| Nucleic acids are the genetic materials of all life,which are the basis for the differences between species and individuals.With the development of nucleic acid analysis technologies（e.g.,nucleic acid sequencing and amplification）,various genes can be identified and quantified,thus forming the basis of medical diagnosis,forensic tests,environmental analysis,and food inspection.Compared with other nucleic acid analysis technologies,amplification-based schemes are favored for nucleic acid testing（NAT）,due to their high efficiency,potential applications and easy accessibility.However,limits still exist during their popularization,such as the relatively expensive instruments and the requirements for special optical components of the current state-of-the-art nucleic acid amplification strategies.Therefore,it is desired to improve the readout technology and the analytical performances of isothermal amplification,which is important for the medical diagnosis and infectious diseases control in resource-limited regions.SYBR Green I（SG）is one of the most popular nucleic acid dyes in the quantitative PCR（q PCR）.Previously,it was demonstrated that the twisted structure of SG would be restricted upon binding with ds DNA,thus liberating its photosensitization activity.The generated singlet oxygen（1O2）could effectively oxidize the chromogenic substrate 3,3’,5,5’-tetramethylbenzidine（TMB）to produce colored product,the signal of which is correlated with the concentration of ds DNA（photosensitization colorimetric assay,PCA）.The proposed PCA features label-free,simple,and fast in quantification of ds DNA,which is thus suitable for nucleic acid amplification,such as PCR.Therefore,in this thesis,we intend to couple PCA with several isothermal amplification schemes to develop new NAT methods.The whole research contains four parts as follows:（1）Coupling of termini transformation of ds DNA with photosensitization colorimetric assay for analysis of a series of nucleic acid-based targets.In this part,a label-free and universal photosensitization colorimetric assay based on target-induced ds DNA termini switching has been developed for the analysis of nucleic acids,proteins,small molecules and metal ions.First,hairpin DNAs with recessed 3′-termini were catalyzed to produce ds DNA with protruding 3′-termini via catalytic hairpin assembly（CHA）.Then,the unassembled hairpin DNAs were selectively digested by exonuclease III.Finally,the rest ds DNA that is positively correlated with target nucleic acids were quantified by photosensitization colorimetric testing.Because of signal amplification,the limit of detection（LOD）for DNA and mi RNA were as low as 9.8p M and 4.2 PM,respectively.Besides,the proposed assay could also be extended to PDGF-BB,cocaine,and Hg2+through aptamer recognition.This strategy was highly sensitive and versatile,the principle of which will be desirable for various biosensor developments and applications.（2）Using photosensitization colorimetric assay for evaluation of modified primers for reducing the non-specific amplifications in recombinase polymerase amplification（RPA）.RPA is a typical and high-performance isothermal amplification protocol,but suffers heavily from the non-specific amplicons due to low temperature amplification.Here,we explored thiophosphate primers,exonucleases,and smart primers to suppress the non-specific artifacts in RPA,the final performance of which was evaluated through photosensitization colorimetric assay.Preliminary results indicated all these endeavors could work,but still far away from satisfactory.Meanwhile,we were indicated that the supplier of the RPA kit made significant progress on improving the performances,i.e.,the non-specific artifacts in RPA were alleviated greatly.Therefore,our attempts on this issue were cancelled at the present stage.However,we believe that the results and experiences on this issue may be helpful for other isothermal amplification systems.（3）RPA coupled with photosensitization colorimetric assay for fast Salmonella spp.testing.Salmonella spp.is one of the most serious foodborne pathogens causing millions of infection cases annually,especially in resource-limited areas.The standard culture method（2-3 days）and current nucleic acid amplification-based testing are not suitable for on-site testing in rural areas with heavy Salmonella spp.burden.Here,we developed a colorimetric recombinase polymerase amplification（RPA）method for fast and sensitive Salmonella spp.testing in 1 h.Specifically,inv A gene from the genomic DNA of Salmonella spp.was amplified isothermally to produce double-strand DNA（ds DNA）amplicons,which were directly quantified by a photosensitization colorimetric assay.The proposed method offered a lowest detectable concentration of5×103 cfu/m L,which is much lower than that of ELISA（105-107 cfu/m L）.The detectable limit could be further pushed down to 3 cfu/m L upon coupling with bacteria pre-enrichment for 6 hrs.Analysis of synthetic milk samples confirmed the high sensitivity（90%）and specificity（85%）of the method for Salmonella spp.testing.Moreover,use of DNA releaser could further simplify the whole testing operation.Because RPA features low temperature amplification（25-42°C）without the need of specific instruments,and the ds DNA-based photosensitization colorimetric assay served as a simple and facile readout for RPA,our method thus allows fast and low-cost Salmonella spp.testing in food samples.（4）Portable device for high throughput nucleic acid quantification and its application in Salmonella testing.In this part,a paper-based array device was developed for high-throughput quantification of nucleic acid amplicons.For this goal,the liquid photosensitization colorimetric assay was transferred to paper substrate,with96 paper substrates arrayed in a typical 96-well manner.Subsequently,a light-emitting diode（LED）array with stable luminous flux and evenly-distributed illumination（via Trace Pro simulation and optimization）was fabricated through 3D printing.The LED and the paper substrates were mounted into a box,with each LED placed just on the top of the paper substrate.For facile color readout,a cell phone was taken for photographing of the paper substrates and the RGB signal was extracted with a self-built App（m PCA）on the phone.The developed portable device was explored for reading out of the PCR amplicons.Using Salmonella as the model analyte,the analytical results of Salmonella contained milk by the proposed device were in good accordance with those by q PCR.Compared with commercial PCRD,the proposed device featured low cost and high throughput,and thus may be a potential choice for future nucleic acid-based POCT. |
