at 4°C.

The volume ratio of CdTe quantum dot solution/Staphylococcus aureus antibody in step (2) is 15:1, and the mass ratio of Staphylococcus aureus antibody/EDC/NHS is 1:6:6.

(3) Functionalization of magnetic nanospheres:

According to the volume ratio of 15:1, take the PBS buffer solution (pH7.4) prepared in the laboratory, with a concentration of 5 mg/mL Staphylococcus aureus antibody, Fe ₃ O ₄ solid powder and cross-linking agent 1-(3-dimethyl Aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), mixed and reacted at room temperature for 1 hour, centrifuged and stored in a refrigerator at 4°C.

The volume ratio of PBS buffer solution (pH7.4)/Staphylococcus aureus antibody in step (3) is 15:1, and the mass ratio of Staphylococcus aureus antibody/Fe ₃ O ₄ solid powder/EDC is 1:6: 6.

(4) Working electrode pretreatment:

Sonicate the bare electrode with deionized water, absolute ethanol and deionized water for 10 minutes respectively, and then repeatedly polish it to the mirror surface with alumina powder with a particle size of 1 μm, 0.3 μm and 0.05 μm, and then place them in ethanol, deionized water, etc. Ultrasonic cleaning in water for 10 min, and finally dry with nitrogen gas for later use.

(5) Modification of the working electrode and preparation of the sensor:

Take the staphylococcus aureus cultured at 37°C and after serial dilution, add the antibody functionalized magnetic beads (Fe ₃ O ₄ -Ab) in step (3), add the rotor at room temperature and stir for 10 minutes, then add the step (2) Antibody-functionalized quantum dots (QDs-Ab), reacted at room temperature for 30 minutes. After the reaction was over, the rotor was taken out and placed on a magnet for adsorption for 20 minutes. Carefully remove the supernatant with a pipette gun, add HNO ₃ (0.1M) made in the laboratory, and sonicate for 40 minutes.

The volume ratio of Staphylococcus aureus, Fe ₃ O ₄ -Ab, QDs-Ab and HNO ₃ is 2:1:1:2.5. After cleaning the electrode surface with PBS, immerse it in 1~2mL HAC/NaAC (pH4.6 ) solution, the rotor stirs evenly.

The above-mentioned fast magnetic separation electrochemical immunosensing and its highly sensitive detection of Staphylococcus aureus are characterized in that: the establishment of the working curve of the fast magnetic separation electrochemical immunosensor and the detection of the actual milk sample by the fast magnetic separation electrochemical immunosensor . The working curve of the prepared fast magnetic separation electrochemical immunosensor is: when the concentration of Staphylococcus aureus is 2.21×10 ² ~2.21×10 ⁷ CFUmL ^-1 , its linear equation is: y=0.9934x-0.3526, and the detection limit is : 83 CFUmL ^-1 . The performance of the prepared fast magnetic separation electrochemical immunosensor also includes good specificity, repeatability and stability as well as the detection of actual milk samples.

Principle: The present invention successfully prepares CdTe quantum dots through the aqueous phase method, and then functionalizes the quantum dots and magnetic nanospheres respectively. The functionalized quantum dots and magnetic nanospheres can specifically bind Staphylococcus aureus to form a sandwich-structure complex to be detected. A magnetic field is applied to the bottom of the test tube, and the complex is enriched at the bottom. By removing the supernatant and washing properly, the target can be completely separated from the impurities, and then the SWV electrochemical detection is performed. According to the obtained data, a standard curve and a linear equation can be obtained to indirectly determine the concentration of Staphylococcus aureus in the sample to be tested, as shown in Figure 1, Figure 2 and Figure 3.

Compared with the prior art, the present invention has the advantages of:

(1) Nanomaterials have a large specific surface area, more surface active sites, and strong bioaffinity. They can be used as carriers to adsorb or support immune molecules, which is conducive to improving many performances of sensors. The immune nano-magnetic beads and quantum dots mainly capture the bacteria through immune recognition, and quickly separate them under the action of a magnetic field without destroying the biological activity of the bacteria. In addition, the specific antigen-antibody combination determines the high sensitivity and specificity of the immunosensor, and is free from interference from other substances. The magnetic separation and electrochemical detection processes are also fast and simple. The whole operation process is simple, fast, practical, and highly sensitive, and provides another safe and effective new method for detecting whether the food contains excessive Staphylococcus aureus.

