Dual potential ratio-type ECL biosensor and its application in MRSA detection

Abstract

The application discloses a double-potential ratio type ECL biosensor, preparation and application thereof in MRSA detection. The application utilizes aptamer to convert bacteria into nucleic acid signal output, utilizes multi-stage nucleic acid cutting to realize amplification of the nucleic acid signal, and utilizes a large number of trivalent iron active centers in a metal organic gel to activate a co-reaction in an electrochemical reaction, so that two-pole luminescent materials can better emit signals. The nucleic acid signal amplification and signal strength improvement can help the platform to detect low-concentration bacteria, improve the sensitivity of the platform, and realize more accurate detection results through mutual correction of two-stage signals, and realize high-speed detection of low-concentration MRSA in various real samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological analysis detection, and particularly relates to a double-potential ratio type electrochemiluminescence sensor and application thereof in efficient detection of methicillin-resistant Staphylococcus aureus MRSA. BACKGROUND

[0002] In the process of social development, bacteria have always been a major threat to human public health safety. Although the discovery of antibiotics has saved countless patients, it has also been severely misused. With the misuse of antibiotics, some bacteria have developed resistance to the effects of antibiotics, and even break down and actively expel drugs, making clinical treatment of related bacteria a very serious challenge. In the face of bacteria with multiple drug resistance, patients are more dangerous when infected with bacteria, so early diagnosis and treatment of infection has become more important. The traditional methods such as culture counting and PCR commonly used in hospitals can achieve very accurate judgment when detecting bacteria, but often require a long bacterial culture time and complex equipment. Considering the very rapid growth and reproduction rate of bacteria, previous detection methods have been difficult to cope with today's situation. Scientists have tried to detect bacteria in the early stage of infection to better eliminate bacteria and reduce drug use on the basis of developing new antibiotics and other biological drugs. Among them, methicillin-resistant Staphylococcus aureus (MRSA) has received widespread attention since its discovery due to its multiple drug resistance. In the 1960s, a British researcher first discovered MRSA, and in the following decades, with the use of antibiotics, the emergence of MRSA has become increasingly frequent. Due to the multiple drug resistance and strong transmission ability of MRSA, it poses a great threat to postoperative patients, and MRSA-infected patients have been found in hospitals in many developed countries, and the mortality rate of patients after being infected with MRSA is more than 50%. Further studies have shown that the infection rate in relatively underdeveloped regions such as Asia, Africa and Latin America may have exceeded 80%. This poses a serious threat to public safety and food safety.

[0003] With the introduction of various advanced detection methods, electrochemiluminescence (ECL) is widely used in various analytical environments due to its high sensitivity and easy-to-operate characteristics, which can also well solve the challenges faced by bacterial detection. At the same time, the electrochemical workstation has good adaptability to various substrate materials, such as semiconductor quantum dots (QDs) that are widely concerned due to their excellent electrochemical properties, metal-organic frameworks (MOFs) that have excellent loading capacity, and luminol that has good electrochemical signals. Among them, the trivalent iron ion active center in the organic iron gel can catalyze the reaction of the co-reactant and the luminescent material to better occur, and the combination of MOF and quantum dots can make the quantum dots aggregate and better participate in the reaction process. SUMMARY

[0004] The application designs a ratio type double potential ECL biosensor assisted by exonuclease III, constructs an ECL biological detection system for bacteria, especially methicillin-resistant Staphylococcus aureus, and provides a thought for efficient and sensitive detection of bacteria, especially methicillin-resistant Staphylococcus aureus. Specifically, the application relates to a ratio type ECL biosensor and application thereof in MRSA detection. To achieve the technical purpose, the application adopts the following technical scheme:

[0005] The double potential ratio type ECL biosensor of the application is obtained by the method comprising the following steps:

[0006] (1) modifying metal organic gel Fe-MOG on the surface of the electrode and modifying probe DNA on the surface of the gel;

[0007] (2) modifying anodic luminous material ABEI-AuNPs on the DNA anodic signal chain and pairing with the probe DNA in step (1);

[0008] (3) modifying cathodic luminous material ZIF-8@CdS on the DNA cathodic signal chain;

[0009] (4) annealing aptamer DNA, trigger chain 1 and trigger chain 2 and naturally cooling to generate a triple helix structure;

[0010] (5) incubating target bacteria and the triple helix structure obtained in step (4), during which the bacteria combine with the aptamer DNA and release two trigger chains;

[0011] (6) centrifuging the product obtained in step (5), taking the supernatant, mixing with free hairpin DNA, the product obtained in step (3) and exonuclease III, and then adding on the surface of the electrode and incubating at 36-38 DEG C for 2-4 hours.

[0012] Step (1) above realizes the modification of metal organic gel on the surface of the electrode body and the modification of a layer of probe DNA on the surface of the gel through amide bond. The carboxyl on the surface of the metal organic gel and the amino on the DNA are connected through amide bond under the assistance of EDC and NHS. Preferably, the metal organic gel Fe-MOG is synthesized by the following method: a mixture of Fe(NO)3·9H2O (0.3mM) and 1,3,5-benzene tricarboxylic acid (H3BTC, 0.1mM) is ultrasonically treated in 1.75mL deionized water-acetonitrile (v / v=1:1) solution, then the mixture is left to react at 70 DEG C for 2h until a brick red gel is formed, and Fe-MOG is prepared. The solvent is removed by freeze-drying treatment and the powder is ground into powder for characterization and further use, and the dry gel is obtained. Of course, Fe-MOG can also be prepared according to the known literature method.

