Evanescent Wave Multicolour Fluorescence Biosensor And Application In Detection Of Heavy Metal Ions

Heavy metal ions are a serious threat to water environmental safety and human health due to their biological toxicity,non-degradable and bioaccumulation.Heavy metal ions often coexist in the environment or other matrix.Therefore,the development of simple,rapid and sensitive simultaneous detection technology for heavy metal ions is of great significance to improve the detection efficiency and reduce the detection cost.The penetration depth of evanescent wave is only tens of nanometers to hundreds of nanometers,which is less interfered by other substances in the matrix.It can greatly improve the signal-to-noise ratio and reduce the influence of solution matrix,which is suitable for the detection of complex matrix.Based on evanescent wave total internal reflection and multicolour fluorescence analysis technology,we successfully built evanescent wave multicolour fluorescence all-fiber biosensor（EWMB）.Combined with the specific recognition of functional nucleic acids and the principle of fluorescence resonance energy transfer（FRET）,we established a simple,rapidand sensitive detection method for single heavy metal ion,and realized the simple,rapid,sensitive and simultaneous detection of multiple heavy metal ions.The main research results are as follows:（1）Combined with the principle of multicolor fluorescence analysis and evanescent wave fiber sensing,using a single fiber probe and the strategy of"multi wavelength laser time-sharing excitation,multi-color fluorescence synchronous acquisition",a kind of evanescent wave multicolor fluorescence all-fiber biosensor（EWMB）is designed,which successfully realizes the simultaneous acquisition and analysis of different wavelength fluorescence signals.Two kinds of fluorescent dyes,Cy5.5 and Cy3,with different excitation and emission wavelengths are used to test the performance of the evanescent wave multicolor fluorescence all-fiber biosensor.The results show that the biosensor can realize the simultaneous detection of two wavelengths of fluorescence,and the crosstalk between the two wavelengths of fluorescence can be ignored.The detection limits of Cy5.5 and Cy3 are 0.15 n M and0.26 n M respectively,with good detection sensitivity.（2）Based on T-Hg²⁺-T structure and FRET principle,using fluorescence labeled poly-A DNA strand（CY-A14）and quencher labeled poly-T DNA strand（BQ-T14）as signal reporter and biorecognition element,respectively,the simple and sensitive detection of Hg²⁺is realized.The influence of several key environmental factors on Hg²⁺biosensor,such as p H,temperature,and ionic strength,was investigated in details.Under optimal conditions,the detection limit of Hg²⁺was 8.5 n M,and the biosensor has high selectivity for Hg²⁺.The recoveries of Hg²⁺in actual water samples are 79.8%～111.4%,and the relative standard deviations（RSD）are below 5.0%.Research results demonstrated that the EWMB can realize the simple,rapid,and sensitive detection of Hg²⁺in actual water samples.（3）Based on the principle of DNAzyme and FRET,a rapid,simple and sensitive method for Pb²⁺detection was established,in which the fluorescent group labeled substrate DNA and quenched group labeled GR-5 DNAzyme are used as signal reporter and biorecognition element,respectively.The Pb²⁺detection limit is 1.2 n M,which is enough to meet the requirements of on-site detection.The method has high selectivity for Pb²⁺.The recoveries of Pb²⁺in four kinds of water samples are 73.8%～119.5%,and the relative standard deviation（RSD）is less than 5.5%.This method has good accuracy and repeatability for the detection of Pb²⁺in actual environmental water samples.Human serum is used as the detection matrix and EWMB is used for the experiment.The detection limit of Pb²⁺in diluted（200-fold）serum matrix is 1.4 n M.The results show that the method can be used to detect Pb²⁺in serum matrix,which further widened the application scope of this Pb²⁺detection method.（4）Based on the principle of FRET and time-resolved effect,a fast and sensitive method for simultaneous detection of Hg²⁺and Pb²⁺is established by using fluorescence labeled complementary DNA strand and quencher labeled functional nucleic acid as signal reporter and biorecognition element,respectively.In addition,we propose a method to avoid interferences between DNAs used in detection,and verify the feasibility of this method.The biosensor has good LOD and wide detection range,that is,the LODs are 4.2 n M and 1.6 n M,and the linear response ranges are 110 n M～750n M and 7.5 n M～150 n M,for Hg²⁺and Pb²⁺respectively.The recoveries of Hg²⁺and Pb²⁺in actual water samples are 85.2%～109.5%and 75.6%～111.1%respectively,and the relative standard deviations（RSD）are below 6.0%,which are within the analytical requirements,indicating that this method is suitable for simultaneous determination of Hg²⁺and Pb²⁺in environmental water samples and have good precision and accuracy.This study provides new methods for the single/simultaneous detection of heavy metal ions,which can meet the requirements of rapid and sensitive detection of heavy metal ions in complex water samples.In the future,it can be further applied to the detection of other target substances（such as other metal ions or proteins）,and become a powerful alternative tool for the analysis of pollutants in environmental samples.