BARCODED METAL NANOWIRES FOR MULTIPLEXED DNA HYBRIDIZATION ASSAYS

Open Access
Author:
Stoermer, Rebecca Louise
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 05, 2006
Committee Members:
  • Christine Dolan Keating, Committee Chair
  • Thomas E Mallouk, Committee Member
  • Paul S Weiss, Committee Member
  • Ming Tien, Committee Member
Keywords:
  • barcoded metal nanowires
  • DNA
  • multiplexed
  • beacons
  • glass
  • silver
  • fluorophores
  • nanowires
  • metal
  • fluorescence
  • tags
Abstract:
ABSTRACT This thesis work was aimed towards developing multiplexed DNA hybridization assays using barcoded metal nanowires. These nanowires are typically several microns in length and ~300 nm in diameter, having segments of different metals along their lengths. They are promising for multiplexed bioanalysis due to the large number of striping patterns that can be synthesized, and the ease of optical read-out using simple reflectance microscopy. Oftentimes silver is used as one of the metals in the striped wires due to its high reflectivity at all visible wavelengths and large reflectance contrast to other metals. Since Ag metal is susceptible to oxidation, the long-term stability of Ag- containing wires in aqueous buffers was investigated and is reported in Chapter 2. It was found that wires stored in hybridization buffer for longer than two weeks began to show significant degradation of Ag segments. When agitated with continuous vortexing, the Ag oxidation progressed more rapidly, rendering the barcoded metal nanowires stored in hybridization buffer unidentifiable in less than one week. Addition of 40 mM citrate as a mild reducing agent increased Ag stability by 17 weeks over those stored in hybridization buffer. Nanowires subjected to continuous vortexing in 40 mM citrate buffer retained Ag segment stability for longer than 2 weeks. Derivatization of the wires with biomolecules such as are used in bioassays affords some additional protection against Ag degradation. Also, it has been discovered that wires coated with rhodamine-tagged DNA oligonucleotides attached via neutravidin-biotin chemistry are stable for 12 days in hybridization buffer and for at least 63 days when 40 mM citrate is added as a reducing agent. Ag deterioration in these experiments was coupled to loss of fluorescence from the labeled DNA, as well as wire breakage. When fluorescently-tagged oligonucleotides are located near metal surfaces, their emission intensity is impacted by both electromagnetic effects (i.e., quenching and/or enhancement of emission) and the structure of the nucleic acids (e.g., random coil, hairpin, or duplex). In Chapter 3, experiments are presented that explore the effect of label position and secondary structure in oligonucleotide probes as a function of hybridization buffer, which impacts the percentage of double-stranded probes on the surface after exposure to complementary DNA. Nanowires containing identifiable patterns of Au and Ag segments were used as the metal substrates in this work, which allowed for direct comparison of different dye positions in a single multiplexed experiment and differences in emission for probes attached to the two metals. The observed metal-dye separation dependence for unstructured, surface-bound oligonucleotides is highly sensitive to hybridization efficiency, due to substantial changes in DNA extension from the surface upon hybridization. In contrast, fluorophore-labeled oligonucleotides designed to form hairpin secondary structures analogous to solution-phase molecular beacon probes are relatively insensitive to hybridization efficiency, since the folded form is quenched and therefore does not appreciably impact the observed distance-dependence of the response. Differences in fluorescence patterning on Au and Ag were noted as a function of not only chromophore identity, but also metal¡Vdye separation. For example, emission intensity for tetramethylrhodamine (TAMRA)-labeled oligonucleotides changed from brighter on Ag for 24-base probes to brighter on Au for 48-base probes. Fluorescence enhancement at the ends of nanowires and at surface defects were also observed, where heightened electromagnetic fields affect the fluorescence. For the research presented in Chapter 4, molecular beacon probes have been combined with barcoded metal nanowires to enable no-wash, sealed-chamber, multiplexed detection of nucleic acids. Probe design and experimental parameters important in nanowire-based molecular beacon assays are discussed. Loop regions of 24 bases and five-base-pair stem regions in the molecular beacon probes gave optimal performance. The results suggest that thermodynamic predictions for secondary structure stability of solution-phase MB can guide probe design for nanowire-based assays. The predicted solution phase ƒ´G for probes in 500 mM buffered NaCl of approximately ¡V4 kcal/mol performed better than those with ƒ´G > ¡V2 or < ¡V6 kcal/mol. Buffered 300 and 500 mM NaCl solutions were selected after comparison of several buffers previously reported for similar types of assays, and concentrations in the range of 300 to 750 mM NaCl was found to be the optimal ionic strength for the hybridization temperature (25 oC) and probe designs used here. The sensitivity of this assay was ~100 pM and was limited by incomplete quenching. Single base mismatches could be discriminated from fully complementary targets. Oligonucleotide target sequences specific for Human Immunodeficiency, Hepatitis C, and Severe Acute Respiratory Viruses were assayed simultaneously in a no-wash, sealed-chamber, multiplexed experiment in which each of three probe sequences was attached to a different pattern of encoded nanowires. Finally, demonstrated in this chapter is that probe-coated nanowires retain their selectivity and sensitivity in a triplexed assay after storage for over three months. With the increasing interest in simultaneous detection of specific DNA hybridization events, the development of methods to measure multiple DNA interactions at one time is of great importance. Conventional microarrays allow thousands of DNA hybridization interactions to be measured at once; however, this method of detection is limited by high cost as well as the stability and characteristic properties of fluorescent dyes. In Chapter 5, barcoded nanowires were investigated as replacements for fluorophores on glass surfaces such as those used in microarrays. Potential advantages of nanowires include ease of reflectance-based optical read-out, the large number of tags available, and ability to distinguish multiple hybridizations occurring in a single DNA spot. A method of attaching DNA to glass microscope slides was employed which includes the use of a carboxy-terminated silane to derivatize glass slides for DNA attachment. Also determined here is the efficiency of using nanowires as tags in complementary DNA hybridization events. An average of ~5 % nonspecific binding was reported for nanowire attachment for all samples. In all chapters, it has been demonstrated that barcoded metal nanowires have promising uses in multiplexed bioanalysis for the detection of multiple analytes simultaneously. The metal surfaces have provided useful information about interactions between fluorophores and metals and DNA hybridization events (as described in Chapter 3). The surfaces, however, are subject to oxidation of silver segments (Chapter 2), and occasional pits in the metal surfaces have led to fluorescence enhancements at those pits, causing non-uniform fluorescence (Chapter 3). Also, non-uniformity in signal intensity results from different underlying metals acting differently on the fluorophores, as well as the distance the fluorophores are positioned from the surface (Chapter 3). To avoid the effects that the metal surfaces impose on fluorophores, nanowires were glass coated before use as substrates for fluorescently labeled bioassays. This work is presented in Chapter 6. Two different glass thicknesses (13.5 nm and 100 nm) were prepared and studied and 100 nm thickness provided the best surface, as the glass coating that resulted was very uniform. Also studied were the protective benefits that the silicon dioxide layers provide by shielding the silver segments from oxidative environments. We note that a 100 nm thick glass coating on the wires provided excellent resistance to dissolution of Ag segments when sonicated in nitric acid for 30 min and retained the same overall length and optical properties. Also shown in Chapter 6 is the feasibility of using glass coated nanowires as substrates in 2-plex assays where a single base mismatch in the target DNA sequence of a gene that affects the function of a tumor suppressor protein was detected.