Electrochemical Aptamer Sensors Based On Nanocomposite Containing Of Enzyme And Their Application In Environmental Analysis

Recently,Kanamycin?Kana?is one of common veterinary medicines used in animal husbandry,agriculture and aquaculture in China and thus residue of veterinary drugs in milk attracted more and more attention.Long-term human consumption of Kana residues can cause hearing loss,tinnitus and ear satiety?ototoxicity?;more seriously,it can lead to permanent hearing loss.Lead,a heavy metal,can be enriched in the human body through food chain,then causing a variety of diseases,such as:leukemia,hypertension and so on.In this paper,based on the properties of aptamers specific binding their target,the sensor interface was fabricated by molybdenum disulfide,graphene oxide,metal nanoparticles,and other nano materials.The electrochemical sensors with the character of high sensitivity,good selective,fast response,stable performance were prepared.The main research contents are as follows:?1?Horseradish peroxidase?HRP?,gold nanoparticles?AuNPs?and complementary DNA strand of kanamycin aptamer?cDNA?were used for preparing conjugates?HRP-Au-cDNA?though Au–thiol interactions between AuNPs and cDNA and gold ammonia bond between AuNPs and HRP.Then the AuNPs and HRP-Au-cDNA complex were characterized by UV-vis absorption spectroscopy and transmission electron microscopy.After the cDNA in the conjugates hybridized with Kana aptamers immobilized on a gold electrode though Au–thiol interactions,hydroquinone?HQ?and H₂O₂ in solution were catalyzed by HRP to generate a voltammetric peak current signal.The Kana was detected by a direct competition mode.The electrochemical behavior of the sensor was investigated by cyclic voltammetry?CV?and differential pulse voltammetry?DPV?.After the cDNA in the conjugates hybridized with kanamycin aptamer immobilized on a gold electrode though Au–thiol interactions,hydroquinone?HQ?and H₂O₂ in solution was catalyzed by HRP to generate voltammetric peak current signal.In the presence of kanamycin,aptamers bind with kanamycin to produce a complex on the surface of the gold electrode.The HRP-AuNPs-cDNA conjugates therefore fall off from the electrode surface and the current signal of voltammetric peak decreases.The aptamer sensor was applied to determine kanamycin by differential pulse voltammetry?DPV?.Under the optimal conditions,the cathodic peak current was highly linearly increased with kanamycin concentrations over the range of 0.010 to 150?g/L,with a detection limit of 0.005?g/L.This aptamer sensor was applied in the determination of kanamycin in milk with high sensitivity and selectivity.?2?MoS₂ nanosheets were prepared by a simple liquid-phase ultrasonic stripping method.A biosensor for sensitive detection of Kana was fabricated based on the property that Kana can specifically bind with its aptamer.A composite composed of MoS₂ nanosheets,AuNPs and hemin?He??denoted as MoS₂-Au-He?were prepared by in-situ growth method and electrostatic interaction.The composites possessed high synergetic catalysis activity towards the electroreduction of hydrogen peroxide.Furthermore,glucose oxidase?GOD?and Au NPs were used as marker of the complementary DNA?cDNA?strand of kanamycin aptamer to prepare a conjugate?referred as cDNA-Au-GOD?by self-assembly that was designed as the signal probe.Then the complex was characterized by UV-vis absorption spectroscopy and transmission electron microscopy.Both cDNA-Au-GOD and MoS₂-Au-He were applied to fabricate aptasensor for kanamycin.MoS₂-Au-He acted as solid platform for kanamycin aptamer and signal transmitters.AuNPs were employed as the supporter of cDNA and GOD which catalyze dissolved oxygen to produce hydrogen peroxide in the presence of glucose.Under optimal condition,the electrochemical characteristics of the biosensor were investigated by differential pulse voltammetry.Then cathodic peak current of H₂O₂ was recorded by differential pulse voltammetry.The electrochemical reduction of H₂O₂ was catalyzed by MoS2-Au-He that was modified onto the surface of a glassy carbon electrode?GCE?.The cathodic peak current of H₂O₂ was highly linearly decreased with an increase of kanamycin concentrations from 1.0 ng/L to 1.0×10⁵ ng/L,with a detection limit of 0.8 ng/L.This aptasensor can be used to detect kanamycin in milk with high sensitivity and selectivity.?3?A kind of petaloid CdS nanoparticles with excellent electrochemical luminescence?ECL?performance were prepared by one-step hydrothermal method and were used as electrochemiluminescence?ECL?emitter.A new conjugate?HRP-AuNPs-apt?was prepared with kanamycin aptamer?apt?.The HRP-AuNPs-apt was used as the probes to amplify ECL signal.A composite composed of AuNPs and chitosan?CS?was coated on the surface of a glassy carbon electrode?GCE?which was modified with CdS nanospheres and CS?denoted as CdS/CS/GCE?.After the kanamycin aptamer in the conjugates immobilized on the modified electrode though Au–thiol interactions hybridized with its complementary DNA chain?cDNA?,HRP in the conjugates will catalyze the redox reaction of hydrogen peroxide to cause ECL signal decreasing due to H₂O₂ as a major coreactant in the ECL emitter of CdS nanospheres.Under the optimal conditions,the ECL signal was highly linearly increased with kanamycin concentrations over the range of 0.001 to 100?g/L,with a detection limit of 0.5 ng/L.A novel ECL method by a direct competitive mode was established for determining Kana.This aptasensor was applied in the determination of kanamycin in real samples and satisfactory results were obtained.?4?A novel,simple,and versatile electrochemiluminescence?ECL?sensing platform was developed to detect Pb?II?ions based on HRP-mimicking DNAzyme to catalyze the reduction of hydrogen peroxide.Poly?diallyldimethylammonium chloride??PDDA?with positive charge was adsorbed onto the surface of graphene oxide?GO?and then CdS quantum dots?QDs?with negative charge were enriched to the surface of GO by electrostatic interaction between CdS QDs and PDDA to prepare a composite?denoted as P-GO@QDs?.The P-GO@QDs were characterized by UV-vis absorption spectroscopy,fluorescence spectroscopy,transmission electron microscopy and field emission scanning electron microscopy and were immobilized on the surface of a glassy carbon electrode.CdS QDs can produce ECL signal using hydrogen peroxide as coreactant.G-rich DNA strand,i.e.T30695,can combine with Pb?II?ions to form a stable parallel G-quadruplex.The G-quadruplex can further combine with hemin to form DNAzyme which have a catalysis towards hydrogen peroxide causing a change in ECL signal.Amino-modified T30695 was attached to P-GO@QDs composite by the reaction between the amino group of T30695 and carboxyl group of CdS QDs.When the concentration of Pb?II?in the solution increased,the amount of DNAzyme increased,then catalyzed more H₂O₂ leading to a decrease of ECL signal.The ECL sensor was established based on the principle.The ECL signal of the biosensor was linearly dependent on the logarithm of Pb?II?concentration from 1.0×10^-14 to 1.0×10^-1111 mol/L with a detection limit of 9×10^-12mol/L.An excellent performance of ECL biosensor indicated that it is promising for Pb?II?detection in real samples.There are not obvious differences found between the proposed method and the conventional spectrophotometric method in which dithizone is used as chromogenic agent.