(2) This experiment combines nanotechnology with electrochemical immunology technology, and through rapid magnetic separation, the quantitative detection of Staphylococcus aureus in milk has good specificity. Compared with traditional microbiological methods, this technology for the detection of Staphylococcus aureus in food greatly shortens the analysis time, is simple to operate, and can better meet the requirements of food hygiene, drinking water safety, environmental protection and control of infectious diseases. Requirements for the rapid detection of Staphylococcus aureus.

Description of drawings

Fig. 1 The preparation of fast magnetic separation electrochemical immunosensor and its detection principle diagram to Staphylococcus aureus;

Fig. 2 SWV curves for detecting different concentrations of Staphylococcus aureus (a) 0, (b) 2.2×10 ² , (c) 2.2×10 ³ , (d) 2.2×10 ⁴ , (e) 2.2×10 ⁵ , (f) 2.2×10 ⁶ , (g) 2.2×10 ⁷ CFU·mL ^-1 ;

Fig. 3 Standard curve diagram of SWV peak current of different concentrations of Staphylococcus aureus.

Detailed ways

The present invention is described in further detail below in conjunction with accompanying drawing embodiment, and concrete operation method is as follows:

(1) Preparation of CdTe quantum dots

26μL of 6mM mercaptopropionic acid (MPA) was added to 50mL of 2mM MCdCl ₂ solution, the pH was adjusted to 9.0 with 1M NaOH solution, mixed with N ₂ for 30min, and then 0.8mL of 0.0625M NaHTe solution was slowly added to form dark yellow CdTe quantum dots. The mass ratio of Cd ²⁺ /MPA/The ^- is 1:3:0.5. The obtained quantum dot solution was refluxed for 10 hours, the attainable particle size was 2.6 nm, and the concentration was 4.7 μM. The prepared quantum dot solution was ultrafiltered at 15,000 g for 10 min at 4°C to remove excess mercaptopropionic acid, rinsed twice, and finally adjusted to 700 μL with PBS of pH 7.4, and placed in a refrigerator at 4°C save.

(2) Functionalization of CdTe quantum dots

The cross-linking reagents used in the present invention include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Specific operation method: Take 470 μL of the synthesized CdTe quantum dot solution, add 30 μL (5 mg/ml) of antibody, mix 1 mg of EDC and NHS, and stir at room temperature for 1 h. After centrifugation, store in a refrigerator at 4°C.

(3) Functionalization of magnetic nanospheres

Take 470 μL of PBS buffer solution (pH7.4) prepared in the laboratory, 1 mg of Fe ₃ O ₄ solid powder, and cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) 1mg, were added to PBS buffer solution (pH7.4). Antibody 30 μL (5 mg/mL) was added. React at room temperature for 1 h, centrifuge and store in a refrigerator at 4°C.

(4) Modification of the working electrode and preparation of the sensor

Take 20 μL of Staphylococcus aureus cultured at 37°C and serially diluted, add 10 μL of antibody functionalized magnetic beads (Fe ₃ O ₄ -Ab) in step (3), add a rotor at room temperature and stir for 10 minutes, then add step ( 2) 10 μL of antibody-functionalized quantum dots (QDs-Ab) was reacted at room temperature for 30 minutes. After the reaction was over, the rotor was taken out and placed on a magnet for adsorption for 20 minutes. Carefully remove the supernatant with a pipette gun, add 25 μL of laboratory-made HNO ₃ (0.1M), and sonicate for 40 minutes. After cleaning the surface of the electrode with PBS, immerse it in 1 mL of HAC/NaAC (pH 4.6) solution and stir well with the rotor.

