DNA Assay Based on Nanoparticle-modified 2D Lipid Bilayer Micropattern Platform

Abstract

Lipids are a group of naturally occurring molecules that contain hydrocarbon and are soluble in nonpolar solvents, which are including diverse group of organic compounds such as fats, waxes, steroids, glycolipids, and phospholipids. They play an essential role in organisms such as storing energy, signaling, and constructing cell membrane structures. Phospholipids as the dominant lipid molecules in cell membranes, contain hydrophilic head groups and hydrophobic tails, which governs the spontaneous self-assembled bilayer structure. Then, phospholipid bilayer can be used as a fluidic membrane for the dynamic reaction or a cell mimicking model membrane for the biological study. Especially, supported lipid bilayers (SLBs) of phospholipids on solid glasses can endow the various physical and chemical functionalities of controlling biomolecules reaction, the modification capabilities to incorporate various biomolecules, and the robust platform to monitor the dynamic reactions by optical devices. DNA, deoxyribonucleic acid, is an essential molecule for organisms to survive and reproduce, which contains genetic codes for inheritance. All the genetic information is encoded by a characteristic sequence of four kinds of bases (i.e. adenine (A), cytosine (C), guanine (G), and thymine (T)) in a DNA strand. Mutation or random change in this DNA sequence by accidental exposure to mutagens or copying errors in DNA replication process, can result in the distortion of inheritance or malfunctioned genetic disorder, which cause genetic diseases or cancers. In the past decades, DNA bioassay has received broad attention due to its potential applications in a diversity of fields, e.g., clinical diagnosis, biomedical engineering, food development, environmental protection, forensic investigation and screening of biowarfare agents. One of main challenges in the development of DNA biosensors is the ultrasensitive, quantifiable, and highly reliable DNA detection without any help of amplification

amplification steps using enzymes, fluorescence dyes, and nanomaterials suffer from the erroneous signals by the amplification of the false signals or background signals, need complexed experimental procedures, and cause the signals to be vulnerable to a variety of environmental factors. To realize such an amplification-free, ultrasensitive DNA detection, an endeavor to discriminate rare true-signals from false-signals is necessary. Here, we employed the plasmonic nanoparticles-tethered fluidic SLB platform. Plamonic nanoparticles can generate highly strong light scattering signals and a molecular binding-involved distance change between nanoparticles at the several nanometers level can be detected by the plasmonic coupling. Moreover, the 2D fluidic lipid bilayer can concentrate target DNAs and improve the efficiency of binding reactions in a 2D lipid bilayer pattern. Accordingly, we firstly studied the characteristic properties and fundamental behaviors of SLB layer and nanoparticles on SLB layer with various experimental conditions. Next, the optimized conditions for the ultrasensitive DNA sensing were obtained. Furthermore, this platform and methodology can be applied to the discrimination of various point mutated single-nucleotide polymorphism (SNP) sequences. In chapter 1, we describe recently developed nanomaterial-tethered SLB platform in a formational standpoint. We summarize representative and convenient methods for the formation of supported phospholipid bilayers on planar hydrophilic substrates or micropatterns and linking methods for connecting between nanomaterials and the surface of the bilayer in a synthetic standpoint. We further focus on applications of nanomaterial-tethered SLB in biosensors to detect target molecules with ultrasensitivity and high target specificity. In chapter 2, we studied ultrasensitive and high-selective DNA bioassay through kinetic analysis of dissociation nanodimers using nanomaterial-tethered SLB platform. Amplification/enzyme-free detection and quantification of DNA at ultra-low concentrations, typically 10s-1,000s of targets in solution, is highly challenging but beneficial by offering a more straightforward, less contamination-prone, temperature control-free, less expensive, more quantitative and highly selective detection method than amplification/enzyme-based methods such as the polymerase chain reaction (PCR). Here, we developed an ultrasensitive, highly reliable bio-analytical method [the dynamic analysis on whole nanoparticle cumulative binding events (DANCE)] that allows for quantitatively analyzing dynamically associating and dissociating dimers generated by recognition of DNA strands with two-dimensionally mobile, photostable, and dark-field-detectable DNA-modified nanoparticles (NPs) on a lipid bilayer micropattern. Our results show that the amplification/enzyme-free DANCE provides the PCR-like sensitivity with high target specificity and excellent quantification capability for 10s-1,000s of DNA strands in a whole sample. In chapter 3, we used nanomaterial-tethered SLB biosensor to analyze sensitively mutant position determination of single base mismatched (SBM) DNA sequences. Although the thermodynamic difference among the mutant position-variable SBM sequences is too minuscule to differentiate them, we can measure and analyze the dissociation constants (koff) of various SBM sequences, respectively, which is obtained by counting dissociating nanodimers. As the sequence length is longer, koff value is gradually smaller due to the stability of duplex via multiple Watson-Crick base-pairing

however, the significant reduction of koff value from 6mer to 7mer sequences were exhibited, which seems to be related to the seven contiguous Watson-Crick base pair rule. By comparison of dissociations among 7mer, 13mer and 15mer DNA duplex systems, we also proved the seven contiguous Watson-Crick base pair rule, even though more than 10mer sequence. Kinetic information of SBM sequences obtained by counting the dissociation events of DNA mediated plasmonic nanodimer, is very sensitive to discriminate mutant point SBM sequences and helpful to understand the mechanism of DNA hybridization dynamics at the single molecular level. Moreover, our research and platform are expected to give much insight into unveiling the dynamic information of various biological reactions among biomaterials such as nucleic acids, proteins, and carbohydrates and the new biological mechanisms in the cellular level.