Terminal Protection Analysis Applied In Biosensor Research

Biomarker is in general a substance used as an indicator of a biological state. It isa characteristic that is objectively measured and evaluated as an indicator of normalbiological processes, pathogenic processes, or pharmacologic responses to atherapeutic intervention. There’re many kinds of biomarkers, among which the mostimportant are specific small molecule, protein and mutant gene.It’s significant todevelop the biosensor for the detection of small molecule, protein and mutant gene.Otherwise, Many important biological processes in vivo are achieved through therecognition and interaction between small organic molecules and bioactivemacromolecules that’re protein, nucleic acid and hydrocarbon. The interaction betweenthem is basis for the signal transduction, energy transmission, metabolism and functionregulation. From the chemical point of view, it’s important to study the interactionespecially between small organic molecule and protein for drug discovery and probescreening. In recent years, there has been developed many methods in studing theinteraction between small molecule and protein, such as fluorescence resonance energytransfer, surface plasmon resonance and fluorescence polarization, etc. However thesemethods have the weaknesses of strong interference, low sensitivity, straitapplicability. It urgently needs to find a highly sensitive and selective technicalmethods. In addition, with accomplishment of the human genome sequence, reseachersgradually focus on considering the methods for detecting individual genotypes. Mostresearch on single nucleotide polymorphism typing of huge quantitive DNA samples isvery expensive. Since that, we have developed a new analytical techniques that isterminal protection assay applied in genotyping analysis and detection of interationbetween small molecule and their receptor protein. The concrete content is as follows:(1) The second chapter firstly describes the proof-of-principle of a terminalprotection assay of small-molecule-linked DNA. Terminal protection assay is based onour new finding that single-stranded DNA (ssDNA) terminally binded to a smallmolecule is protected from the degradation by exonuclease I (Exo I) when the smallmolecule moiety is bound to its protein target. This finding converts the binding ofsmall molecules to proteins into the presence of a specific DNA sequence, whichenables us to detect the interaction between small organic molecules and their proteintargets utilizing various DNA sequence amplification and detection technologies. On the basis of selective assembly of single-walled carbon nanotubes (SWNTs) withsurface-tethered small-molecule-linked ssDNA not protected by protein binding, anovel electrochemical strategy for terminal protection assay has been developed.Through detecting the redox signal mediated by SWNT assembly on a16-mercaptohexadecanoic acid-blocked electrode, this strategy is able to ensuresubstantial signal amplification and a low background current. This strategy isdemonstrated for quantitative analysis of the interaction of folate with a tumorbiomarker of folate receptor (FR), and a detection limit of3pM FR is readily achievedwith desirable specificity and sensitivity(2) The third chapter further reports a new finding of generalized terminalprotection that small molecule-DNA chimeras are protected from degradation byvarious DNA exonucleases, when the small molecule moieties are binded to theirprotein targets. This generalization translates small molecule-protein interaction assaysinto the detection of DNA of various structures, affording a useful mechanism for theanalytics of small molecules. On the basis of this mechanism, a label-free biosensorstrategy has been developed for a homogeneous assay of protein-small moleculeinteractions based on the fluorescence staining detection. Also, a label-free SNPgenotyping technique is desighed based on polymerase extension of a single nucleotidewith a small molecule label. The developed techniques are demonstrated using a modelprotein-small molecule system of biotin/streptavidin and a model SNP system ofhuman β-globin gene around the position of codon39. The results revealed that theprotein-small molecule interaction assay strategy shows dynamic responses in theconcentration range from0.5to100nM with a detection limit of0.1nM, and the SNPtyping technique gives dynamic responses in the concentration range from0.1to200nM with a detection limit of0.02nM. Besides desirable sensitivity, the developedstrategies also offer high selectivity, excellent reproducibility, low cost, and simplifiedoperations.