Electrochemical Biosensors Using Aptamers For Aminoglycoside Antibiotic

Food safety is related to human health and life safety,but also related to social harmony and economic development.However,with the increasing globalization of the economy and the growing international trade in food,the major food safety incidents that endanger human health and life safety have occurred frequently.Therefore,food safety has become a global public health problem.It is an important research topic for analytical researchers that antibiotics,hormones and other harmful substances caused by food safety problems.In order to prevent and treat animal disease,the cultivators heavily use of antibiotics and other chemical drugs in poultry,livestock and aquatic products,which causing the antibiotic residues in animal tissues.Once consumers eat these animal derived foods containing antibiotic residues,it will cause toxic reactions and even death.Aminoglycoside antibiotics are more commonly used antibiotics,which are easily appeared in food especially animal derived food that resulting in excessive antibiotics and causing a variety of injuries to the human body.Therefore,there is an urgent need to establish a simple,sensitive,rapid and accurate method for the detection of aminoglycoside antibiotic residues.At present,the mainly methods of aminoglycoside antibiotics detection are about microbial,immunoassay,chromatography,photometry,electrochemical and so on.In these methods,some need more complex sample preparation and the operation is a time-consuming process,some need large-scale equipment and expensive,and some methods are relatively low sensitivity.Electrochemical analysis is widely used because of its rapid detection,simple device,easy miniaturization,high sensitivity,and is not affected by the turbidity and color of the substrate.Aptamers are small fragments of single stranded nucleotides,which can be DNA or RNA.They can be selectively combined with target molecules.Aptamers are also called "chemical antibodies" because of its nature are similar to antibodies.Compared with the antibodies,aptamers have the advantages of stable,easy to preserve,strong specificity,flexible modification,small molecular weight,controllable binding conditions and wide range of target molecules.Therefore,aptamers have been widely used in the field of environmental detection,clinical diagnosis,food safety detection and other fields.Electrochemical sensor using aptamer has the advantages of simple operation,less sample requirement,fast response and so on.It is applied to the detection of aminoglycoside antibioticsIn order to improve the sensitivity of aminoglycoside antibiotics,we construct two kinds of electrochemical sensors using aptamers.Firstly,the two-dimensional graphene and gold nanoparticles are modified onto glassy carbon electrode by one-step electrochemical deposition.On the one hand,gold nanoparticles/graphene composite nanomaterial increases the specific surface area of the electrode.On the other hand,it serves as a carrier of aptamer and a fully complementary double stranded DNA.The use of exonuclease III selective cleavage of double stranded DNA not only reduces the background current of the sensor and improves the sensitivity of the sensor,but also makes the sensor regenerative,signal amplification and can be recycled.The following are the main contents of this paper:1.A novel and highly sensitive electrochemical biosensor using aptamer for the determination of kanamycin in milk samples was prepared.Firstly,gold nanoparticle/graphene(GNPs/GR)composite nanomaterials with ultrasonic dispersion were modified on the surface of glassy carbon electrode(GCE)by one-step electrochemical co-deposition.GNPs/GR composite nanomaterials increased the area of the electrode.At the same time,self-assembly was used to immobilize the single-stranded with mercapto groups and kanamycin aptamers on the surface of the modified electrode to form an electrochemical sensor.Methylene blue(MB)was used as an electrochemical probe.The electrochemical characterization and kanamycin quantitative determination were performed by electrochemical cyclic voltammetry(CV),electrochemical impedance spectroscopy(EIS)and differential pulse voltammetry(DPV).When the sensor bond with different concentrations of the target kanamycin,it induced some kanamycin aptamers fell off the sensor surface.Due to the decrease of the number of double stranded on the surface of the sensor,the number of MB adsorbed on single-stranded DNA was reduced and the oxidation current of MB was decreased significantly.The logarithm of the oxidation peak current of MB and the concentration of kanamycin was linear in the range of 0.1 to 1000 pmol/L,and the detection limit(S/N = 3)of kanamycin was 0.03 pmol/L.The sensor had good reproducibility(relative standard deviation was 2.3%,n = 11),stability and reproducibility.2.A novel and highly sensitive electrochemical biosensor using aptamer based on cyclic amplification of exonuclease III was constructed.The hybridized streptomycin aptamers double strand were fixed on the gold electrode by mercapto self-assembly technique to make the aptamer sensor.Since the exonuclease III only had a cleavage effect on double-stranded DNA,but not cleavaged single-stranded DNA.Therefore,the background current of the sensor was reduced,so that the sensor could be regenerated to play the role of amplification.With thionine(TH)as a redox indicator,the TH oxidation current is small when exonuclease III cleavaged the double strand from the 3' end of the aptamer sensor into fragmented DNA fragments fell off the surface of the electrode before the sensor bond to streptomycin.When the sensor bond to streptomycin,part of the aptamers fell off the electrode surface,the number of double-stranded DNA decreases.Exonuclease III could only cleavage part of the double-stranded DNA so that the amount of TH adsorbed on the single strand increased and the TH oxidation current increased.The linear relationship between the oxidation peak current of TH and the logarithm of streptomycin concentration was in the range of 1.0 to 10000 pmol/L,and the detection limit(S/N = 3)was 0.3 pmol/L.The aptamer sensor reproducible(relative standard deviation RSD 3.8%,n = 11),high sensitivity,specificity and good stability.It could be used for the analysis of streptomycin in milk samples.