The invention successfully prepares CdTe quantum dots through an aqueous phase method, and then functionalizes the quantum dots and magnetic nanospheres respectively. The functionalized quantum dots and magnetic nanospheres can specifically bind Staphylococcus aureus to form a sandwich-structure complex to be detected. A magnetic field is applied to the bottom of the test tube, and the complex is enriched at the bottom. By removing the supernatant and washing properly, the target can be completely separated from the impurities, and then the SWV electrochemical detection is performed. A standard curve and a linear equation can be obtained according to the obtained data, and the concentration of Staphylococcus aureus in the sample to be tested can be determined indirectly.

Testing of Standards

Put the Fe ₃ O ₄ -Ab/ S.aureus/ QDs-Ab sensor prepared by the method of specific example 2 in the standard solution of Staphylococcus aureus for reaction, the concentration of the standard solution is 2.21×10 ² , 2.21×10 ³ , 2.21×10 ⁴ , 2.21×10 ⁵ , 2.21×10 ⁶ , 2.21×10 ⁷ CFUmL ^-1 , (the standard solvent is PBS buffer solution, pH7.4), the working buffer system is HAC/NaAC (pH4.6 ), the response current obtained by square wave pulse voltammetry (SWV) was measured. The working curve of the prepared electrochemical immunosensor is: when the concentration of Staphylococcus aureus is 2.21×10 ² ~2.21×10 ⁷ CFUmL ^-1 , its linear equation is: y=0.9934x-0.3526, and the detection limit is: 83CFUmL ^{- 1} . The prepared fast magnetic separation electrochemical immunosensor is easy to operate, and also includes good specificity, repeatability and stability.

specificity verification test

Select other three bacteria: Escherichia coli, Vibrio parahaemolyticus and Salmonella typhi as comparison objects to determine the specificity of the sensor, drop the same concentration (1×10 ⁴ CFUmL ^-1 and 1×10 ⁵ CFUmL ^-1 ) different species of bacteria on the surface of the electrode, when dropping E. coli, Vibrio parahaemolyticus and Salmonella typhi, the data value of the peak current is less than 1μA, and there is no significant change between different concentrations , while the peak current values corresponding to Staphylococcus aureus are 4.07μA and 4.79μA, and the value has increased significantly. Such a huge change is mainly due to the specificity of the antibody for antigen recognition, which once again shows that the immunosensor has a very high selectivity.

Detection of milk samples

In order to verify the practical application value of the immunosensor, the milk added with Staphylococcus aureus was selected as the test sample (the milk was a fresh product purchased from a local supermarket), and 1 mL of milk was taken and diluted to 10 mL with PBS (pH 7.4). The samples were divided into three parts and different concentrations of Staphylococcus aureus (1.2×10 ² , 1.2×10 ³ and 1.2×10 ⁴ CFU·mL ^-1 ) were added, and the immunosensor was used to detect the above-mentioned treated samples respectively, and the detection results The recovery rates were 93.7%, 92.8% and 104.4%, indicating that the immunosensor has high detection accuracy for Staphylococcus aureus in actual samples, and the results are accurate and reliable.

The above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention shall also belong to the scope of the present invention. protected range.

Claims (6)

1. the preparation method of quick Magneto separate electrochemistry immuno-sensing, is characterized in that carrying out according to following step:

(1) preparation of CdTe quantum:

6mM mercaptopropionic acid (MPA) joins 2mMCdCl ₂in solution, by the NaOH solution of 1M, PH is adjusted to 9.0, pass into N ₂mixing 30min, more slowly add 0.0625MNaHTe solution, form dark yellow CdTe quantum; The quantum dot solution backflow 10h obtained, can reach particle diameter is 2.6nm, concentration 4.7 μMs; By the quantum dot solution made by 15000g ultrafiltration 10min under 4 DEG C of conditions to remove excessive mercaptopropionic acid, and rinse twice repeatedly, finally use the PBS constant volume of pH7.4, and the refrigerator being placed in 4 DEG C is preserved;

Wherein in step (1) each reactant according to Cd ²⁺/ MPA/HTe ^-thing mass ratio is that 1:3:0.5 adds;

(2) CdTe quantum functionalization:

Get synthetic CdTe quantum solution (4.7 μMs), concentration is the Staphylococcus aureus antibody of 5mg/ml, and cross-linking reagent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) solid particle, 1h is stirred under mixed room temperature, after centrifuging, place the Refrigerator store of 4 DEG C;

Wherein in step (2), the volume ratio of CdTe quantum solution/Staphylococcus aureus antibody is 15:1, and the mass ratio of Staphylococcus aureus antibody/EDC/NHS is 1:6:6;

(3) magnetic nano-balls functionalization:

Be the PBS buffer solution (pH7.4) that 15:1 gets laboratory and prepares according to volume ratio, concentration is 5mg/mL Staphylococcus aureus antibody, Fe ₃o ₄pressed powder and crosslinking chemical 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), at room temperature hybrid reaction 1h, places the Refrigerator store of 4 DEG C after centrifuging;

Wherein in step (3), the volume ratio of PBS buffer solution (pH7.4)/Staphylococcus aureus antibody is 15:1, Staphylococcus aureus antibody/Fe ₃o ₄the mass ratio of pressed powder/EDC is 1:6:6;

(4) working electrode pre-service:

By bare electrode successively with deionized water, absolute ethyl alcohol and deionized water ultrasonic 10min respectively, repeatedly polish to minute surface with the alumina powder that particle diameter is 1 μm, 0.3 μm, 0.05 μm successively again, then be placed in ethanol, deionized water for ultrasonic cleaning 10min respectively, finally dry up with nitrogen for subsequent use;

(5) modification of working electrode and the preparation of sensor:

Cultivate under getting 37 DEG C of conditions, the staphylococcus aureus after gradient dilution, add the antibody functionalized magnetic bead (Fe of step (3) ₃o ₄-Ab), after adding rotor stirring reaction 10min under room temperature, add the quantum dot (QDs-Ab) that step (2) is antibody functionalized, under room temperature, react 30min; After reaction terminates, take out rotor, standing adsorption 20min on magnet; Carefully take out supernatant liquor with liquid-transfering gun, and add testing laboratory self-control HNO ₃, and ultrasonic 40min (0.1M);

Wherein staphylococcus aureus, Fe ₃o ₄-Ab, QDs-Ab and HNO ₃each volume ratio is 2:1:1:2.5, behind PBS cleaning electrode surface, is immersed 1 ~ 2mLHAC/NaAC(pH4.6) in solution, rotor stirs.

2. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, is characterized in that, it is characterized in that wherein in step (1) each reactant according to Cd ²⁺/ MPA/HTe ^-thing mass ratio is that 1:3:0.5 adds.

3. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, it is characterized in that, the volume ratio that it is characterized in that CdTe quantum solution/Staphylococcus aureus antibody in wherein step (2) is 15:1, and the mass ratio of Staphylococcus aureus antibody/EDC/NHS is 1:6:6.

4. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, it is characterized in that, the volume ratio that it is characterized in that PBS buffer solution (pH7.4)/Staphylococcus aureus antibody in wherein step (3) is 15:1, Staphylococcus aureus antibody/Fe ₃o ₄the mass ratio of pressed powder/EDC is 1:6:6.

5. the preparation method of quick Magneto separate electrochemistry immuno-sensing according to claim 1, is characterized in that, it is characterized in that wherein step (5) staphylococcus aureus, Fe ₃o ₄-Ab, QDs-Ab and HNO ₃each volume ratio is 2:1:1:2.5, behind PBS cleaning electrode surface, is immersed 1 ~ 2mLHAC/NaAC(pH4.6) in solution, rotor stirs.

6. quick Magneto separate electrochemistry immuno-sensing according to claim 1 is to staphylococcus aureus high-sensitivity detecting method, it is characterized in that: the foundation of quick Magneto separate electrochemical immunosensor working curve and quick Magneto separate electrochemical immunosensor are to the detection of actual milk sample; Prepared quick Magneto separate electrochemical immunosensor working curve is: when staphylococcus aureus concentration is 2.21 × 10 ²~ 2.21 × 10 ⁷cFUmL ^-1time, its linear equation is: y=0.9934x-0.3526, and detection is limited to: 83CFUmL ^-1.