Colorimetric Methods For MG Detection Based On Aptasensors

Malachite green（MG）was widely used in aquaculture due to its strong insecticidal and antibacterial effects and infection prevention.However,MG has been found to be teratogenic,carcinogenic and mutagenic on human bodies.Therefore,it is important to develop rapid and accurate detection methods for MG residue.In this dissertation,three colorimetric aptasensors were established,and have been successfully applied to the detection of MG in fish and aquaculture water samples.The main research results were described as follows:Section 1,a facile colorimetric aptasensor for highly sensitive detection of MG based on RNA aptamer and AuNPs was developed.AuNPs agglomerated in salt with the solution color changed from red to blue.However,the complexation of RNA aptamer with AuNPs prevented the nanoparticles from aggregation in a high-salt solution with red color of solution because of the electrostatic interaction between RNA aptamer and AuNPs.In the presence of MG,RNA aptamer specifically bound with MG,causing AuNPs aggregation with blue in color in salt.With the increases of MG concentrations,the values of A₅₂₀20 decreased and the values of A₆₉₀90 increased,and the color of the solutions gradually changed from red to blue.Taking advantage of this sensing technique,MG was detected by naked eyes or UV-Vis spectroscopy within one hour.The results showed that the linear range of MG was 0.6¹2.5μmol L^-11 at the optimum conditions of0.2 mol L^-11 NaCl,10μmol L^-11 RNA and 7 nmol L^-11 AuNPs.A linear equation ofΔ(A₆₉₀/A₅₂₀)=0.06C-0.01 was obtained with a correlation coefficient（R²）of 0.993.The detection limit was 0.04μmol L^-1（3α/κ,n=9）.Furthermore,the developed method was successfully applied to the detection of MG in aquaculture water with recoveries ranging from 92%to 108%.The results indicated that the developed method hold significant potentials for trace MG detection.Section 2,a facile colorimetric method was developed based on the inhibition of peroxidase-like activity of Fe₃O₄NPs with DNA aptamer.Magnetic Fe₃O₄ nanoparticles（Fe₃O₄NPs）was found to own strong peroxidase-like character which can catalyze the oxidation of 3,3’,5,5’-tetramethylbenzidine（TMB）by H₂O₂ with a color change from colorless to dark blue.However,the peroxidase-like activity can be significantly inhibited by MG-DNA aptamer,making the solution light blue.In the presence of MG,the aptamer would preferentially and specifically bind to MG with free Fe₃O₄NPs releasing,and the peroxidase-like activity of Fe₃O₄NPs recovered.MG content can be determined according to the color change of the solution or the absorbance value at 650 nm.Based on the colorimetric method,MG was detected with a linear range from 0.1 to 1μmol L^-1and a detection limit of 9.5 nmol L^-1（3σ,n=9）.The colorimetric method was applied to the detection of MG in fish and aquaculture water with recoveries ranging from 80%to 120%（RSD<6.4%）.The results indicated that the developed colorimetric method could be used to detect MG rapidity,sensitivity and specificity.Section 3,a new specific and sensitive colorimetric aptasensor based on the inhibited peroxidase-like activity of gold nanoparticles（AuNPs）by CTAB was developed for the detection of malachite green（MG）.With strong peroxidase-like activity,AuNPs can catalyze the oxidation reaction of 3,3’,5,5’-tetramethylbenzidine（TMB）and H₂O₂ with a blue color in solution,but the catalytic activity was found to be significantly inhibited by hexadecyl trimethyl ammonium bromide（CTAB）through the aggregation of AuNPs.After mixed with RNA-aptamer,the negatively charged RNA bound with positively charged CTAB,which would greatly enhance the catalytic activity of AuNPs.However,in the presence of MG,RNA-aptamer specifically bound with MG,leading to the aggregation of AuNPs again with a light blue in solution.MG concentrations ranging from 0.01 to 2μmol L^-11 were quantified through the absorbance at 650nm and the color changes of solution.The detection limit was 1.8 nmol L^-1.The aptasensor showed high specificity and accuracy for MG sensing in freshwater and seawater samples with recovery ranging from 80%to 120%.The results were similar to those of HPLC and ELISA,illustrated that the developed aptasensor was highly adaptable.