Ratiometric Fluorescence Sensors For The Detection Of Mercury Ion And Application In Farmland Irrigation Water

Heavy metal pollution has become an important issue threatening the modernisation of agriculture and the health of people in China.As one of the most toxic heavy metals,mercury(?)ion(Hg²⁺)can enter the agricultural environment,and inhibits nitrification and ammonification in soil,influences the crop growth and enriches in crops,which may further enter the human body via food chain to cause serious damage to the nervous system.Researchers hence develop various techniques for the detection of Hg²⁺,such as atomic spectroscopy,inductively coupled plasma mass spectrometry(ICP-MS),electrochemical sensor,and fluorescence method.Atomic spectrometry and ICP-MS are accurate and reliable but require the expensive instrument and skilled operation;electrochemical sensor and fluorescence method are cheap,easy to operate and suitable for the on-site analysis.Currently,fluorescence method has been widely used for the detection of Hg²⁺in various samples;however,the agricultural analysis suffers from the complex matrix and various influencing factors,and thus it is crucial to improve the accuracy of fluorescence methods.To address these issues,we aim to develop efficient ratiometric fluorescence sensors for the accurate and rapid determination of Hg²⁺in agricultural environment.In this thesis,to improve the accuracy of fluorescence sensors,we developed novel methods for the preparation of fluorescent probes,and combined fluorescence methods with ratiometry to construct a series of ratiometric fluorescence sensing systems to achieve accurate detection of Hg²⁺in agricultural irrigation water,and a smartphone-based portable fluorescence detection device was developed,which was can be applied to the rapid and accurate analysis of Hg²⁺.The main research elements are as follows.(1)A ratiometric fluorescence sensing method based on bioenzyme and internal filtration effect(IFE)mechanism was developed to detect Hg²⁺with the enhanced sensitivity and accuracy.Gold nanoclusters(AuNCs)capped with L-proline were used as fluorescent donor,and 2,3-diaminophenylazine(DAP)acted as fluorescent acceptor.DAP was generated by oxidating o-phenylenediamine(OPD)with laccase(LACC),and the catalytic activity of LACC was closely related to the concentration of Hg²⁺.Based on this principle,the inhibited catalytic activity of LACC by Hg²⁺was used to modulate the fluorescence intensity ratio of DAP to AuNCs.The relationship between the fluorescence intensity ratio of DAP to AuNCs and Hg²⁺concentration was established.Under the optimal conditions,the linear range of the fabricated sensing system for Hg²⁺detection was 0.8-35?M with a detection limit of 0.27?M.The method was used for the detection of Hg²⁺in tap water and irrigation water,offering satisfactory results.(2)To reduce the cost and improve the reliability of detection,silicon quantum dots(SiQDs)was synthesized with a house-hold microwave oven to achieve rapid(8 min)and efficient preparation of SiQDs,reducing the time and cost;platinum nanoparticles(Pt NPs)were used as nanoenzyme to replace LACC to improve the stability of sensor.A SiQDs/Pt NPs/OPD/H₂O₂-based ratiometric fluorescence sensing system was then constructed with the principle of fluorescence resonance energy transfer(FRET)for the accurate detection of Hg²⁺.The resultant linear range for Hg²⁺was 20-3000 n M with a detection limit as low as 10.5 n M under the optimal conditions,and the sensing system was successfully applied to the irrigation water analysis,and the result was consistent with that of ICP-MS.(3)To improve selectivity of sensing,DNA was introduced to achieve the specific recognization of Hg²⁺by forming T-Hg²⁺-T structure;meanwhile,exonuclease III(Exo III)was used for signal amplification.Rox-labelled T-rich DNA strand was assembled on the surface of SiQDs via electrostatic interaction,and the fluorescence of Rox was quenched by SiQDs;upon addition of Hg²⁺,Exo III sheared DNA double strand formed with T-Hg²⁺-T,and released Hg²⁺and Rox,thereby resoring the fluorescence of Rox.The quenchment of Rox fluorescence was derived form the energy transfer between Rox and SiQDs that SiQDs as the acceptor was not to be affected.Exo III shearing process occurs cyclically,allowing the amplified response signal.The detection hence can be achieved based on the relationship between the fluorescence intensity ratio of Rox and SiQDs and Hg²⁺concentration,allowing a linear range of 0.02-10 n M with a detection limit of 0.01 n M.The developed sensing syatem was successfully used for the highly specific analysis of Hg²⁺in irrigation water in agricultural fields.(4)In order to realize the practical application of ratiometric fluorescence method,a portable ratiometric fluorescence analysis device based on image acquisition and RGB colour analysis with smartphone was developed.The device consisted of two parts,including a miniature test cassette as the host and a smartphone as the software carrier for fluorescence image acquisition and RGB analysis.The detector box integrated components such as LED light sources,sample compartments and filters,and useed a designed printed circuit board to realize the control of light sources etc.A ratiometric fluorescence sensing system based on AuNCs and SiQDs was proposed that Hg²⁺quneched the fluorescence of AuNCs while that of SiQDs kept stable.Meanhile,a fluorescence transition from pink to blue under different concentrations of Hg²⁺can be observed.The developed device achieved rapid and accurate analysis of Hg²⁺,which was expected to be applied to field analysis of Hg²⁺in irrigated farmland water.