Rapid Determinating The Staphylococcal Enterotoxin B By Colorimetry Based On The Growth Of Gold Nanoparticles Regulated By DNAzyme

Biosensing technique is a high technology formed by interdisciplinary penetration.As a new type of detection device,biosensor can transform the concentration of target into detectable optical and electric signal by identifying component and transducer.Colorimetric sensors are widely used in the field of analysis and detection,because the optical signal can be detected simply and quickly.In recent years,the application of nanomaterials in biosensor has become a hot spot.Gold nanoparticles is a typical example,because of its unique optical properties,greatly improve the sensitivity of colorimetric sensors.Staphylococcal enterotoxin B(SEB)can cause a variety of diseases at very low concentration,such as burn sepsis and staphylococcal toxic shock syndrome.Therefore,the detection of SEB has important clinical significance.Present work constructed a colorimetric method for the determination of SEB based on the growth of gold nanoparticles.This assay is sensitive,inexpensive and rapid,which provides a new tool for the clinical diagnosis of related disease.This method can also detect different target with different identifying components,therefore,it is a general type of biosensing techniques.Objective:Present study established an universal colorimetric assay with color change coupling corresponding aptamer as recognition element via hemin/G-quadruplex DNAzyme-guided growth of gold nanoparticles.The capability of this constructed colorimetry detection platform waas evaluated with the target of SEB as a representative of analytes.Methods:1.The growth of gold nanoparticles: The HAu Cl4 was reduced to gold nanoparticles by hydrogen peroxide solution at various concentrations in the MES buffer.Then the status of nanoparticles was characterized.2.The generation of DNAzymes with the activity of hemin/G-quadruplex horseradish peroxidase: The probe G were hybridized with the sequences SEB binding aptamer in reaction buffer.When SEB and hemin were existed,SEB matched the aptamer sequences specifically,then the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzymes was formed by combination of probe G and hemin.3.Colorimetric determination of SEB: The strategy of target-induced DNAzyme was used to control the generating speed of gold nanoparticles,then the SEB was determinated.4.Optimization of method: In order to increase signal-to-noise ratio,various parameters were systematically investigated and optimized,including MES buffer concentration,gold(III)chloride trihydrate concentration,hydrogen peroxide concentration,measurement time and the sequences of aptamer and probe G.5.Performance evaluation of the constructed new colorimetric method: Under the optimized conditions,a series of experiment was performed to evaluate the detection limit,linear range,selectivity.6.Real sample detection of SEB: Serums samples containing SEB were examined using this developed colorimetric sensor.Results:1.The ultimate status of synthetic gold nanoparticles were investigated by UV-Vis spectra,DLS and TEM,respectively.The UV-Vis absorption spectra of the blue-and red-colored nanoparticle solutions exhibited maximum absorptions at 604 nm and 557 nm.DLS analysis revealed the average hydrodynamic diameters of the resultant nanoparticles were 282.4 nm and 61.9 nm,and the polydispersity indices were 0.347 and 0.094,respectively.The size and morphology of the gold particles were furtherly characterized via TEM.Typical TEM images showed that the gold nanoparticles were relatively uniform and quasi-spherical or aggregated and less-regular-shape.2.Agarose gel electrophoresis showed that little ds DNA was observed with the increasing of SEB's concentration.The results of UV-Vis spectra showed that the more DNAzyme was obtained with higher concentration of SEB.3.DNAzyme was activated by SEB,which slowed down the kinetics of Au NPs growth.The blue was appeared and the detection of SEB was realized with the change of color.4.Optimization of method: Based on a series of experiment,various parameters were optimized as follow: The MES buffer concentration was 2 m M;gold(III)chloride trihydrate concentration was 0.3 m M;hydrogen peroxide concentration was 120 ?M;measurement point was 15 min after starting the reaction;sequences of G3 and APTSEB1 were chosen.5.The performance of the colorimetric assay was evaluated with the optimized experimental conditions.The decreased absorbance intensity(-?A550)exhibited a good liner correlation with the logarithm values of SEB concentration at a range of 0.1 pg m L-1-500 pg m L-1(R2 = 0.9895).The detection of SEB with a quite low naked-eye detection limit down to 1 pg m L-1.Significant decrease in absorbance measured at 550 nm was induced only by the presence of SEB.There was almost no red-shift of the LSPR peaks with high concentration of BSA,thrombin and Ig G.6.Three concentrations of SEB(10 pg m L-1,100 pg m L-1,and 1000 pg m L-1)were prepared with diluted human serum,respectively.These results had no significant differences in that of that of same concentrations of SEB prepared with buffer solution,which indicated that this sensor can detect clinical samples effectivelyConclusion:A colorimetric detection platform was developed successfully based on hemin/G-quadruplex DNAzyme-guided growth of gold nanoparticles.This colorimetric sensor with enzyme-free can detect SEB easily,which had high sensitivity,wide detection range and good specificity.