Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

DNA hybridization is of tremendous importance in biology, bionanotechnology, and biophysics. Molecular beacons are engineered DNA hairpins with a fluorophore and a quencher labeled on each of the two ends. A target DNA can open the hairpin to give an increased fluorescence signal. To date, the majority of molecular beacon detections have been performed only in aqueous buffers. We describe herein DNA detection in nine different organic solvents, methanol, ethanol, isopropanol, acetonitrile, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol, and glycerol, varying each up to 75% (v/v). In comparison with detection in water, the detection in organic solvents showed several important features. First, the molecular beacon hybridizes to its target DNA in the presence of all nine solvents up to a certain percentage. Second, the rate of this hybridization was significantly faster in most organic solvents compared with water. For example, in 56% ethanol, the beacon showed a 70-fold rate enhancement. Third, the ability of the molecular beacon to discriminate single-base mismatch is still maintained. Lastly, the DNA melting temperature in the organic solvents showed a solvent concentration-dependent decrease. This study suggests that molecular beacons can be used for applications where organic solvents must be involved or organic solvents can be intentionally added to improve the molecular beacon performance.University of Waterloo || Natural Sciences and Engineering Research Council |

Protection and Promotion of UV Radiation-Induced Liposome Leakage via DNA-Directed Assembly with Gold Nanoparticles

This is the peer reviewed version of the following article: Dave, N., & Liu, J. (2011). Protection and Promotion of UV Radiation-Induced Liposome Leakage via DNA-Directed Assembly with Gold Nanoparticles. Advanced Materials, 23(28), 3182–3186, which has been published in final form at https://doi.org/10.1002/adma.201101086. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.DNA-functionalized gold nanoparticles and liposomes are assembled by a linker DNA. The gold nanoparticles can either protect the liposome from UV radiation induced leakage or can promote the leakage, depending on the DNA sequence and the alignment of the particles.University of Waterloo || Natural Sciences and Engineering Research Council |

Biomimetic sensing based on chemically induced assembly of a signaling DNA aptamer on a fluid bilayer membrane

The adenosine aptamer was split into two halves and linked to a fluid liposome surface; addition of adenosine resulted in aptamer assembly, which did not occur if the split aptamer was attached to silica nanoparticles, demonstrating the feasibility of using aptamer probes to study diffusion within lipid membranes.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council || Ontario Ministry of Research and Innovation |

DNA functionalized soft materials: preparation, biophysical properties and analytical applications.

Bio-nanotechnology is the use of biomolecules to control both the structure and property of nanomaterials. No biomolecule has been employed more often than DNA as exemplified in the numerous demonstrations of DNA-directed assembly of nanomaterials. DNA has been used to covalently functionalize and assemble soft nanoparticles (e.g. liposomes) and hard nanoparticles (e.g. gold and silica nanoparticles) into a variety of hierarchical nanostructures. The majority of previous work however has focused on the latter, i.e., the assembly of “hard” nanoparticles such as gold nanoparticles (AuNPs) as oppose to the assembly of soft materials. The primary focus of this thesis is to add to the growing field of DNA-directed assembly of soft materials owing to the promise of such materials in a variety of analytical and biomedical applications. The first class of soft materials considered are liposomes which interestingly can be deformed by relatively weak intermolecular forces. In addition, DNA anchored to its surface can readily diffuse laterally within the lipid bilayer while DNA attached to inorganic nanoparticles remain fixed in position. We systematically consider the effect of varying the liposome structure, size, charge, and fluidity on liposome assemblies, in chapter 2. In addition, the interesting properties of liposomes are highlighted by a side-by-side comparison to DNA-functionalized gold nanoparticles, offering fundamental insights into DNA-directed assembly. Furthermore, hybrid DNA-directed assemblies composed of both AuNPs and liposomes are described in Chapter 3. In particular, the photothermal effects of such DNA-coupled liposome and AuNP assemblies were modulated by controlling the distance between liposome and AuNP allowing such systems to have potential application in drug-delivery. In chapter 4, the utility of liposomes is demonstrated as we exploit the fluidity of its diffuse bilayer with split aptamer functionalization for the rapid and selective detection of metabolites. The second class of soft material of interest in this thesis are hydrogels, which are cross-linked hydrophilic polymers. Because hydrogels are swollen in water, they can be used to immobilize biomolecules such as DNA for a myriad of applications. In chapter 5, the preparation and characterization of DNA-functionalized polyacrylamide hydrogels are presented. The use of such a DNA-modified hydrogel for the simultaneous detection and removal of mercury from water is subsequently demonstrated

