Novel Biosensing Methods Based On Fluorescent Copper Nanoparticles And Metal-organic Frameworks

Biosensing technology is a novel interdisciplinary subject which is integrated by chemistry,informatics,life science and other disciplines.It shows many desirable advantages such as low-cost,good selectivity,online analysis.Recently,due to these excellent features,biosensing technology has been widely applied in analytical detectionandmedicaldiagnosis.Comparedwithconventionalmaterials,nanomaterials which attract great attentions nowadays exhibit different physical and chemical properties for its small size effect,quantum size effect,surface effect and macroscopic quantum tunneling effect.The appearance and superior properties of nanomaterials have found a new path for the development of biosensing technology.The combination of nanomaterials and biosensing technology has vigorously promoted the rapid development of chemical analysis,medicine,material science and other fields.In this thesis,on the basis of fluorescent copper nanoparticles（CuNPs）and metal-organic frameworks（MOFs）,we design three novel fluorescent biosensors with quick response and effective cost to detect potassium ion（K⁺）,oligonucleotide sequences associated with human immunodeficiency virus（HIV）,adenosine triphosphate（ATP）,respectively.The details are as following:（1）In chapter 2,based on the specific recognition between aptamers and its target K⁺,we developed a label-free K⁺biosensor with high sensitivity.The principle was inspired by exonuclease I（Exo I）and terminal protection,we achieved the analysis of K⁺concentration by CuNPs signal.We employed a single-strand DNA（ssDNA）that contained two parts,one was 3′-terminus K⁺aptamers,the other was 5′-terminus poly thymine（polyT）.If the 3′-terminus K⁺aptamers recognized targets,they could fold into G-quadruplexes after binding with K⁺.These G-quadruplexes displayed effective resistance to nuclease digestion that toward ssDNA from 3′-terminus to 5′-terminus.Because of the successful 3’-terminal protection,5′-terminus polyT remained and provided the substrate to trigger the successful formation of CuNPs.As a result of the in situ synthesis,the reaction system showed strong fluorescence emission.In the absence of K⁺,3’-terminus parts failed to undergo the target-induced conformational change and to fold into G-quadruplexes.For there were no second structures to resist the digestion,ssDNA including 5’-terminus polyT part was cleaved into mono-or oligonucleotide fragments by Exo I.Without template to construct CuNPs,the low fluorescent signal was detected.This proposed approach showed satisfactory potentiality in detecting real samples with low detection limit,good sensitivity,low-cost,desirable specificity.（2）In chapter 3,a novel fluorescent biosensor based on copper metal-organic frameworks was constructed for the detection of oligonucleotide sequences that associated with human immunodeficiency virus.Cu₃（BTC）₂ is a new kind of porous material that consisted of center copper site and trimesic acid,it can distinguish single-strand DNA（ssDNA）and double-strand DNA（dsDNA）for the different adsorbent capacity toward them.The degradation of materials was also explored in this chapter.Due toπ-πstacking,Cu₃（BTC）₂ could absorb FAM-labeled ssDNA and quench the FAM through photoinduced electron transfer（PET）.While the complementary sequences were introduced to form dsDNA after hybridization,as the low adsorption,dsDNA was free instead of being fixed on the Cu₃（BTC）₂ so that FAM retained strong fluorescence emission.On the basis of this discovery,we designed a rapid and efficient sensing platform for the detection of HIV sequences.During the experiment,we discussed the degradation of Cu₃（BTC）₂,as the strong coordination ability of carboxyl and amino with Cu²⁺,these two functional groups in amino acids can replace the organic compound and break the coordination bonds between Cu²⁺and trimesic acid,leading to degradation.As a result,the ssDNA which was absorbed on the MOFs become free and the signal of FAM recovered.This degradation experiment offers potential for rapid detection of small-sized amino acids.（3）In chapter 4,ATP is a type of energy storage and conversion important substance in vivo which is known as“molecular currency”as well as the inhibitor of S1 nuclease.Based on the inhibition and the assistance from ssDNA specific endonuclease S1,a label-free biosensing system for specifically detecting ATP was proposed through the 602 nm fluorescent signal from CuNPs.A ssDNA consisted of30 thymine（T30）worked as the template for synthesis of CuNPs.S1 nuclease could cleave T30 into mono-or oligonucleotide fragments.Without T30 template,system couldn’t synthesize CuNPs that lead to low fluorescence.When ATP was introduced to incubate with S1 nuclease,the activity of enzyme was inhibited and failed to hydrolyze T30.In the presence of T30,the synthesis of CuNPs was successfully triggered and the strong fluorescence could be detected.The fluorescence intensity was positively correlated with ATP concentration,the target could be detected in the range of 0-0.2 mM.The result revealed that this label-free and low-cost biosensing system can be applied to specifically detect ATP.This biosensing system can effectively avoid complex strands design and successfully achieve the detection of ATP.