Construction Of An Aptasensor For Detecting Enrofloxacin Based On TdTase And RCA Two Isothermal Nucleic Acid Amplification Techniques

Enrofloxacin(ENR)is a synthetic fluoroquinolone antibacterial drug that has been widely used in the prevention and treatment of various animal infectious diseases.Especially in recent years,the application in the prevention and treatment of aquatic animal diseases is developing rapidly.However,the extensive use of ENR may cause residues in animal-derived foods,which seriously affect human health.Among the traditional detection methods,like the high-performance liquid chromatography,requiring complicated sample preparation,and the expensive large instruments take a long time.In this study,the nucleic acid aptamer of enrofloxacin was assembled on the surface of gold nanogold as a recognition nanobioprobe,and the aptamer sensor for ENR was constructed by combining the magnetic beads(Microbeads,MBs)separation technique.Firstly,the amplification of the ENR recognition signal is mediated by the terminal deoxynucleotidyl transferase(TdTase)-mediated growth of the nano-interface DNA at room temperature,which is used to establish a simple and easy-to-use detection of ENR residues in food.Secondly,in this study,a novel tdt-generated RCA sensing and analysis method with universal primer for quantitative detection of ENR was implemented by combining TdTase mediated nano-interface DNA growth and RCA amplification technology.This paper mainly includes the following three parts:(1)Firstly,AuNPs with a particle size of 15 nm was prepared by the classical reduction method,and the aptamer1 of the sulfhydryl group was modified at the 5'-end of the AuNPs surface.The 3'-terminal modified amino aptamer2 was assembled on the surface of a 1 ?m aldehyde-based magnetic bead and characterized by ultraviolet-visible absorption spectroscopy.Secondly,the target molecules were identified by a two-stage aptamer sandwich structure and characterized by a fluorescence spectrophotometer.Further,both probes participate in TdT amplification,verifying that TdTase extends only at 3'-OH and results in a growth product of single-stranded DNA(ssDNA)without acting at the 5' end.Probes and ssDNA were characterized by agarose gel electrophoresis and atomic force microscopy.The results indicate that TdTase can efficiently grow the 3'-OH end of DNA at the nano-interface,with a chain length of 850 nm.(2)Further,we applied this DNA growth strategy in combination with aptamer technology to the aptamer sensor construction study of ENR.A TdTase-based aptamer sensor was established for the quantitative detection of ENR.In the nucleic acid amplification technology,the commonly used PCR technology requires strict temperature control and special equipment.TdTase-based amplification technology eliminates the need for complex sample preparation,amplification of templates,and special equipment to achieve DNA growth and elongation at room temperature.The dual recognition mechanism of the two probes helps to improve the specificity of the detection.When the target is captured,the naked eye can directly observe the color of the nanogold in the supernatant after the sandwich reaction,and then determine the concentration of the target molecule.It is introduced so that the composite structure in the solution can be separated and washed directly under the action of magnetic response.The results show that the sensor has a good response performance to ENR concentration range of 0.01-0.1 mg/mL.At the same time,it can clearly distinguish the control signals of ciprofloxacin(CFX),ofloxacin(OFX)and norfloxacin(NFX).(3)The common RCA technique requires specific primers to be designed for each target molecule.When detecting a variety of target molecules,it is necessary to design a variety of corresponding primers.Nano-biological probes with AuNPs as the carrier need to assemble probes and primers at the same time on the surface of AuNPs,which reduces the assembly efficiency in the presence of multiple DNA chains.In this study,TdT and RCA technologies were further combined to establish a new TdT-based sensing analysis method with universal primer RCA for quantitative detection of ENR.Under the catalysis of TdTase,a single-stranded DNA containing only A base is appropriately amplified at the DNA end assembled on the surface of the AuNPs,and the poly A is hybridized as a primer and a polyT contained in the circular template.A subsequent RCA amplification is initiated.AuNPs is assembled with only one DNA to improve assembly efficiency and reduce detection cost.The TdT amplification at the DNA end generates primers to trigger RCA.The reaction is carried out under isothermal conditions without special equipment,eliminating the need for primer design.The results showed that the sensory analysis method can clearly distinguish the control signals of ciprofloxacin,ofloxacin and norfloxacin,and it is good in the range of 0.01 ~ 1000 ng /mL(Y = 0.1590 + 0.0337 * X).Linear relationship.Statistical analysis showed that the detection limit of ENR was 0.5 pg / mL,and the versatility of the system was achieved by simultaneously distinguishing multiple target molecules.