Amplifying the Macromolecular Crowding Effect Using Nanoparticles

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemistry Society copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Zaki, A., Dave, N., & Liu, J. (2012). Amplifying the Macromolecular Crowding Effect Using Nanoparticles. Journal of the American Chemical Society, 134(1), 35–38. https://doi.org/10.1021/ja207661zThe melting temperature (Tm) of DNA is affected not only by salt but also by the presence of high molecular weight (MW) solutes, such as polyethylene glycol (PEG), acting as a crowding agent. For short DNAs in a solution of low MW PEGs, however, the change of excluded volume upon melting is very small, leading to no increase in Tm. We demonstrate herein that by attaching 12-mer DNAs to gold nanoparticles, the excluded volume change was significantly increased upon melting, leading to increased Tm even with PEG 200. Larger AuNPs, higher MW PEGs, and higher PEG concentrations show even larger effects in stabilizing the DNA. This study reveals a unique and fundamental feature at nanoscale due to geometric effects. It also suggests that weak interactions can be stabilized by a combination of polyvalent binding and the enhanced macromolecular crowding effect using nanoparticles.University of Waterloo || Canada Foundation for Innovation || Ontario Ministry of Research and Innovation || Natural Sciences and Engineering Research Council |

Stimuli-responsive releasing of gold nanoparticles and liposomes from aptamer-functionalized hydrogels

This is an author-created, un-copyedited version of an article accepted for publication/published in Nanotechnology. IOP Publishing Ltd is not responsible for any errors or omissions in this version of the manuscript or any version derived from it. The Version of Record is available online at https://doi.org/10.1088/0957-4484/22/49/494011Controlled release of therapeutic agents is important for improving drug efficacy and reducing toxicity. Recently, hydrogels have been used for controlled release applications. While the majority of the previous work focused on releasing the cargo in response to physical stimuli such as temperature, light, electric field, and pH, we aim to trigger cargo release in the presence of small metabolites. In our system a DNA aptamer that can bind to adenosine, AMP, and ATP was used as a linker to attach either DNA-functionalized gold nanoparticles or liposomes to DNA-functionalized hydrogels. In the presence of the metabolite, both the nanoparticle and liposome cargos were released. The effect of salt, temperature, target concentration, and drying has been systematically studied. Interestingly, we found that the gel can be completely dried while retaining the DNA linkages and adenosine induced release was still achieved after rehydration. Our work demonstrates that aptamers can be used to control the release of drugs and other materials attached to hydrogels.University of Waterloo || Canadian Foundation for Innovation || Natural Sciences and Engineering Research Council |

Electrostatically Directed Visual Fluorescence Response of DNA-Functionalized Monolithic Hydrogels for Highly Sensitive Hg2+ Detection

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Applied Materials and Interfaces, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/am101068cHydrogels are cross-linked hydrophilic polymer networks with low optical background and high loading capacity for immobilization of biomolecules. Importantly, the property of hydrogel can be precisely controlled by changing the monomer composition. This feature, however, has not been investigated in the rational design of hydrogel-based optical sensors. We herein explore electrostatic interactions between an immobilized mercury binding DNA, a DNA staining dye (SYBR Green I), and the hydrogel backbone. A thymine-rich DNA was covalently functionalized within monolithic hydrogels containing a positive, neutral, or negative backbone. These hydrogels can be used as sensors for mercury detection since the DNA can selectively bind Hg2+ between thymine bases inducing a hairpin structure. SYBR Green I can then bind to the hairpin to emit green fluorescence. For the neutral or negatively charged gels, addition of the dye in the absence of Hg2+ resulted in intense yellow background fluorescence, which was attributed to SYBR Green I binding to the unfolded DNA. We found that, by introducing 20% positively charged allylamine monomer, the background fluorescence was significantly reduced. This was attributed to the repulsion between positively charged SYBR Green I by the gel matrix as well as the strong binding between the DNA and the gel backbone. The signal-to-background ratio and detection limit was, respectively, improved by 6- and 9-fold using the cationic gel instead of neutral polyacrylamide gel. This study helps understand the electrostatic interaction within hydrogels, showing that hydrogels can not only serve as a high capacity matrix for sensor immobilization but also can actively influence the interaction between involved molecules.University of Waterloo || Natural Sciences and Engineering Research Council |

Dissociation and Degradation of Thiol-Modified DNA on Gold Nanoparticles in Aqueous and Organic Solvents