[0013] After the Fe-MOG was prepared, the modified probe DNA was prepared: it was dispersed in PBS buffer (2 mg / mL) and dropped on the electrode surface until a thin film was formed on the electrode surface, the electrode was inserted into a solution containing EDC and NHS for 30 min to activate the carboxyl group on the surface of Fe-MOG, then 10 μL of 3 μM amino-modified DNA was added to the electrode surface and incubated at 4°C for 3 h, and then 10 μL of amino PEG was added to the electrode surface for 30 min to prevent non-specific binding. Finally, the ABEI-AuNP modified with probe DNA was added to the electrode for 3 h, and the DNA concentration of each step was 3 μM. The amino group of the probe DNA is modified because the carboxyl group of the base material can pair to form an amide bond.

[0014] The sequence of the probe DNA is designed as SEQ ID NO. 1 in the sequence listing as follows:

[0015] 5'-GACAAGCGT TTTCTGTGATGAACTA TTTTTTTTTT-3'

[0016] The above step (2) can realize the linkage of the anode material and the electrode. Preferably, the isoluminol-modified gold nanoparticles, i.e. the anode light-emitting material ABEI-AuNPs, are prepared by reducing chloroauric acid with isoluminol at room temperature. Specifically, the following method is used for synthesis: 9 mL of HAuCl4 (25 mM) solution is mixed with 45 mL of ultrapure water and stirred vigorously. Then 5 mL of ABEI (isoluminol) (4 mM) solution is added and reacted at room temperature for 2 h; finally, another 6 mL of HAuCl4 stock solution is added and the reaction is continued for 2 h until the mixture turns wine red. The method of salt aging is used to modify the thiolated DNA to the surface of gold nanoparticles. 6 μL of 10 μM DNA anode signal chain is mixed with 10 μL of 10 nM AuNPs, then 0.05 M sodium citrate and 0.01% Tween-20 buffer are added and incubated for 3 h, then 20 μL of sodium citrate is added and reacted for 12 h, NaCl solution is continuously added within three days to make the final NaCl concentration 0.3 M, and finally the unbound DNA, gold nanoparticles and excess salt are removed by centrifugation and dispersed in PBS buffer. Of course, the preparation of ABEI-AuNPs can also be prepared according to the known literature method.

[0017] The sequence of the DNA anode signal chain is designed as SEQ ID NO. 2 in the sequence listing as follows:

[0018] 5'-TTTTTTTTTT TAGTTCATCACAGAAA TTGGGCGGTAC-3'

[0019] The base positions of the DNA anode signal chain which are complementary to the probe DNA are underlined in the corresponding sequence, and when they are complementary, one strand is in the direction of 3'-5', and the other is in the direction of 5'-3'.

[0020] Preferably, the cathode light-emitting material ZIF-8@CdS in step (3) is prepared by first reacting 2-methylimidazole and zinc acetate in methanol to prepare ZIF-8, then dispersing the washed and dried ZIF-8 in an ethanol solution and adding thioacetamide and cadmium chloride to react at 60-80°C. Specifically, the following method is used for synthesis: 0.5948 g of zinc acetate hexahydrate and 0.6568 g of 2-methylimidazole are dissolved in 50 mL of methanol, respectively, and after ultrasonic dissolution, they are mixed under stirring, and after high-speed stirring for one hour, the reaction is stopped, and after centrifugation at 10000 rpm for 5 min, the precipitate is washed with methanol and continues to be centrifuged and collected, and after repeating three times, the precipitate is placed in a 60°C oven and dried overnight. 100 mg of the precipitate is dissolved in 20 mL of ethanol, then 30 mg of thioacetamide and 20 mg of hydrated cadmium chloride are added, and after stirring at 70°C for 1 h, the product is collected, washed and dried overnight. The final product is dissolved in ultrapure water (2 mg / mL), then the modified thiol DNA anode signal chain is added and the supernatant is shaken. Finally, it is centrifuged and dispersed in PBS buffer. The thiol group on the DNA is used to form a sulfur-cadmium bond with the CdS on the outer surface of ZIF-8@CdS, so as to realize the connection of the modified ZIF-8@CdS on the DNA anode signal chain.

[0021] wherein the sequence of the DNA anode signal chain is designed by the applicant and is SEQ ID NO. 3 in the sequence listing, as follows:

[0022] 5'-TTTTTTTTTTCATCACAGAAAACGCAATGGTCTTTCTGTGATGCGGTTG-3'

[0023] Preferably, step (4) can be to add trigger chain 1 (6 μL, 100 μM), trigger chain 2 (6 μL, 100 μM) and aptamer (6 μL, 100 μM) to 42 μL of TE buffer, and after annealing at 90°C for 10 minutes, a triple helix structure is formed by natural cooling to room temperature, and then stored in a 4°C refrigerator for standby.

