Instantaneous attachment of an ultrahigh density of nonthiolated DNA to gold nanoparticles and its applications.

The last 16 years have witnessed the landmark development of polyvalent thiolated DNA-functionalized gold nanoparticles (AuNP's) possessing striking properties within the emerging field of nanobiotechnology. Many novel properties of this hybrid nanomaterial are attributed to the dense DNA shell. However, the question of whether nonthiolated polyvalent DNA-AuNP could be fabricated with a high DNA density and properties similar to those of its thiolated counterpart has not been explored in detail. Herein, we report that by simply tuning the pH of the DNA-AuNP mixture an ultrahigh capacity of nonthiolated DNA can be conjugated to AuNP's in a few minutes, resulting in polyvalent DNA-AuNP conjugates with cooperative melting behavior, a typical property of polyvalent thiolated DNA-functionalized AuNP's. With this method, large AuNP's (e.g., 50 nm) can be functionalized to achieve the colorimetric detection of sub-nanometer DNA. Furthermore, this fast, stable DNA loading was employed to separate AuNP's of different sizes. We propose that a large fraction of the attached DNAs are adsorbed via one or a few terminal bases to afford the high loading capacity and the ability to hybridize with the complementary DNA. This discovery not only offers a time- and cost-effective way to functionalize AuNP's with a high density of nonthiolated DNA but also provides new insights into the fundamental understanding of how DNA strands with different sequences interact with AuNP's.

Aptamer-functionalized hydrogel microparticles for fast visual detection of mercury(II) and adenosine.

With a low optical background, high loading capacity, and good biocompatibility, hydrogels are ideal materials for immobilization of biopolymers to develop optical biosensors. We recently immobilized mercury and lead binding DNAs within a monolithic gel and demonstrated ultrasensitive visual detection of these heavy metals. The high sensitivity was attributed to the enrichment of the analytes into the gels. The signaling kinetics was slow, however, taking about 1 h to obtain a stable optical signal because of a long diffusion distance. In this work, we aim to understand the analyte enrichment process and improve the signaling kinetics by preparing hydrogel microparticles. DNA-functionalized gel beads were synthesized using an emulsion polymerization technique and most of the beads were between 10 and 50 µm. Acrydite-modified DNA was incorporated by copolymerization. Visual detection of 10 nM Hg(2+) was still achieved and a stable signal was obtained in just 2 min. The gel beads could be spotted to form a microarray and dried for storage. A new visual sensor for adenosine was designed and immobilized within the gel beads. The adenosine aptamer binds its target about 1000-fold less tightly compared to the mercury binding DNA, allowing a comparison to be made on analyte enrichment by aptamer-functionalized hydrogels.

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.

Amplifying the macromolecular crowding effect using nanoparticles.

The melting temperature (T(m)) 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 T(m). We demonstrate herein that by attaching 12-mer DNAs to gold nanoparticles, the excluded volume change was significantly increased upon melting, leading to increased T(m) 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.

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

Controlled 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.

Dissociation and degradation of thiol-modified DNA on gold nanoparticles in aqueous and organic solvents.

Gold 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.

Electrostatically directed visual fluorescence response of DNA-functionalized monolithic hydrogels for highly sensitive Hg²+ detection.

Hydrogels 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 Hg(2+) 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 Hg(2+) 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.

Programmable assembly of DNA-functionalized liposomes by DNA.

Bionanotechnology involves the use of biomolecules to control both the structure and property of nanomaterials. One of the most studied examples is DNA-directed assembly of inorganic nanoparticles such as gold nanoparticles (AuNPs). However, systematic studies on DNA-linked soft nanoparticles, such as liposomes, are still lacking. We herein report the programmable assembly and systematic characterization of DNA-linked liposomes as a function of liposome size, charge, fluidity, composition, DNA spacer, linker DNA sequence, and salt concentration for direct comparison to DNA-directed assembly of AuNPs. Similar to the assemblies of AuNPs, sharp melting transitions were observed for liposomes where the first derivative of the melting curve full width at half-maximum (fwhm) is equal to or less than 1 °C for all of the tested liposomes, allowing sequence specific DNA detection. We found that parameters such as liposome size, charge, and fluidity have little effect on the DNA melting temperature. Cryo-TEM studies showed that programmable assemblies can be obtained and that the majority of the liposomes maintained a spherical shape in the assembled state. While liposome and AuNP systems are similar in many aspects, there are also important differences that can be explained by their respective physical properties.

DNA-functionalized monolithic hydrogels and gold nanoparticles for colorimetric DNA detection.

Highly 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.

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.

Regenerable DNA-functionalized hydrogels for ultrasensitive, instrument-free mercury(II) detection and removal in water.

Mercury is a highly toxic environmental pollutant with bioaccumulative properties. Therefore, new materials are required to not only detect but also effectively remove mercury from environmental sources such as water. We herein describe a polyacrylamide hydrogel-based sensor functionalized with a thymine-rich DNA that can simultaneously detect and remove mercury from water. Detection is achieved by selective binding of Hg(2+) between two thymine bases, inducing a hairpin structure where, upon addition of SYBR Green I dye, green fluorescence is observed. In the absence of Hg(2+), however, addition of the dye results in yellow fluorescence. Using the naked eye, the detection limit in a 50 mL water sample is 10 nM Hg(2+). This sensor can be regenerated using a simple acid treatment and can remove Hg(2+) from water at a rate of approximately 1 h(-1). This sensor was also used to detect and remove Hg(2+) from samples of Lake Ontario water spiked with mercury. In addition, these hydrogel-based sensors are resistant to nuclease and can be rehydrated from dried gels for storage and DNA protection. Similar methods can be used to functionalize hydrogels with other nucleic acids, proteins, and small molecules for environmental and biomedical applications.