Bacillus Amyloliquefaciens Spore-based Functional Microspheres For DNA Detection

DNA detection is of great significance in basic and practical application research fields,such as disease diagnosis,drug screening,DNA damage detection,and forensic analysis.Therefore,development of novel DNA detection methods with simple operation and high sensitivity has become one of the scientific research hotspots.According to abundant natural functional groups on the surface of spore?the dormant cell of bacillus?,we constructed respectively two Bacillus amyloliquefaciens spore-based functional microspheres,and investigated their applications in DNA detection:First,fluorescent intercalators?such as SYBR Green I?are widely used to detect target DNA in real samples based on hybridization reaction.The basic principle is that these fluorescent reagents show no fluorescence signal in free state,but there are strong fluorescence signals when they combined with ds DNA.This typical DNA detection method features simplicity and low cost.However,in the actual operation process,the free ss DNA in the sample and residual probe ss DNA can still combine with these fluorescent reagents to produce strong noise signal,reducing the sensitivity of target DNA detection and greatly affecting the detection result.We self-assembled Ti⁴⁺ions onto the surface of Bacillus amyloliquefaciens spore microspheres through chemisorption,successfully constructed a highly dispersed Ti⁴⁺@spore microspheres.The research demonstrated that Ti⁴⁺@spore functional microspheres showed different affinity to ss DNA and ds DNA.Therefore,Ti⁴⁺@spore microspheres could selectively adsorb ss DNA and remove ss DNA in the sample and redundant probes ss DNA during the hybridization reaction by centrifugation,which could reduce the noise caused by ss DNA and greatly improve the signal-to-noise ratio of ds DNA detection and the sensitivity of DNA detection.and present high specificity.The experimental results showed that the linear range of this Ti⁴⁺@spore-based DNA detection method was 0.5 pmol L^-1–2500 pmol L^-1,and the limit of detection was50.0 fmol L^-1?SNR=3?.A series of repeatability measurements of 500 pmol L^-1 gave reproducible results with a relative standard deviation of 4.15%?n=11?,demonstrating that this method has a good prospect in biological application.Second,microspheres?such as magnetic beads,silicon dioxide,and metal oxide?have been widely used as supports for DNA detection?such as liquid phase biochip technique?because of their stability and simple operation.ZIF-8,a metal-organic framework?MOF?material,can be assembled on the surface of Bacillus amyloliquefaciens spore through layer-by-layer method to form ZIF-8@spore microspheres.Results showed that the specific surface area of this biological microspheres was significantly increased from 11.79 m² g^–1 of spore to 987.42 m² g^–1;In addition,ZIF-8@spore microspheres presented strong adsorption ability to streptomycin through the interaction between MOF and protein.As a principle-of-proof,ZIF-8@spore microspheres were used for DNA detection.First,streptavidin was adsorbed on the surface of ZIF-8@spore microspheres through the interaction with ZIF-8.Subsequently,biotin-labeled probe DNA was immobilized on the surface of microspheres by the interaction between streptavidin and biotin.Finally,the target DNA and signal DNA labeled with the fluorophore 6-FAM were conjugated to the microspheres through hybridization reaction for fluorescence analysis.Due to the increase of specific surface area,ZIF-8@spore microsphere could combine with abundant probe DNA,which enhanced the efficiency of hybridization with target DNA,and improved the sensitivity of DNA detection.The experimental results showed that the linear range of this ZIF-8@spore-based DNA detection method was 0.05 nmol L^-1–10 nmol L^-1,and the limit of detection was 10.0 pmol L^-1?SNR=3?.A series of repeatability measurements of 1 nmol L^-1 gave reproducible results with a relative standard deviation of 3.88%?n=11?,indicating that this ZIF-8@spore microspheres present a greet perspective in liquid phase biochip technique.