Study On Aptasensor And Biomimetic Nanodelivery For Staphylococcus Aureus

Objective:The increasing prevalence of S.aureus infections and the widespread of drug-resistant bacteria pose a serious threat to global public health.Therefore,timely,rapid and accurate detection of S.aureus and effective treatment are important for food safety,environmental monitoring,clinical treatment and prevention of the spread of drug-resistant bacteria.This study focuses on the current dilemma of S.aureus detection and treatment by constructing several highly sensitive SA biosensors based on specific biorecognition element aptamers and detection signal amplification strategies,and constructing a biomimetic nanodelivery based on bacterial outer membrane vesicles for the delivery of PNA and to explore the antisense therapeutic effect of PNA on S.aureus.Methods:（1）Construction of a fluorescent aptasensor for S.aureus based on a novel simple amplification strategy.The biotin-modified aptamer binder to four FAM-modified pc DNAs via base-pairing to form heterodimeric sequence（biotin-aptamer&FAM-pc DNAs）and was immobilized on streptavidin modified MNP_S by strong affinity interactions between biotin and streptavidin to form biotin-aptamers&FAM-pc DNAs@streptavidin-MNP_S detection probes.When the aptamer specifically recognized and binder to S.aureus,the four FAM-pc DNAs beacon probes in the biotin-aptamers&FAM-pc DNAs@streptavidin-MNP_S detection probes were replaced and released into the supernatant,and the concentration of S.aureus can be quantified by measuring the fluorescence intensity of the replaced beacon probes FAM-pc DNAs（λexc/em=492/520 nm）;（2）Construction of an electrochemical aptasensor for S.aureus based on redox biocapacitance.Chi cathodic electrodeposition was first performed on the surface of GCE,followed by Cat anodic grafting on the Chi film.The redox capacitor amplification system was constructed using Cat-Chi film and Fc-Ru³⁺double mediators.The aldehyde-modified aptamer was immobilized on the electrode surface by the Schiff base reaction with the amino group of Chi.When the aptamer specifically recognized and binder S.aureus,the S.aureus limited the migration of ions in the double dielectric and blocked charge transfer from the electrode.CV detects and records the reduction in peak current signal caused by S.aureus;（3）Construction of a highly sensitive electrochemical sensor for S.aureus based on multiple amplification strategy.Biotin-aptamer 1 binder to streptavidin-modified MNP_S to form the streptavidin-MNP_S@biotin-aptamer 1 capture probe.Biotin-aptamer 2 binder to four pc DNAs-SH to form the hybrid sequence pc DNAs-SH&biotin-aptamer 2,which was then bound to the MNP_S to form pc DNAs-SH&biotin-aptamer 2@streptavidin-MNP_S detection probes through the strong affinity interactions between biotin and streptavidin.The S.aureus which was captured by the streptavidin-MNP_S@biotin-aptamer 1 capture probe in the sample could displaces pc DNAs-SH in the pc DNAs-SH&biotin-adaptor 2@streptavidin-MNP_Sdetection probe.Then we could detect pc DNAs-SH using an electrochemical sensor based on the Cat-Chi-Fc-Ru³⁺redox capacitor amplification system,with capacitance change（Q₀-Q）/Q₀ as the analytical index;（4）Biomimetic bionanodelivery of PNA and its antisense treatment of S.aureus.MSN were first prepared by sol-gel method.The EV secreted by S.aureus was extracted using an affinity resin-based chromatography kit and coated on the MSN by simple sonication（50 W,3 min）.The stability,the biocompatibility（hemolysis assay）and internalization of the MSN@EV were evaluated.The PNA drug was encapsulated by simple physical adsorption of MSN,and the encapsulation rate and drug loading capacity were calculated.The in vitro release and in vitro antimicrobial effects of the constructed PNA@MSN@EV biomimetic nanodelivery system was initially explored.Results:（1）The proposed fluorometric aptasensor based on a novel simple amplification strategy of replacing four beacon probes with one S.aureus has good specificity,ultra-high sensitivity（1.23 cfu/m L）and wide linear range（1～10⁸ cfu/m L）.The recovery test further demonstrated that the proposed fluorometric aptasensor can detect S.aureus in spiked blood samples;（2）The electrochemical aptasensor based on Cat-Chi-Fc-Ru³⁺redox capacitance signal amplification strategy constructed in this study showed good performance with fast detection（25 min）,wide linear range（10～10⁸ cfu/m L）,high sensitivity（2 cfu/m L）,and also good regeneration,stability and specificity.The electrochemical aptasensor can also directly quantify S.aureus in whole blood samples;（3）The proposed electrochemical sensor based on MNP_S enrichment and separation system,an S.aureus replacement four beacon probes and Cat-Chi-Fc-Ru³⁺redox capacitor amplification system showed single cell detection capability（<1 cfu/m L）,strong specificity and it is stable and reusable.The recovery experiments further demonstrated that the constructed electrochemical sensor could be used for the detection of blood samples;（4）The MSN prepared by the sol-gel method has a uniform particle size（100 nm）and a clear pore size（hexagonal）,which could be used to load PNA with good encapsulation efficiency（62.90%）and drug loading（7.74%）.The concentration of EVs extracted by affinity resin chromatography was about 1.74 mg/m L.All characterization showed that EV could be well coated on MSN through simple ultrasonic treatment（50 W,3 min）,and its stability and blood compatibility was good.In vitro antibacterial test showed that PNA@MSN@EV exhibited enhanced antibacterial activity against S.aureus and revealed strong bactericidal activity at equivalent PNA concentration（8μM）,which was significantly higher than that of free PNA.Conclusion:（1）The novel and simple amplification strategy can significantly improve the sensitivity of existing fluorescent aptasensor so that it can be used as a powerful tool for early detection of pathogens and provide new ideas for clinical exploration of pathogenic bacteria diagnostic methods.（2）The Cat-Chi-Fc-Ru³⁺redox capacitor-amplification based electrochemical aptasensor with good detection performance and fast detection speed for timely response and real-time monitoring,demonstrating its potential application in POCT systems and integration in portable,implantable and/or wearable devices.（3）The highly sensitive electrochemical sensor based on multiple amplification strategy enables single cell level monitoring and is stable and reusable,removing interference from the complex environment of the sample,making it widely applicable to food safety,environmental monitoring and clinical diagnosis.（4）The EV-coated MSN biomimetic nanodelivery system provides a novel strategy to enhance PNA for the treatment of bacterial infections,and the technology is expected to the delivery of antibiotics that are traditionally less effective or susceptible to resistance.