Study On Gold Nanoparticles Based Nucleic Acid Molecular Probes And Its Application In Biochemical Analysis

Nucleic acid molecule probes are comprised of nucleic acid sequences to recognize targets by base pairing,metal ion binding and aptamer capture to produce optical,electrical or magnetic signal changes.In the different signal output-modes of detection methods,the optical probe nucleic acid molecules have get the depth and extensive research,because of its high sensitivity,wide range of targets,sundry measure methods,simple experimental operation and so on.Gold nanoparticles(AuNPs)are biocompatible,which make them suitable for covalent binding with biorecognization molecules.Their surface plasmon resonance absorption in the UV-visible area will change with the shape of the AuNPs,the distance between the particles,the solution dielectric constant,the temperature,and other factors.These changes will leads to the color change of AuNPs solution.At present,AuNPs-based colorimetric method has been widely used to detect inorganic metal ions,DNA,proteins,tumor cell and so on.However,since the detection signal of nucleic acid molecule probes to the target is generally achieved through 1:1 transmission,so the detection sensitivity is limited within a certain range.With the development of the disease diagnosis and scientific research,greater sensitivity and selectivity techniques are required.In this dissertation,we combined the AuNPs based nucleic acid molecule optical probe with some new signal amplification technology to detect tumor cells,Hg2+and telomerase activity.The main researches of this dissertation aresummarized as follows:1.Highly sensitive Colorimetric Cancer Cell Detection Based on dual Signal AmplificationFacile and efficient detection of cancer cells at their preclinical stages is one of the central challenges in cancer diagnostics.A direct,rapid,highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer.In this work,we developed,for the first time,an easy and intuitive dispersion-dominated colorimetric strategy for cancer cell detection based on combining multi-DNA released from an aptamer scaffold with cyclic enzymatic amplification,which was triggered by aptamer DNA conformational switch and demonstrated by non-crosslinking gold nanoparticles(Au NPs)aggregation.First,five kinds of messenger DNAs(mDNAs)were aligned on the cancer cell aptamers modified on magnetic beads(MBs)to form mDNAs-Apt-MBs biocompatible nanosensors.In the presence of target cells,the aptamer would bind to the receptors on the cell membranes and mDNAs would be released,resulting in the first amplification that one biological binding event would cause the release of multiple kinds of mDNAs simultaneously.After magnetic separation,the released mDNAs were introduced into the cyclic enzymatic amplification to cleave more single strand DNA(ssDNA)fragments.Instead of modification of Au NPs,these fragments and mDNAs could be adsorbed on the surface of Au NPs to prevent particles aggregation and ensure the stability and color of solution in high salt environments.The linear response for HL-60 cells in a concentration range from 10 to 104 cells was obtained with a detection limit of 4 cells in buffer solution.Moreover,the feasibility of the proposed strategy was demonstrated in diluted serum sample.This dual signal amplification method can be extended to other types of cancer cells,which has potential application on Point-of-care cancer diagnosis.2.A Colorimetric/Fluorescent Dual-mode Sensor for Hg2+ DetectionA sensitive colorimetric/fluorescent dual-mode sensor based on hybridization chain reaction(HCR)combining multifunctional Au NPs is presented for the detection of Hg2+ in aqueous solution.In Hg2+ absent solution,the surface of Au NPs was covered by hairpin auxiliary DNAs and a single strand DNA(ssDNA),which prevented Au NPs from salt-induced aggregation.At the same time,the fluorescence intensity of the dye-labeled hairpin probes was significantly quenched by Au NPs.In the presence of Hg2+,T-Hg2+-T coordination chemistry between helper DNA and the hairpin probes was induced which triggered the formation of extended double-stranded DNA(dsDNA)polymers via HCR.The formed dsDNA polymers were stiffer which couldn't attach to Au NPs,resulting in a red-to-blue color change along with salt-induced aggregation of Au NPs for colorimetric sensing.Meanwhile,the fluorescence of dye-labeled DNA turns on.Due to the HCR amplification effect,a sensitive detection of Hg2+ was achieved with detection limit of 0.1 nM.Colorimetry is suitable for the analysis in salt solution with concentration lower than 100 mM.It serves as an intuitive method that Hg2+ down to 1.0 nM could be identified by naked eyes.For high-concentration salt solutions such as industrial effluent,fluorescent sensing acts as a better choice.Both the color and fluorescence changes of the proposed sensor exhibited high selectivity against other metal ions.Lake water was collected and analyzed using the dual-mode sensor,the results confirmed the practicability of the proposed approach.3.Enzymatic Signal Amplification for Sensitive Colorimetric Detection of Telomerase Activity in Cancer cellsA facile and sensitive strategy for eolorimetrie detection of telomerase activity in cancer cells was designed by enzymatic amplification coupled with recognition molecular beacon.The linker DNA was used to assemble two sets of DNA-modified AuNPs,inducing the aggregation of AuNPs,producing a purple solution.The telomere repeat units of(TTAGGG)n are extended at the end of telomerase substrate primer in the presence of the active telomerase.So,one elongation product can open more than one molecular beacon via strand displacement reaction to realize first amplification.Subsequently,the opened molecular beacon was recycled by the aid of nicking endonuclease to continuously cut linker.DNA,leading to a second amplification.Consequently,the cleaved fragments of linker DNA can no longer assemble into DNA-AuNPs,resulting in a red color.Owing to the double amplification strategy,the proposed method allowed the detection of telomerase activity in cell extracts equivalent to 25 HeLa cells without PCR amplification.This method is a sensitive and convenient method for visualized detection of telomerase activity.