[0024] wherein the sequence of the aptamer DNA is SEQ ID NO. 4 in the sequence listing, the sequence of trigger chain 1 is SEQ ID NO. 5 in the sequence listing, and the sequence of trigger chain 2 is SEQ ID NO. 6 in the sequence listing, as follows:

[0025] 5'-ATGCGGTTGGTTGCGGTTGGGCATGATGTATTTCTGTGATGCGGTTGTTTTT-3';

[0026] 5'-CATCATGCCCAACCGCAATTTTT-3';

[0027] 5'-CAACCGCATCACAGAAATTTTTT-3'.

[0028] Preferably, step (5) determines the concentration of the bacterial species by dilution plate coating method and ultraviolet absorbance value, and the absorbance is detected after the bacteria are cultured in LB medium for 24 hours, and then the concentration of the bacterial suspension is prepared according to the concentration curve. The bacteria are grown in LB medium at 37℃ for 24 hours, and then the concentration of the bacteria is quantified by optical density. Subsequently, centrifugation is performed at 8000 rpm for 10 minutes, and the bacteria are dispersed in PBS buffer, washed repeatedly for 3 times, and stored in a refrigerator at 4℃ for standby. Then, the three-stranded structure in step (4) is centrifuged to obtain the supernatant, and the bacteria after the reaction are removed. The absorbance should be measured at regular time intervals during the bacterial culture to determine the change of the concentration of the bacteria, and after the bacterial culture is completed, the impurities in the culture medium can be removed by centrifugation and TAE buffer washing.

[0029] Of course, in the process of assembling the biosensor, the reaction conditions such as reaction time, magnesium ion concentration, exonuclease III concentration and the ratio of the two kinds of gold nanoparticles can be adjusted to achieve better sensor performance.

[0030] Preferably, the free hairpin DNA sequence in step (6) is designed as SEQ ID NO. 7 in the sequence list, as follows:

[0031] 5'-GTACCGCCCAACCGCTTGTCATGCGGCGGTTGGGCATGATG-3'

[0032] The application provides application of the double-potential ratio type ECL biosensor in bacterial detection, and the target bacteria are Salmonella enteritidis, methicillin-resistant Staphylococcus aureus, Salmonella typhi, Bacillus subtilis or Escherichia coli.

[0033] Specifically, when the ECL biosensor of the application is applied to bacterial detection, the following method steps can be used: (1) dispersing metal organic gel Fe-MOG in 2 mg / mL PBS buffer and dropping on the surface of the electrode body to form a thin film, then inserting the electrode into a solution containing EDC and NHS to react to activate the carboxyl group on the surface of Fe-MOG, and then dropping 10 μL of 3 μM probe DNA modified with amino on the surface of the electrode and incubating at 4℃ for 3 hours;

[0034] (2) 6 μL of 10 μM DNA anode signal chain was mixed with 10 μL of 10 nM ABEI-AuNPs, and then 0.05 M sodium citrate and 0.01% Tween-20 buffer was added and incubated for 3 h, followed by adding 20 μL of sodium citrate and reacting for 12 h, and NaCl solution was continuously added within three days to make the final NaCl concentration 0.3 M, and finally centrifugation was performed to remove the unbound DNA and dispersion was performed in PBS buffer to obtain ABEI-AuNPs modified with DNA anode signal chain, which was added dropwise on the electrode and incubated at 4°C for 3 h;

[0035] (3) ZIF-8@CdS was dissolved in ultrapure water to form a solution of 2 mg / mL, and then the thiol-modified DNA cathode signal chain was added and the supernatant was shaken, centrifuged and dispersed in PBS buffer;

[0036] (4) Trigger chain 1, trigger chain 2 and aptamer DNA were annealed at 90°C for 10 min and then naturally cooled to room temperature to form a triple helix structure, which was stored in a refrigerator at 4°C;

[0037] (5) The gradient concentration bacteria were incubated with the triple helix structure obtained in step (4) at 37°C for 2 h, and then the supernatant was obtained by centrifugation;

[0038] (6) The supernatant in step (5) was mixed with the product obtained in step (3) and exonuclease III, and then was added dropwise on the electrode surface treated in step (2) and incubated at 37°C for 3 h, and the cyclic cleavage and release of the anode material and the connection of the cathode material to the electrode surface were started. During the period, the two trigger chains were opened to free the hairpin and the cathode signal hairpin, and were cut by exonuclease III, and the cut walker chain and the cathode signal chain were released. The walker chain, the probe chain on the electrode surface and the anode signal chain formed a triple helix structure and triggered the enzyme cutting reaction, so that the anode signal chain was cut and the anode material was released from the electrode surface. The cathode signal hairpin was cut to generate the cathode signal chain, and the electrode surface probe chain was combined with the cathode signal chain after the anode signal chain was cut, so that the cathode luminescent material was connected to the electrode, which realized the decrease of the anode signal and the increase of the cathode signal.

[0039] The application also provides a detection system for detecting MRSA, which comprises the double-potential ratio type ECL biosensor described above.

