Highly Sensitive Detection Of Bisphenol A By Using DNA Hybridization Induced Quenching Of Fluorescence With Biochar

Bisphenol A,known as 2,2～(4-hydroxyphenyl)propane,or BPA,is an exogenous endocrine disruptor and an additive in synthetic plastic packaging products.It is one of the most serious environmental contaminants,often present at low levels in various water sources.At present,human life is inseparable from plastic products,which makes BPA extremely easy to enter the human body,and it will seriously damage the functions of various organs and endocrine,immune,nervous and other systems.Therefore,it is urgent to detect BPA in water environment.Aptamers are oligonucleotide fragments screened in vitro that can specifically bind proteins or other small molecules.The base pairs are generally 25-80.Aptamer has many important advantages,such as strong molecular affinity,convenient and rapid synthesis,good stability and high resolution effect.As a new type of molecular element recognition,the sensor technology based on aptamer has shown good results and has a promising future.At present,aptamer sensors have been widely used in environmental detection,agriculture,medicine and other fields,showing unique advantages,and all have good detection effect.Magnetic bead is a kind of ball with good dispersion and regular round shape,which can make a fast induction to the magnetic field and has strong magnetic properties,be used to separate,wash and capture target molecules.It can covalently couple DNA,polypeptide,protein and other biological ligands to its surface under the interaction of specific chemical reagents.Carboxylated magnetic beads are modified carboxyl groups to the surface of magnetic microspheres,which can be directed to biological ligands with amino groups.The average particle size is about 2.8 μm.Biochar(BC)is a carbon-rich solid product obtained by pyrolysis of biomass materials(such as wood or leaves,etc.)under anoxic or anaerobic conditions.It has advantages such as convenient preparation conditions,low price,large specific surface area,good stability and strong adsorbability.Moreover,the functional groups of biological carbon are abundant and can be obtained under different preparation conditions.After being dissolved in water,it can be well dispersed in aqueous solution by ultrasonic treatment,so it can be modified by biomolecules(such as DNA,antibodies or proteins)in aqueous state.Therefore,if biochar can quench fluorescence,it has inestimable prospects in the application of biosensors as an alternative to expensive fluorescence quenchers such as graphene.In this paper,the biochar nano fluorescence quencher with complementary oligonucleotide chain coupling,and BPA specific aptamer labeled with FAM as recognition elements coupled with carboxyl,magnetic beads,both through the principle of complementary base pairing build fluorescence biosensor(MB-Apt-BC)to detect BPA.To explore the construction of nano fluorescence quenching components(BCcDNA),the feasibility of the fluorescent biosensor(MB-Apt-BC)detection of BPA mechanism,build the best condition of the sensor(that is,the complementary DNA base length,concentration of BC-cDNA,hybridization temperature of MB-Apt and BC-cDNA,hybridization time of MB-Apt and BC-cDNA),the best incubation time of the sensor with BPA,under the above optimum experiment conditions,gradient concentration of BPA to explore the performance of the sensor,and its detection specificity,anti-interference of cation and ions and detection reliability in real water were analyzed.The following conclusions are drawn:(1)A kind of biochar nanoparticles(BC)was prepared by pyrolysis of bagasse,which was suitable to be used as fluorescence quenching agent and rich in carboxyl group.The BC-cDNA was constructed by chemical bond coupling with complementary oligonucleotide chain(cDNA).(2)The MB-Apt fluorescent probe was constructed by chemical bond fixation method,and the fluorescence biosensor(MB-Apt-BC)was successfully constructed by hybridization with the nano fluorescence quencher(BC-cDNA).when the concentration of BC-cDNA was 120ng/m L,hybridization time was 1 hour,the hybridization temperature was 37℃ and the incubation time of MB-Apt-BC with BPA was 40 minutes,the BPA detection effect was the best.(3)In the detection of BPA by MB-Apt-BC biosensor,With the gradient change of BPA concentration,the recovery degree of sensor fluorescence intensity also shows a linear change in the range of 0-50 ng/m L,and the minimum detection concentration was 1.12 ng/m L(4.9n M).The constructed MB-Apt-BC fluorescence biosensor can realize the specific detection of BPA,and can accurately detect BPA when added to various cation and cation ions,indicating that the fluorescence biosensor has good antiinterference characteristics;the BPA detection method was proved to be reliable by calculating the recovery rate of the actual water sample.