| Heavy metals have been widely used in industry and agriculture due to their excellent physical,chemical and mechanical properties.At the same time,they cause inevitable heavy metal pollution problems by a large number of production and application.The pollution is formed under the effect of many natural and human factors.In addition,heavy metals are quite recalcitrant to degradation and are difficult to eliminate when ingested.The development of effective detection techniques for heavy metals,is of great importance for the environment,food safety and human health safety.In recent years,the heavy metal ligand-based approaches are applied to rapid detection of heavy metals by a number of researchers.The strong signal conversion and amplification abilities of the system,lead to impressive promotion of the assay sensitivity.Meanwhile,the rapid binding and response ability between heavy metal and its ligand ensure the processing can be finished in a short period of time.Combining with multiple solid substrates(ELISA microplate,chromatographic test strip),signal display materials(fluorescent and chemiluminescent nanomaterial)and the portable testing instruments,the approach can be used in on-site testing.In this study,we mainly focus on the research of two heavy metal ions(Hg2+ and Cu2+).We combine their corresponding new specific ligands(antibody,protein or nucleic acid)with nanomaterials technology,electrochemical sensing technology and Raman analysis technology,and develop rapid detection methods for Hg2+ and Cu2+.The main contents include the following aspects:(1)New anti-Hg2+ monoclonal antibody(McAb)is used as the Hg2+ specific ligand,and a green immunochromatographic electrochemical assay for Cu2+ detection in water and milk samples has been constructed.The new anti-Hg2+ McAb recognized a coating antigen protein in the absence of Hg2+(green coating antigen),the recognition between the new McAb and coating antigen was uniquely influnced by individual Hg2+ ions without the need for a chelator.By this we design an immunochromatographic test strip using dually labeled gold nanoparticles(AuNPs)along with electrochemical detection.The developed method do without the sample pretreatment and the use of coating antigen without Hg2+ confer the assay with green properties.The application of HRP-AuNPs signal amplification probe,chromatographic test strip system and electrochemical detection,resulting in good features such as rapidity,accuracy and high sensitivity.The detection limit for Hg2+ is 0.03 ng mL-1,which is far lower than The Environmental Protection Agency(EPA)and The Ministry of Health of the P.R.China regulated the maximum allowed contamination level of Hg2+ in drinking water(2 ng mL-1)and milk(10 ng mL-1).(2)High sensitivity is one of the high-performance parameters that is pursued by many researchers in target analytes assay.Surface-enhanced Raman scattering(SERS),an extremely sensitive analytical technique which is widely used in chemical and biological analyses.We use new Cu2+-specific recognition element:multiple antibiotic resistance regulator(MarR)protein as Cu2+ specific ligand for SERS analysis of Cu2+based on a dual hot-spot model.MarR as specific bridging molecules in a SERS hot-spot model,and Cu2+ induces formation of MarR tetramers,resulting in the formation of SERS hot spots.In comparison with traditional hot-spot model,the formation of dual hot-spot by introducing signal amplification probe increase the sensing platform sensitivity 50-fold.The detection limit for Cu2+ in water samples is 0.18 nM,which is much lower than the United States EPA limit of Cu2+ in drinkable water(20 μM).The high sensitivity,high specificity,and rapid detection capacity of the SERS assay therefore provide a combined advantage over current assays.(3)On the basis of previous work,a multipath colorimetric assay for Cu2+detection has been constructed utilizing MarR protein as Cu2+ specific ligand coupling with gold nanoparticle probes.Cu2+ can specifically induce the aggregation of MarR coated AuNPs,and the colorimetric assay for Cu2+ can be achieved by a red-to-blue color change.The three detection approaches(the naked eye-,UV-vis-and smartphone-based approaches)with complementing each other in terms of their own advantages provides greater flexibility and more options for different requirements and conditions.Under optimal conditions,the Cu2+ concentration in water samples was quantified in less than 5 min with limits of detection for the naked eye,UV-vis and smartphone-based approaches of 1 μM,405 nM and 61 nM,respectively.Our strategy has great potential for application in on-site monitoring of Cu2+ with respect to excellent selectivity,simpleness,speediness and multipath transition detector,and the unique response of MarR towards copper ions may provide a new approach to Cu2+ sensing.(4)A rapid,high sensitive and high selective detection method for Cu2+ in aqueous solution was constructed by using signal-amplified lateral flow test strip(SA-LFTS)based on Cu+-catalyzed click chemistry and hybridization of single-stranded DNA(ssDNA).Alkyne and azide modified ssDNA acted as specific elements for Cu2+ recognition.The hybridization between two ssDNA with partially complementary sequences which is labelled on different AuNPs probe,resulting in more AuNPs are capturing at the test line,thus signal amplification is achieved.Under optimal conditions,Cu2+ can be detected with a linear range of 5 nM-20 μM,limit of detections of 5 nM and 3.7 nM by visual observation and quantitative analysis,respectively.The sensitivity of the method is 80-times higher than that of traditional test strip.The method is further applied to determine of Cu2+ in tap water and river water and the results results were confirmed by comparison with those obtained by inductively coupled plasma mass spectrometry,and this comparison indicated the developed method has good accuracy for Cu2+.Our assay exhibits excellent selectivity because of the great specificity of the Cu+-catalyzed alkyne-azide click chemistry,and the results can be determined by naked eye,make the assay exhibit a wider application prospect in Cu2+ field-based testing. |
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