[0040] The application is applied to the specific detection of MRSA, the target bacteria and the triple-stranded structure are incubated for 2 hours, during which the bacteria bind with the aptamer DNA and release two trigger chains; then the released trigger chains are centrifuged to separate the supernatant from the free hairpin DNA, the cathode signal hairpin modified with ZIF-8@CdS and exonuclease III, and then added dropwise on the electrode surface; the two trigger chains open the free hairpin and the cathode signal hairpin (cathode signal chain) and are cut by exonuclease III, releasing the cut walker chain and the cathode signal chain; the walker chain and the probe chain on the electrode surface, the anode signal chain form a triple-stranded structure and trigger the enzyme cutting reaction, so that the anode signal chain is cut and the anode material is released from the electrode surface; after the cathode signal hairpin is cut, the cathode signal chain is generated, and after the anode signal chain is cut, the probe chain on the electrode surface is combined with the cathode signal chain, so that the cathode luminescent material is connected with the electrode, which realizes the decrease of the anode signal and the increase of the cathode signal.

[0041] The application adopts exonuclease III, metal organic gel, metal organic framework and other auxiliary methods, can realize multi-stage signal amplification, and realizes the detection of trace bacteria. Meanwhile, in real samples, selective detection of various bacteria can also be realized. Based on the technology of the application, by adjusting different aptamers and other conditions, specific binding with different types of bacteria can be realized to realize detection, so the target bacteria of the application include but are not limited to enteritis salmonella, methicillin-resistant staphylococcus aureus, typhoid salmonella, bacillus subtilis and escherichia coli and the like. In the application, electrochemiluminescence signals can appear at two potentials. Before the reaction occurs, only the anode exists, after the reaction is completed, the anode signal decreases and the cathode signal appears and increases with the increase of the concentration of the target, realizing the dual-potential ECL detection.

[0042] The principle of the application is analyzed as follows:

[0043] 1. Aptamer recognition of bacteria and nucleic acid amplification

[0044] The affinity of the aptamer to the bacteria is higher, so the aptamer can bind with the bacteria and release two trigger DNAs in the presence of the bacteria, then the released trigger DNAs bind with the free hairpin DNA and the cathode signal hairpin DNA modified on the surface of ZIF-8@CdS to cut and release the walker chain and the cathode signal chain, then the walker and the probe DNA on the electrode surface and the anode signal chain form a triple-stranded structure and trigger enzyme cutting to release the anode material. In this way, multi-stage cutting of nucleic acid is realized.

[0045] 2. Ratio type signal detection of MRSA based on dual-potential electrochemiluminescent material

[0046] The walker chain generated in the reaction can be combined with the double-stranded structure on the electrode surface and trigger the enzyme cutting, after the anode signal chain is cut, the isoluminol modified gold nanoparticles are released away from the electrode surface and lead to the decline of the anode signal; at the same time, due to the release of the cut anode signal chain, the probe DNA enters the single-stranded state, at this time, the cut cathode signal chain will be complementary to it, so that the ZIF-8@CdS introduced into the electrode and the enhancement of the cathode signal are realized.

[0047] The present application utilizes aptamer to convert bacteria into nucleic acid signal output, utilizes multi-stage nucleic acid cutting to realize nucleic acid signal amplification, utilizes a large number of trivalent iron active centers in metal organic gel to activate the co-reaction in the electrochemical reaction, and can help the two-pole luminescent material to better emit signals. Such nucleic acid signal amplification and signal strength enhancement can help the platform to detect low-concentration bacteria, improve the sensitivity of the platform, and realize more accurate detection results by using the ratio type signal output to correct each other through two-stage signals, realize high-speed detection of low-concentration MRSA in various real samples. The multi-stage amplification of DNA in the present application speeds up the enzyme cutting reaction speed. The biosensor platform of the present application has a good linear relationship in the range of 10 0 to 10 7 cfu / mL, and the detection limit (LOD) is 1 cfu / mL, and 10 5 cfu / mL of MRSA can cause a significant ECL response, and the ECL signals of other non-target bacteria are almost the same as the blank signal, so the present application has high selectivity for MRSA detection. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Principle diagram of the ratio type electrochemiluminescence platform for detecting MRSA;

[0049] Figure 2 The multi-stage nucleic acid amplification and two-pole material replacement process are verified by polyacrylamide gel chromatography;

[0050] Figure 3 Characterization of metal organic gel material;

[0051] Figure 4 Characterization of isoluminol modified gold nanoparticles;

[0052] Figure 5 Figure 6 Characterization of ZIF-8@CdS;

[0053] Figure 7 Feasibility verification of the detection platform;

[0054] Figure 8 Condition optimization of the MRSA detection process in Example 1;

[0055] Figure 9 is a plot of the concentration of MRSA versus the log of the ECL ratio in Example 1 and the linear relationship;

[0056] Figure 10 is the detection and selectivity verification of MRSA in real samples in Example 1;

[0057] Figure 11 is the stability of MRSA detection in Example 1. DETAILED DESCRIPTION

[0058] The following examples are further illustrations of the present application and are not intended to limit the scope of the application, as defined in the appended claims, which make reference to the content of the present specification. One skilled in the art can and will know and appreciate any simple change or substitution based on the essential spirit of the present application and should be aware that such change or substitution should fall within the scope of the present application.