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/la200241dGold nanoparticles functionalized with thiol-modified DNA have been widely used in making various nanostructures, colorimetric biosensors, and drug delivery vehicles. Over the past 15 years, significant progress has been made to improve the stability of such functionalized nanoparticles. The stability of the gold–thiol bond in this system, however, has not been studied in a systematic manner. Most information on the gold–thiol bond was obtained from the study of self-assembled monolayers (SAMs). In this study, we employed two fluorophore-labeled and thiol-modified DNAs. The long-term stability of the thiol–gold bond as a function of time, salt, temperature, pH, and organic solvent has been studied. We found that the bond spontaneously dissociated under all tested conditions. The dissociation was favored at high salt, high pH, and high temperature, and little DNA degradation was observed in our system. Most organic solvents showed a moderate protection effect on the gold–thiol bond. The stability of the gold–thiol bond in the DNA system was also compared with that in SAMs. While there are many similarities, we also observed opposite trends for the salt and ethanol effect. This study suggests that the purified DNA-functionalized gold nanoparticles should be freshly prepared and used in a day or two. Long-term storage should be carried out at relatively low temperature in low salt and slightly acidic buffers.University of Waterloo || Natural Sciences and Engineering Research Council |

DNA-Functionalized Monolithic Hydrogels and Gold Nanoparticles for Colorimetric DNA Detection

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Applied Materials & Interfaces, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see Baeissa, A., Dave, N., Smith, B. D., & Liu, J. (2010). DNA-Functionalized Monolithic Hydrogels and Gold Nanoparticles for Colorimetric DNA Detection. ACS Applied Materials & Interfaces, 2(12), 3594–3600. https://doi.org/10.1021/am100780dHighly sensitive and selective DNA detection plays a central role in many fields of research, and various assay platforms have been developed. Compared to homogeneous DNA detection, surface-immobilized probes allow washing steps and signal amplification to give higher sensitivity. Previously research was focused on developing glass or gold-based surfaces for DNA immobilization; we herein report hydrogel-immobilized DNA. Specifically, acrydite-modified DNA was covalently functionalized to the polyacrylamide hydrogel during gel formation. There are several advantages of these DNA-functionalized monolithic hydrogels. First, they can be easily handled in a way similar to that in homogeneous assays. Second, they have a low optical background where, in combination with DNA-functionalized gold nanoparticles, even ∼0.1 nM target DNA can be visually detected. By using the attached gold nanoparticles to catalyze the reduction of Ag+, as low as 1 pM target DNA can be detected. The gels can be regenerated by a simple thermal treatment, and the regenerated gels perform similarly to freshly prepared ones. The amount of gold nanoparticles adsorbed through DNA hybridization decreases with increasing gel percentage. Other parameters including the DNA concentration, DNA sequence, ionic strength of the solution, and temperature have also been systematically characterized in this study.University of Waterloo || Natural Sciences and Engineering Research Council || Ministry of Higher Education of Saudi Arabia |

Assembly of DNA-Functionalized Gold Nanoparticles with Gaps and Overhangs in Linker DNA

This document is the Accepted Manuscript version of a Published Work that appeared in final form in The Journal of Physical Chemistry C, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jp111073wDNA-directed assembly of gold nanoparticles (AuNPs) has been extensively studied because of its important applications in analytical chemistry, materials science, and nanomedicine. In a typical system, two DNA-functionalized AuNPs are assembled via a linker DNA to form large aggregates. In the majority of the previous reports, the linker DNA is fully base paired with no gaps or overhangs present. Introducing such nonbase-paired regions in the linker DNA has been recently shown to be important for making stimuli-responsive materials and in crystallization of such AuNPs. In this work, we systematically studied the effect of introducing gaps and overhangs in the linker DNA to understand the kinetics of assembly and the melting transition of these aggregates. We found that the assembly kinetics decreased with increasing linker DNA length. The melting temperature decreased with the loss of base stacking by introducing gaps as well as the steric effect of overhangs. Additional insights were obtained by measuring the melting curves of the free DNAs in the absence of AuNPs. For example, it appeared that DNA base stacking at the nick site was favored in assembled nanoparticles compared to that in free DNA. Our results indicate that, while it is possible to form AuNP assemblies with linker DNAs containing various types of unpaired regions, these kinetic and thermodynamic factors need to be considered when designing related sensors and materials.University of Waterloo || Natural Sciences and Engineering Research Council |