Designed Tetrahedral DNA Nanostructureand Its Applications In Biosensor

In this dissertation, we focused on designing tetrahedral DNA nanostructures probeand its application in biosensors. The sensitivity of biomolecular detection is limitednot only by the affinity of the biomolecules but also by the interfacial properties of thebiosensor. At macroscopic interfaces, the number of probes is large, where has stronginterstrand entanglement, moreover, mass transport rate is very slow. At micro-ornanoscopic interfaces, mass transport is fast, nevertheless, the limited space availablein nanosensors restricts the effective (hybridizable) number of immobilized probemolecules. By incorporating tetrahedral DNA nanostructures into macroscopic goldinterfaces, not only the number of probes is large, but also the lateral space betweenthe DNA probes could be finely controlled, which has an advantage of fast bindingkinetics and sensitive detection. In addition, tetrahedral DNA nanostructure has amuch thicker layer, and places the probes in a solution-phase-like environment withenhance capture-target probe binding affinity. Therefore, based on tetrahedral DNAnanostructure, we have developed programmable engineering of a trans-scalebiosensing interface for ultrasensitive DNA detection, electrochemical biosensor formicroRNA detection, and studied electron transport of methylene blue (MB), andenrichment of circulating tumor cells (CTCs). The main results are described asfollowing:Firstly, it is challenge to design programmable engineering of a biosensing interfacein the field of biomolecule detection. To address the problem,we designed varioussize of the tetrahedral DNA nanostructures owing to the self-assembled DNAnanostructures with precise control, which allowed “soft lithographic” strategy toengineer the gold interface. This trans-scale surface engineering could pattern DNAnanostructures with nanometer precision on macroscopic gold electrodes, and finelytune the lateral space between the DNA probes which are anchored on thesenanostructures. Moreover, we systematically investigated the relationship betweenlateral distance of the DNA probes and the performance of the biosensor, and improved both the kinetic and thermodynamic process of the biomolecule recognitionon the interface, and then significantly improved the sensitivity and kinetics of theelectrochemical biosensor.Secondly,we developed a target-responsive, DNA nanostructure-based E-DNA sensorfor microRNA analysis. MicroRNAs become a promising biomarker because of theirfunctions in regulationg many cellular processes, however, because of the short sizeand low abundance of microRNA, it is challenging to develop fast, inexpensive, andsimple biosensor to detect them. In our degign, the use of DNA tetrahedron ensuredthe stem-loop structure in well controlled density with improved reactivity, and couldsensitively and specifically detect microRNA-141as low as1fM.Thirdly,we combined the tetrahedral DNA nanostructure probes and hybridizationchain reaction (HCR) amplification for ultrasensitive DNA and microRNAdetection.The detection limits for DNA and microRNA are100aM and10aM,respectively.Compared to the widely used supersandwich amplification, the detectionlimits areimproved by3orders of magnitude. And the sisitivity was100-fold morethan the third E-DNA biosensor.Fourthly, functional DNA tetrahedron nanostructure modified with sulfur at threevertices can be rapidly and firmly assembled at gold surfaces, which ensures theorientation of probe and controlls the distance between neighbor probes, advoidinginterstrand entanglement, so we could study the electron transport of MB at differentposition with precise control. We first reported that MB would not intercalate theπ-stack base pairs of DNA when MB is covalently linked to the DNA at the locationofnicks, and without intercalation, the DNA-mediated charge-transportreactionisprohibited, which offers thepotential of application in molecule switch. Because of thedifferent signals of MB between two positions, we designed a tetrahedral DNAnanostructure-based stem-loop gene sensor, which positively responded to the targetprobe, leading to a signal-on sensor, which could avoid false positives. Furthermore,this gene sensor is simple, synthesized by one step, and can sensitively and selectivelydiscriminate single-nucleotide polymorphism (SNP).Finally, we designed a self-assembled tetrahedron as a rigid scaffold to organizemultivalent DNA aptamers SYL3C, targeting EpCAM,which is overexpressed on thesurfaces of mostCTCs, with precisecontrolof the number, distance, positionandorientation for enhance CTCs enrichment. Aptamer conjugated tetrahedron showedspecially binding to CTCs, and multivalent aptamers on tetrahedron showed higher binding avidities and more efficient enrichment than mono-aptamer. This methodbased on DNA nanostructure, had more biologic compatibility and was harmless tocells.The majority of the isolated cellsremained viableand the viability rateswereallcalculated more than85%, which could be directly cultured and propagatein vitrowithout separating magnetic beads. The cultured isolated cells hadnormalcellularmorphology and the same expression of EpCAM and mRNA as MCF-7cells culturedby media. Moreover, these multivalent aptamers could efficiently isolate MCF7fromheterogeneous mixtures and whole blood, with more than70%capture efficiency,which was more20%than monovalent aptamer. In addition, these multivalentaptamers conjugated on tetrahedral DNA nanostructure could detect CTCs in thewhole blood from breast cancer patients.