[0059] Salmonella enterica (CMCC(B)50335) was provided by China Center for Type Culture Collection. Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300), Salmonella typhi (ATCC 14028), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) were purchased from China General Microbiological Culture Collection Center.

[0060] Example 1

[0061] The nucleic acid multi-stage amplification process was verified on a 10% polyacrylamide gel chromatogram. The aptamer releases two trigger chains in the presence of bacteria, which open two hairpin DNA - free hairpin and cathode signal hairpin - and are cut by enzymes to generate walker chains and cathode signal chains, followed by the formation of a triple helix structure of the walker with the probe DNA and the anode signal chain and the cutting of the anode signal chain, and finally the combination of the probe DNA and the cathode signal chain. Subsequently, electrophoresis was performed in 1xTAE buffer at 96V for 12 hours, and the final electrophoresis result was displayed by a gel imager, as shown in Figure 2 .

[0062] 1.1 Preparation of the ECL detection platform:

[0063] Anodic light emitting material ABEI-Au and electrode surface linkage: 9 mL of HAuCl4(25 mM) solution was mixed with 45 mL of ultrapure water and stirred vigorously. Then 5 mL of ABEI (4 mM) solution was added and reacted at room temperature for 2 h; finally, another 6 mL of HAuCl4stock solution was added and the reaction continued for 2 h until the mixture turned wine red. After mixing 6 μL of 10 μM DNA with 10 μL of 10 nM AuNPs, 0.05 M sodium citrate and 0.01% Tween-20 buffer was added and incubated for 3 h, then 20 μL of sodium citrate was added and reacted for 12 h, NaCl solution was continuously added within three days to make the final NaCl concentration 0.3 M, and finally the unbound DNA, gold nanoparticles and excess salt were removed by centrifugation and dispersed in PBS buffer.

[0064] 1.2 Cathodic light emitting material ZIF-8@CdS surface modified DNA: 0.5948 g of zinc acetate hexahydrate and 0.6568 g of 2-methylimidazole were dissolved in 50 mL of methanol respectively, and after ultrasonic treatment to complete dissolution, they were mixed under stirring. After high-speed stirring for one hour, the reaction was stopped, centrifuged at 10000 rpm for 5 min, washed with methanol and continued to centrifuge to collect the precipitate. After repeating three times, the precipitate was placed in a 60°C oven and dried overnight. 100 mg of the precipitate was dissolved in 20 mL of ethanol, then 30 mg of thioacetamide and 20 mg of hydrated cadmium chloride were added, and after stirring at 70°C for 1 h, the product was collected, washed and dried overnight. The final product was dissolved in ultrapure water (2 mg / mL), then thiol-modified DNA was added and the supernatant was shaken. Finally, it was collected by centrifugation and dispersed in PBS buffer.

[0065] 1.3 Metal-organic gel preparation: Fe-MOG was prepared by ultrasonic treatment of a mixture of Fe(NO)3·9H2O (0.3 mM) and 1,3,5-benzenetricarboxylic acid (H3BTC, 0.1 mM) in 1.75 mL of deionized water-acetonitrile (v / v = 1:1) solution, and then reacted at 70°C for 2 hours until a brick red gel was formed.

[0066] 1.4 Detection application: The metal organic iron gel powder was dispersed in PBS buffer (2 mg / mL) and dropped on the electrode surface until a thin film was formed on the electrode surface, the electrode was inserted into a solution containing EDC and NHS for 30 min to activate the carboxyl group on the surface of Fe-MOG, then 10 μL of 3 μM amino-modified DNA was added to the electrode surface and incubated at 4°C for 3 h, and then 10 μL of amino-PEG was added to the electrode surface for 30 min to prevent non-specific binding. Finally, the DNA-modified ABEI-AuNP was added to the electrode for 3 h, and the DNA concentration of each step was 3 μM. Trigger strand 1, trigger strand 2 and aptamer DNA (6 μL, 100 μM) were added to TE buffer (10 mM, 5 mM Mg 2+ ) and annealed at 90°C for 10 min, then naturally cooled to room temperature to form a triple-stranded structure, and stored in a refrigerator at 4°C for standby. The bacteria were grown in LB medium at 37°C for 24 hours, then the bacterial concentration was quantified by optical density. Subsequently, centrifuged at 8000 rpm for 10 min, dispersed in PBS buffer, washed repeatedly for 3 times and stored in a refrigerator at 4°C for standby. Then the bacteria were mixed with the triple-stranded structure and incubated and centrifuged to remove the supernatant after the reaction. The reaction supernatant, free hairpin DNA, exonuclease III and DNA-modified cathode signal chain were mixed and dropped on the modified electrode surface; the cycle cutting was started to release the anode material and the cathode material was connected to the electrode surface.

