| Point-of-care testing(POCT)is a technique that can quickly get results on site via portable devices and do not require professionals.Due to its advantages of simplicity,rapidity and miniaturization of instruments,point-of-care testing has been widely utilized in various fields,such as clinical examination,food safety,environment monitoring and so on.The POCT of disease biomarkers and environment pollutant have a great demand with the increasing attentions of people to personal health monitoring and environment management.At present,the commercial POCT device is very limited.It is urgent to develop sensitive,reliable and easy to use methods for detecting disease biomarkers and and heavy metal ions.In this thesis,the novel POCT methods were established for detection of disease biomarkers and Hg2+via portable devices.The main research contents can be summarized into the following three parts.1.Research on the chemiluminescence point-of-care detection of cancer cellsA simple,rapid and point-of-care strategy was proposed for detecting cancer cells based on the signal amplification of cyclic strand displacement reaction(SDR)via chemiluminescence detector.The trigger DNA of SDR was released by the specific binding between cancer cells CCRF-CEM and aptamer.The stem-loop structure of DNA H1 was opened to expose the stem of H1 when cDNA hybridized with the MBs-H1.The stem of H1 hybridized with HRP-tagged H2 to form more stable H1/HRP-H2 duplex,replacing cDNA which will trigger another cycle of SDR.More and more HRP-H2 complexes were enriched onto the surface of magnetic beads.After magnetic separation and washing,MBs-H 1/HRP-H2 complexes mixed with luminol/H2O2 solution.The HRP catalyzed the chemiluminescence of luminol/H2O2 solution.Due to the magnetic enrichment and signal amplification of SDR,CCRF-CEM cells were sensitively detected by portable chemiluminescent detector.A excellent linear relationship between chemiluminescence signal and the number of CCRF-CEM cells was obtained in the range of 100 to 5×104 cells.The detection limit is as low as 85 cells/mL.Therefore,it is a sensitive and facile POC method for chemiluminescent detection of cancer cells.2.Research on the chemiluminescence point-of-care detection of circulating tumor DNAA chemiluminescence POC methods based on the in-situ formation of G-quadruplex/hemin DNAzyme was developed for detecting circulating tumor DNA via portable chemiluminescence detector.The triplex molecule beacon which was double-labeled hemin is used as the molecular recognition probe.Hemin dimer has a low peroxidase activity,which can induce low chemiluminesence signal in the presence of luminol and H2O2.Upon circulating tumor DNA hybridizing with the loop of triplex molecular beacon,hemin dimer was destroyed due to the release of the G-rich stem-forming oligonucleotide.The peroxidase activity of hemin obviously enhanced,resulting in a high chemiluminesence signal.In the presence of K+,a G-quadruplex/hemin DNAzyme can be formed to greatly improve the peroxidase catalytic activity.The circulating tumor DNA can be quantitatively detected by monitoring the change in chemiluminesence signal.Therefore,it provides a simple and fast chemiluminescence POC method for the detection of circulating tumor DNA.3.Research on the gas pressure point-of-care detection of Hg2+A sensitive and rapid POC method was developed for detecting Hg2+based on the specific T-Hg2+-T binding via digital portable pressure meter.The T-rich DNA1 and T-rich DNA2 were modified on the surface of magnetic beads(MBs)and platinum nanoparticles(PtNPs)to form MBs-DNA1 and DNA2-PtNPs complex respectively.In the presence of Hg2+,DNA2-PtNPs were enriched on the surface of MBs-DNA1 via T-Hg2+-T binding.After magnetic separation and washing,the MB-DNA1/PtNPs-DNA2 complex mixed with H2O2 in the sealed wells of 96 well plate.Then,PtNPs effectively catalyzed the decomposition of H2O2 to generate oxygen,leading to an increase in pressure of sealed wells.The gas pressure was linearly related with the concentration of Hg2+in the range between 10 pM and 100 nM with a detection limit of 2.79 pM,which is more sensitive than most of the previous reports.Therefore,it offers a facile,inexpensive,rapid and portable way to detect Hg2+. |
