Development Of Novel Optical Biosensing Technology Using Nucleic Acid And Peptide

In recent years, people are increasingly interested in the optical sensingtechnologies due to the advantages of their speed, the immunity of the signal toelectrical or magnetic interference, the potential for higher information content andmultiplexed assay. Therefore, the optical sensing strategies might hold great potentialin clinic biomarkers detection, drug discovery, environmental and food monitoring,and public safety. Considering some key points in the biomolecular detectionespecially small molecules, enzyme, protein detection, a series of optical sensingmethods were developed with high sensitivity, high specificity, speediness andsimpleness in the thesis. Peptide and nucleic acid as bio-recognition and transductionevents combined with optical sensing strategies to develop the methods to detect smallmolecules, enzymes and proteins. The details are described as follows:(1) Based on the fact that small molecular-protein complexes affect the enzymeactivity, in chapter2, a homogenous fluorescence assay was developed for sensitivedetection of small molecule-protein interaction. A small molecule-labeled DNA/Fok Itransducer is structured which included a DNA heteroduplex, the Fok I recognition site,and a single strand hybridizing with fluorescent probes to amplify signals. In theabsence of target protein, small molecule label couldn’t influence the Fok I activity, so,the DNA/Fok I transducer cyclically cleaves the input fluorescent probes.On thecontrary, in the presence of target protein, the small molecule-protein interaction couldinhibit the Fok I activity, the DNA/Fok I transducer don’t cyclically cleaved the inputfluorescent probes and a weak fluorescence produced. The method is applied to widedissociation constant from subnanomoles to micromoles, such as folate receptor andrecombinant human dihydrofolate reductase, and the detection limit are5pM and10nM separately. In addition, the strategy can also be used to identify unknownsmall-molecule ligands that bind to the protein target at the same site as thesmall-molecule label on the DNA/Fok I transducer with competitive assay.Aminopterin was a model which has the same recognition site with foltate to folatereceptor to screen the unknown small molecular ligand via competition with falate.This developed strategy is highly sensitive for detecting small molecular-proteininteraction via signal amplification and can be further adapted for multiplexed andhigh-throughput applications using multicolor fluorescence-quenched probes and microplate formats with the aid of automated robotic delivery systems. This developedstrategy may hold great promise as a sensitive, specific, robust, and high-throughputplatform for the identification and quantification of small molecules and theirinteractions with target proteins of varying affinities and drug discovery.(2) Taking account of the drawbacks of the method in the chapter2, for instancethe complexity of DNA/Fok I transducer design and expensive cost, a newchemiluminescence strategy is developed to detect protein or small molecule-proteininteraction based small molecular-protein complexes inhibit polymerase and nickingendonuclease activity in chapter3. A small molecule labeled DNA machine isconstructed with a heteroduplex recognized by Bst DNA polymerase, a single strandcontaing the half recognition site of Nt.BstNBI nikase, the complementary sequencewith chemiluminescence reporters and as the template of Bst DNA polymerase at thesame time. The polymerase-induced reaction replicates a single strand that yields theduplex including the nicking site for Nt.BstNBI. Scission of the replicated strandresults a new replication site for polymerase and the concomitant displacement ofchemiluminescence reporters. In contrast, in the presence of target protein, the smallmolecule-protein interaction inhibitors the activity of polymerase and nickingendonuclease and little chemiluminescence reporters produced, so very weakchemiluminescence signals are available. This method is used to detection folatereceptor and the detection limit is1pM. This developed strategy holds manyadvantages such as simple design, low cost, high sensitivity and it maybe becomeanother choice for the target protein detection, small molecule-protein interactionstudy or drug discovery.(3) According to the fact that the aggregation of gold nanoparticles induce theplasmon resonance absorption peak shift, a novel biosensing strategy was developedfor sensitivity and specificity screening of histone-modifying enzymes based onassembly of peptide-decorated AuNPs mediated by an antibody specificallyrecognizing the modified peptide in chapter4. In the method, peptides werebio-recognition events and in the present of histone-modifying enzymes, the substratepeptide is modified at a specified site with a certain group such as methyl or acetyl.After the addition of an antibody specific to this modified peptide, the peptidessubjected to enzymatic modifications can be recognized by the antibody. This triggersa network-like assembly of the peptide modified AuNP and thus induces a significantvariation in the plasmon resonance absorption peak with a visualized color change.The detection limit is0.2nM for histone methyl transferase. Compared with the present methods, the characters of the strategy are better and may be a robust platformfor the epigenetics study.(4) According to the theory that plasmonic coupling between AuNPs can enhancethe electromagnetic fields in the interstitial spots of AuNPs by many orders ofmagnitude, which evokes dramatic enhancement of the Surface Enhanced RamanScattering (SERS) signal, a novel SERS strategy is developed based on assembly of thepeptide-decorated AuNPs into network structures mediated by peptide assembly for theextracellular proteinases activity detection in chapter5. The method is simply designedand only one peptide is required. The peptide composed with assembly peptide andsubstrate peptide and roman dyes are decorated on the gold nanoparticles. In thepresence of extracellular proteinases, the substrate peptide was hydrolyzed and theassembly peptide is exposed and it is able to assemble nanostructure to crosslink thegold nanoparticles. The result induces the interparticle plasmonic coupling and theenhancement of the Surface Enhanced Raman Scattering (SERS) signal. In the method,different Raman dyes are used for multiplex assays of the aggregation events of AuNPstriggered by different enzymatic reactions. Elastase and matrix metalloproteinase(MMP-7) as models, the detection limits are0.0005U/mL and30pM separately.