[0067] As Figure 2The polyacrylamide gel analysis of nucleic acid reaction process is shown. Lanes 1, 2, and 3 are aptamer and two trigger chains, respectively, and after reaction, a three-stranded structure is generated, resulting in the disappearance of the original bands and the generation of an up-shifted band (Lane 4). After incubation of the three-stranded structure with bacteria and separation, a relatively obvious band appears at the position of the original trigger chain, which indicates that the bacteria can open the three-stranded structure and release the trigger chain in the process of binding with the aptamer (Lane 5). Subsequently, Trigger 1 opens the hairpin DNA in Lane 6 and generates a new band (Lane 7), and then EXO III (Exonuclease III) reacts with the opened hairpin DNA, resulting in the disappearance of the original band and the formation of a down-shifted new band (Lane 8). In order to verify whether the newly generated band is the target walker, the ordered walker chain is added to Lane 9 for verification, but the position does not completely correspond to Lane 8. Considering that EXO III may bind to the DNA chain and affect the running gel results, the ordered walker chain and EXO III are incubated and then added to Lane 10, and the band indicates that it is consistent with the hairpin DNA cleavage result. Lanes 11-15 are similar to Lanes 6-10, which are the reaction process of trigger chain 2 and cathode signal hairpin. After the combination of the two chains (Lane 12), the enzyme cleavage reaction occurs (Lane 13), and finally the ordered cleavage product cathode signal chain and EXO III are incubated and compared (Lane 15), and it is found that the band position is consistent. In the subsequent reaction, the anode signal chain, the probe DNA, and the cathode signal chain can form a relatively stable new structure (Lane 16 double-stranded structure) (Lane 17 three-stranded structure), and without the introduction of exonuclease, the strand displacement does not occur with the cathode signal chain (Lane 18), which ensures that the background signal is at a relatively low level. After the introduction of exonuclease, a new band is generated (Lane 19), which is consistent with the position of the target double-stranded DNA (Lane 20). The above gel electrophoresis results show that the DNA sequences involved in this paper can react according to the design scheme.

[0068] As Figure 3 The characterization of metal organic gel materials, such as Figure 3 As shown in A, after the formation of Fe-MOG, the C=O peak of carboxyl appears at 1631 cm -1 This indicates that the gel can provide sufficient binding sites for amino-modified DNA. At the same time Figure 3 X-ray photoelectron spectroscopy (XPS) spectrum in C shows the presence of C, O, and Fe elements in Fe-MOG. Further detection of high-resolution XPS spectrum of iron element. Figure 3As shown in Figure D, Fe-MOG shows two typical Fe2p peaks at 725.3 and 711.5 eV, respectively, which are the characteristic peaks of Fe2p3 / 2 and Fe2p1 / 2, respectively. 1 / 2 and Fe 2p 3 / 2 peaks, indicating that the main iron in the gel is Fe3+, which is consistent with its brick red color. This proves that the trivalent iron active center we need exists in large quantities in the synthesized gel, and there are more free carboxyl groups that can serve as DNA binding sites.

[0069] As shown in Figure Figure 4 , the hydrodynamic size of ABEI-reduced gold nanoparticles shows an average size of about 18 nm by dynamic light scattering (DLS), while Figure 4 A also shows that the ABEI-gold nanoparticle size is relatively uniform and has good dispersion.

[0070] As shown in Figure Figure 5 , in the process of synthesizing ZIF-8@CdS, we first synthesized ZIF-8 nanoparticles Figure 5 A, C), from the scanning diagram, it can be seen that its structure is a flat polygonal structure, and further observation by TEM image shows that it has a clear polyhedral structure and no protruding structure on the outer surface, which is similar to the expected structure of ZIF-8. As shown in Figure 5 B, D, it can be seen that after modification by CdS particles, the surface of the nanoparticles has changed significantly, and granular substances appear on the outer surface, which is consistent with the ideal CdS particles, and the dispersion and uniformity are relatively good.

[0071] As shown in Figure Figure 6 , after introducing CdS on the surface of ZIF-8, no obvious change in infrared spectrum occurred Figure 6 A). As shown in Figure Figure 6 B, the XRD pattern of ZIF-8@CdS shows multiple diffraction peaks at 2θ(°) = 24.9°, 28.2°, 43.8°, 47.8° and 51.9°, which correspond to different crystal planes of CdS. In addition, the diffraction peaks at 2θ(°) = 10.4°, 12.8°, 14.7°, 16.5° and 18.1° correspond to the crystal planes of ZIF-8 with high crystallinity. These results show that CdS is uniformly attached to the surface of ZIF-8. As shown in Figure Figure 6 C, the XPS measurement spectrum of ZIF-8 shows a main C1s peak at 284.6 eV and a Zn2p peak at about 1000 eV, and a N1s peak at about 400 eV. For the CdS sample, the main Cd 3d peak is about 410 eV, and the S2p peak is about 160 eV. Finally, in the composite material, we can see the presence of the above characteristic peaks, which proves that CdS and ZIF-8 coexist in the composite material. Figure 6In D, a new peak at 400 nm appeared in the complex, which was consistent with the UV peak position of cadmium sulfide, indicating that the complex had the spectral signals of both raw materials.

