Detection of Metal Ions and Biomolecules Based on Fluorescent Metal Nanoclusters Templated by DNA Aptamer

Author:Zhang Bao Zhu

Supervisor:wei chun ying


Degree Year:2019





Nucleic acid aptamers are widely applied for constructing biology and chemistry sensors due to their high affinity and high specificity with their targets.Ag NCs and Cu/Ag NCs have been applied as fluorescent probes and photoluminescent and electroluminescent materials for bio-labeling,bio-imaging,and ultrasensitive biological detection due to their good photostability,low toxicity,excellent biocompatibility,and ultrasmall size.The fluorescent Ag nanoclusters using DNA as the scaffold(DNA-Ag NCs and DNA-Cu/Ag NCs)have attracted special attention,because of tunable fluorescence properties from blue to near-infrared emission by varying the base sequences or strand lengths of the oligonucleotides.Ag+ and Cu2+ ions bind selectively to the heterocyclic bases of the DNA oligonucleotides rather than to their phosphate and sugar groups.In addition,DNA-template stabilized Ag NCs and Cu/Ag NCs are easy to bind aptamers.Based these ideas,the label-free and turn-on fluorescent probes have been constructed for the detection of metal ions and biomolecules based on aptamer DNA-Ag NCs/Cu Ag NCs.DNA template consists of two segments: one is the Ag NCs/ Cu Ag NCs-nucleation segment at the two termini,and the other is the aptamer segment in the middle of DNA template,There are several other connecting bases between them.The best DNA-template is selected by changing sequences and number of nucleation and connecting segments and choosing the fittest aptamer.The detecting principle is as follow: The two darkish DNA-Ag NCs/Cu Ag NCs are at the two termini of the templates in absence of tagets.However,in presence of targets,the conformational change of the target aptamer due to the high specificity and affinity of the target aptamer to target makes the two darkish DNA-Ag NCs/Cu Ag NCs close,resulting in increasing largely the fluorescence of DNA-Ag NCs/Cu Ag NCs.So the target detection is realized.The main contents and results are as follows:(1)Turn-on detection for Pb2+ based on fluorescent DNA-Ag NCs.Three DNA-templates named C-PS2.M,C-T30695 and C-AGRO100 consisting of the same nucleation segment(C-rich sequence)and three different aptamers were designed.C-PS2.M-DNA-Ag NCs is chosen as the best probe for the most sensitive to Pb2+ among three DNA-Ag NCs.The average diameter of C-PS2.M-DNA-Ag NCs is about 1 nm.The limit of detection is 3.0 n M with a good linear range from 5 to 50 n M(R = 0.9862).Furthermore,it has been used for detection of Pb2+ in real water samples and the certified reference material for lead.(2)Turn-on detection for Hg2+ based on fluorescent DNA-Ag NCs.Two DNA-templates named C-Hg2+-Aptamer-1 and C-Hg2+-Aptamer-2 consisting of the same nucleation segment(C-rich sequence)and two different aptamers were designed in term of the characteristics of coordination for T-Hg2+-T.The aptamers of C-Hg2+-Aptamer-1 and C-Hg2+-Aptamer-2 comprise the same nucleation segment(C-rich sequence)at the termini,T-rich and a few of C sequence and T-rich sequence,respectively.Hg2+ can be detected by C-Hg2+-Aptamer-1-Ag NCs and C-Hg2+-Aptamer-2-Ag NCs,The average diameter of C-Hg2+-Aptamer-1-Ag NCs is about 2 nm.The limit of detection is 0.25 n M for C-Hg2+-Aptamer-1-Ag NCs and 0.8 n M for C-Hg2+-Aptamer-2-Ag NCs with the same good linear range 2-18 n M(R(1)= 0.9985,R(2)= 0.9972).Furthermore,it has been successfully applied for detection of Hg2+ in the tap and the lake water samples.(3)Turn-on detection for ATP based on fluorescent DNA-Cu/Ag NCs.The DNA-template named BT3T3 was first designed according to ATP aptamer,then eight DNA-templates were devised by changing the sorts and number of connecting base sequences between aptamer and nucleation sequence and optimizing nucleation sequences based the template of BT3T3.BT5T5-Cu/Ag NCs is the best candidate for ATP detection.The average diameter of BT5T5-Cu/Ag NCs is about 2 nm.However,when ATP was added,the percentage of BT5T5-Cu/Ag NCs with particle size of 1-2 nm increased,probably due to the dispersion of BT5T5-Cu/Ag NCs by ATP.There is a good linear relationship within 2-18 m M(R = 0.9975)and the limit of detection is 6.983 m M.ATP in fetal bovine serum was detected successfully with the proposed probe.The detection effect was better with BT5T5-Cu/Ag NCs than BT5T5-Ag NCs.In addition,The activity of adenosine deaminase(ADA)was detected using BT5T5-Cu/Ag NCs/ATP complex for catalyzing the conversion of adenosine to inosine by the removal of an amino group,and was detected as low as 5 U/L.Two DNA-templates named TBA1 and TBA2 according to optimizing DNA-template for the detection ATP and combining with the detection characteristics of thrombin that the aptamer sequences binding to TB were replaced and the nucleation sequence at 5’ and 3’ termini were unchanged were designed.TBA1-Cu/Ag NCs is a good candidate to detect thrombin with good sensitivity and seletivity.Thrombin was detected as low as 2 U/L with a good liner ranger 2-10 U/L.Furthermore,thrombin in fetal bovine serum solution was also detected.(4)Determination of Ag+,biothiols and acetylcholinesterase activity.A label-free fluorescent sensor has been constructed for the detection of Ag+,biothiols and acetylcholinesterase activity based on the fluorescence change of TO/G-qudruplex complex due to the conformation convertion of DNA reacted with Ag+,and DNA-Ag+ seized by biothiols and thiocholine,a hydrolysis product of ATCh chloride in the presense of AChE.Three G-rich sequences named HTG,C-MYC and C-KIT were designed and HTG was the fittest candidate for the detection by optimization.The detection limit of Ag+ in the range of 0.5-6.0 μM is 34 n M(R =0.9978);The detection limit of Cys was 25 n M in the range of 0.25-4.5 μM(R =0.9965);and AChE was detected as low as 0.05 U/L in the range of 0.05-1.3 U/L.The proposed method was also applied in screening of AChE inhibitors,for example,tacrine,its IC50 was 18.6 n M.Ag+ was detected successfully using the proposed method in the tap water and the lake water.Hence,the proposed sensor can be extended to monitor Ag+,biothiols and the activity of AChE in the environment,to screen AChE inhibitors,to discover new drugs,to monitor toxic pesticides for bio-safety assessment.In summary,The probes based on DNA-Ag NC and DNA-Cu/Ag NCs have some good advantages such as simplicity,low cost,high sensitivity,good selectivity,low toxicity,and good biocompatibility.Therefore,it possesses potential application value in environmental monitoring and biological sample detection.