Alexander H. Mo

Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

Significance We describe the first, to our knowledge, integrated dynamic DNA nanotechnology and 2D material electronics to overcome current limitations for the detection of DNA single-nucleotide polymorphism (SNP). Electrical detection of DNA has been advancing rapidly to achieve high specificity, sensitivity, and portability. However, the actual implementation of DNA detection is still in infancy because of low specificity, especially for analytically optimal and practically useful length of target DNA strands. Most of the research to date has focused on the enhancement of the sensitivity of DNA biosensors, whereas the specificity problem has remained unsolved. The low specificity is primarily attributed to the nonspecific binding during hybridization of the probe and the target DNA. Here, we have addressed these limitations by designing a functional prototype of electrical biosensors for SNP detection. Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine.

Designing Hollow Nano Gold Golf Balls

Hollow/porous nanoparticles, including nanocarriers, nanoshells, and mesoporous materials have applications in catalysis, photonics, biosensing, and delivery of theranostic agents. Using a hierarchical template synthesis scheme, we have synthesized a nanocarrier mimicking a golf ball, consisting of (i) solid silica core with a pitted gold surface and (ii) a hollow/porous gold shell without silica. The template consisted of 100 nm polystyrene beads attached to a larger silica core. Selective gold plating of the core followed by removal of the polystyrene beads produced a golf ball-like nanostructure with 100 nm pits. Dissolution of the silica core produced a hollow/porous golf ball-like nanostructure.

Energetically Biased DNA Motor Containing a Thermodynamically Stable Partial Strand Displacement State

Current work in tuning DNA kinetics has focused on changing toehold lengths and DNA concentrations. However, kinetics can also be improved by enhancing the completion probability of the strand displacement process. Here, we execute this strategy by creating a toehold DNA motor device with the inclusion of a synthetic nucleotide, inosine, at selected sites. Furthermore, we found that the energetic bias can be tuned such that the device can stay in a stable partially displaced state. This work demonstrates the utility of energetic biases to change DNA strand displacement kinetics and introduces a complementary strategy to the existing designs.

Asymmetric Colloidal Janus Particle Formation Is Core-Size-Dependent

Colloidal particles with asymmetric surface chemistry (Janus particles) have unique bifunctional properties. The size of these particles is an important determinant for their applications in diverse fields from drug delivery to chemical catalysis. The size of Janus particles, with a core surface coated with carboxylate and a partially encapsulating silica shell, depends upon several factors, including the core size and the concentration of carboxylate coating. The role of the carboxylate coating on the Janus particle size is well-understood; however, the role of the core size is not well defined. The role of the carboxylated polystyrene (cPS) core size on the cPS-silica Janus particle morphology (its size and shape) was examined by testing two different silica sizes and five different cPS core sizes. Results from electron microscopy (EM) and dynamic light scattering (DLS) analysis indicate that the composite cPS-silica particle acquires two distinct shapes: (i) when the size of the cPS core is much smaller than the non-cPS silica (b-SiO2) sphere, partially encapsulated Janus particles are formed, and (ii) when the cPS core is larger than or equal to the b-SiO2 sphere, a raspberry-like structure rather than a Janus particle is formed. The cPS-silica Janus particles of ∼100-500 nm size were obtained when the size of the cPS core was much smaller than the non-cPS silica (b-SiO2) sphere. These scalable nanoscale Janus particles will have wide application in a multifunctional delivery platform and catalysis.

Dual-Functionalized Theranostic Nanocarriers.

Nanocarriers with the ability to spatially organize chemically distinct multiple bioactive moieties will have wide combinatory therapeutic and diagnostic (theranostic) applications. We have designed dual-functionalized, 100 nm to 1 μm sized scalable nanocarriers comprising a silica golf ball with amine or quaternary ammonium functional groups located in its pits and hydroxyl groups located on its nonpit surface. These functionalized golf balls selectively captured 10-40 nm charged gold nanoparticles (GNPs) into their pits. The selective capture of GNPs in the golf ball pits is visualized by scanning electron microscopy. ζ potential measurements and analytical modeling indicate that the GNP capture involves its proximity to and the electric charge on the surface of the golf balls. Potential applications of these dual-functionalized carriers include distinct attachment of multiple agents for multifunctional theranostic applications, selective scavenging, and clearance of harmful substances.

Tunable and Regenerative DNA Zipper Based Spring

We report a DNA zipper based actuator device termed ‘DNA- spring’ with tunable and repeated cycles of extension and contraction ability. DNA zipper is a double-stranded DNA system engineered to open upon its specific interaction with appropriately designed single strand DNA (ssDNA), opening of the zipper is driven by binding energy differences between the DNA strands. The zipper system is incorporated with defined modifications to function like a spring, capable of delivering ∼9 pN force over a distance of ∼13 nm, producing ∼116 kJ/mol of work. The spring described here consists of four DNA oligonucleotides totaling 244 bases that utilize ssDNA to trigger its extension and contraction. Time-lapse fluorescence and fluorescent DNA gel electrophoresis analysis is utilized to evaluate and confirm the spring action. A second zipper incorporated into the spring provides the ability to couple/decouple to an object/substrate. Such devices would have wide application, including for conditionally triggered molecular delivery systems and as actuators in nano-devices.

DNA Based Dual-Spring Cross Shaped Nanoactuator

DNA is an attractive platform for nanotechnology applications because of its size, specificity, and designability. However constructing DNA-based platforms that can do work is difficult. We have developed a DNA-based cross-shaped nanoactuator system that cycles between an extended and contracted confirmation relying on strand displacement reactions. The actuator contains 4 structural strands with two unique DNA “zipper” sequences. Each zipper sequence employs traditional adenosine-thymine nucleotides as well as non-traditional inosine-cytidine nucleotides. The I-C bond consists of only 2 hydrogen bonds as opposed to the typical 3 hydrogen bonds found in G-C bonds. The actuator is extended by inserting two ssDNA which are the natural complements to the zipper sequences. The natural complements have a stronger binding affinity to one side of the zipper than both zipper strands have to each other, thus unraveling and allowing the actuator to extend. The two contraction strands contain sequences which are a natural complement to parts of the opening strand. When they bind to the extension sequences, the zippers are able to rebind and this contracts the actuator. Proper assembly and function of the devices was confirmed using fluorescent DNA gel electrophoresis, AFM imaging, and time-lapsed fluorescence.View Large Image | View Hi-Res Image | Download PowerPoint Slide