[0072] As Figure 7 shown, the polished electrode had a relatively small impedance and a high redox current because it was not modified (curve a). When the metal-organic iron gel was added to the electrode surface, the interfacial impedance increased significantly, and the redox current decreased, with the semicircle increasing significantly, which was consistent with expectations (curve d). The DNA chain on the modified electrode carried a large amount of negative charge, which significantly affected the repulsive force of the electrode surface on [Fe(CN)6] -3 / -4 , resulting in a significant increase in electron transfer resistance and a significant decrease in electrochemical current (curve c). After filling the remaining space on the electrode with amino-PEG, the impedance further increased, and the current continued to decrease (curve d). Subsequently, the DNA modified with ABEI gold complementary paired with the DNA on the electrode surface, successfully modifying the electrode surface, resulting in a decrease in interfacial impedance and an increase in redox current, revealing the importance of AuNPs in improving surface conductivity (curve e). After introducing exonuclease and initiating the walker chain to cut the DNA chain modified with ABEI-gold nanoparticles, the release of ABEI-gold nanoparticles resulted in an increase in interfacial impedance and a decrease in redox current (curve f). Finally, introducing DNA modified with ZIF-8@CdS to the electrode also produced similar effects, resulting in an increase in resistance and a decrease in current (curve g). In addition, to study the ECL changes of our sensing platform, the ECL changes were measured using the step-by-step modification method for some important steps. As Figure 7 C shows that the electrochemical signals of the two electrodes are changing significantly as the reaction progresses, with the anodic signal increasing with the modification of ABEI-gold, decreasing after cutting, and finally increasing with the introduction of ZIF-8@CdS-DNA, which is also consistent with expectations, indicating that the biosensor we designed has good performance. The two lines, right high left low before the reaction, left high right low after the reaction. Figure 7 D is the specific numerical change of the signal.

[0073] As Figure 8As shown, in order to obtain the best detection effect, it is necessary to optimize the key factors affecting the reaction process before detection, such as: magnesium ion concentration, enzyme cutting time, enzyme concentration and co-reagent (hydrogen peroxide) concentration. Without changing other conditions: the final signal first increases and then decreases with the increase of magnesium ion concentration, so the optimal magnesium ion concentration is 10 mM; the final signal intensity is better when the exonuclease cutting time is controlled at 4h; the final signal increases with the increase of enzyme concentration, but the change is not obvious after reaching a certain concentration, so the enzyme concentration is controlled at 0.3 U / μL; the hydrogen peroxide concentration is controlled at 10 mM. Under the above optimized conditions, the biosensor is optimized.

[0074] As shown in Figure 9 , the dual-potential ratio type ECL sensor detects different concentrations of MRSA. As shown in Figure 9 A, the electrochemiluminescence intensity changes with the change of the concentration of MRSA. With the increase of the concentration of MRSA, the anodic signal gradually decreases, and the cathodic signal continuously rises. As shown in Figure 9 B, the linear relative is poor, and the linear regression equation in Figure 9 C is obtained after mutual correction of the anodic and cathodic signals: y=0.269xLogC-0.965 (R 2 =0.998), indicating that this biosensor platform has a good linear relationship in the range of 10 0 to 10 7 cfu / mL, and the detection limit (LOD) is 1 cfu / mL. The bacteria with a final concentration of 10 0 -10 7 cfu / mL are added to the serum sample and reacted, as shown in Figure 9 D-9F, and the ECL signal detection is carried out after the reaction is completed, as shown in Figure 9 F, it can be seen that the detection result still has excellent linearity, and the signal change of the anode and the cathode also meets the expectation. Although the trend of the change of the anode and the cathode signal alone is not significant enough, the concentration curve can be corrected after the introduction of the logarithm of the ratio, which also proves that the ratio type ECL biosensor platform has great potential in future development.

[0075] As shown in Figure 10 D, the detection results have high consistency in a variety of common real samples (10% serum, 10% urine, 10% sewage, 1% milk, PBS buffer), which is expected to realize the detection of a variety of real samples in the future. At the same time, in this experiment, we use MRSA and other four non-target bacteria (including Salmonella typhimurium, Salmonella enteritidis, Escherichia coli and Bacillus subtilis) as interference factors, and the detection results in PBS buffer Figure 10 B). The experimental results show that, 105 MRSA at 100 cfu / mL caused a significant ECL response, while the ECL signals of other non-target bacteria at the same concentration were almost the same as the blank signal.

[0076] As shown in Figure 11 To determine whether the repeatability of the sensor is reliable, 10 5 MRSA at 100 cfu / mL was subjected to continuous cyclic potential scanning in the range of -1.3 to +0.6 V. As shown in Figure 11 The average degree of 15 ECL intensity curves obtained by continuous cyclic scanning was high (RSD = 0.305), indicating the stability of the ECL signal.

[0077] It should be noted that the above-mentioned technical content of the present application is only an explanation and clarification to enable those skilled in the art to understand the essence of the present application, so the technical content is not used to limit the essential protection scope of the present application. The essential protection scope of the present application shall be subject to the description in the claims. Those skilled in the art should know that any modification, equivalent replacement and improvement made on the basis of the essential spirit of the present application shall be within the essential protection scope of the present application.

Claims

1. A dual potential ratio ECL biosensor, which is obtained by a method comprising the following steps: (1) modifying a metal organic gel Fe-MOG on the surface of an electrode and modifying a probe DNA on the surface of the gel, wherein the sequence of the probe DNA is SEQ ID NO. 1 in the sequence listing, and the metal organic gel Fe-MOG is prepared by adding trimesic acid and ferric chloride into an acetonitrile solution and then heating; (2) modifying an anodic luminous material ABEI-AuNPs on a DNA anodic signal chain and pairing the DNA anodic signal chain with the probe DNA in step (1) complementarily, wherein the sequence of the DNA anodic signal chain is SEQ ID NO. 2 in the sequence listing; (3) modifying a cathodic luminous material ZIF-8@CdS on a DNA cathodic signal chain, wherein the sequence of the DNA cathodic signal chain is SEQ ID NO. 3 in the sequence listing; (4) annealing an aptamer DNA, a trigger strand 1 and a trigger strand 2 and then naturally cooling to form a triple-stranded structure, wherein the sequence of the aptamer DNA is SEQ ID NO. 4 in the sequence listing, the sequence of the trigger strand 1 is SEQ ID NO. 5 in the sequence listing, and the sequence of the trigger strand 2 is SEQ ID NO. 6 in the sequence listing; (5) incubating a target bacterium with the triple-stranded structure obtained in step (4), during which the bacterium binds to the aptamer DNA and releases the two trigger strands; (6) centrifuging the product obtained in step (5) to obtain a supernatant, mixing the supernatant with free hairpin DNA, the product obtained in step (3) and exonuclease III, and then adding the mixture to the surface of the electrode and incubating at 36-38℃ for 2-4 hours, wherein the sequence of the free hairpin DNA is SEQ ID NO. 7 in the sequence listing.

2. The dual potential ratio ECL biosensor of claim 1, wherein, The anodic luminous material ABEI-AuNPs in step (2) is prepared by reducing chloroauric acid with isoluminol at room temperature.

3. The dual potential ratio ECL biosensor of claim 1, wherein, The cathodic luminous material ZIF-8@CdS in step (3) is prepared by first preparing ZIF-8 by reacting 2-methylimidazole and zinc nitrate in methanol, then dispersing the washed and dried ZIF-8 in an ethanol solution and adding thioacetamide and cadmium chloride, and then reacting at 60-80℃. The aptamer DNA, the trigger strand 1 and the trigger strand 2 in step (4) are annealed at 85-96℃ and then cooled to room temperature.

4. The dual potential ratio ECL biosensor of claim 1, wherein, The incubation in step (5) is performed at 36-38℃ for 1-3 hours.

5. The dual potential ratio ECL biosensor of claim 1, wherein, 6.The dual potential ratio ECL biosensor according to any one of claims 1-5, used in the detection of bacteria. The bacteria are Salmonella enteritidis, methicillin-resistant Staphylococcus aureus, Salmonella typhi, Bacillus subtilis or Escherichia coli.

7. Use according to claim 6, wherein The method comprises the following steps:

8. The use according to claim 6, wherein (1) dispersing the metal organic gel Fe-MOG in a 2 mg / mL PBS buffer and dropping the dispersion on the surface of an electrode body to form a thin film, then inserting the electrode into a solution containing EDC and NHS to react and activate the carboxyl groups on the surface of the Fe-MOG, and then dropping 10 μL of 3 μM probe DNA modified with an amino group on the surface of the electrode and incubating at 4℃ for 3 h; ​ (2) 6 μL of 10 μM DNA anodic signal strand was mixed with 10 μL of 10 nM ABEI-AuNPs, and then added into the buffer of 0.05 M sodium citrate and 0.01% Tween-20 and incubated for 3 h, followed by adding 20 μL of sodium citrate and reacting for 12 h, continuously adding NaCl solution within three days to make the final NaCl concentration 0.3 M, finally centrifuging to remove the unbound DNA and dispersing in PBS buffer to obtain ABEI-AuNPs modified with DNA anodic signal strand, which was added dropwise on the electrode and incubated at 4°C for 3 h; (3) ZIF-8@CdS was dissolved in ultrapure water to form a solution of 2 mg / mL, and then the thiol-modified DNA cathodic signal strand was added and shaken, and the supernatant was collected by centrifugation and dispersed in PBS buffer; (4) The trigger strand 1, trigger strand 2 and aptamer DNA were annealed at 90°C for 10 min and then naturally cooled to room temperature to form a triple-stranded structure, which was stored in a refrigerator at 4°C; (5) The gradient concentration of bacteria was incubated with the triple-stranded structure obtained in step (4) at 37°C for 2 h, and then centrifuged to obtain the supernatant; (6) The supernatant in step (5) was mixed with the product obtained in step (3) and exonuclease III, and then added dropwise on the electrode surface treated in step (2) and incubated at 37°C for 3 h.

9. A detection system for detecting MRSA, comprising the dual potential ratio type ECL biosensor of any one of claims 1-5.

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