Concentric DNA Amplifier That Streamlines In-Solution Biorecognition and On-Particle Biocatalysis.

Colloidal nanoparticle biosensors capable of on-particle biocatalysis are powerful tools for amplified detection of biomolecules. The development and practical uses of such concentric amplifiers can be complicated because of the on-particle biorecognition that involves varying interfacial factors at the biomolecule-nanoparticle interfaces. Herein, we reason that a nanoparticle biosensor equipped with an in-solution biorecognition element may be better fabricated, predicted, controlled, and performed. The in-solution biorecognition shall also be streamlined with the on-particle biocatalysis so that the overall analytical and kinetic performance is not compromised. As a testbed, we introduce a concentric DNA amplifier driven by an enzyme-powered three-dimensional DNA nanomachine, where a DNA walker can be instantly assembled onto a spherical nucleic acid (SNA) track through a polyadenosine anchor. As such, the free DNA walker can participate in reactions in a homogeneous solution before assembling to the SNA track. The instant and stable assembly enabled by both adsorption and complementary base pairing also ensures rapid on-particle biocatalysis. We demonstrate that the in-solution biorecognition effectively eliminates the binding hindrance encountered by the on-particle biorecognition and thus significantly reduced energy barriers for the detection of nucleic acids and proteins. Because of the in-solution biorecognition, our system can also be plugged readily into complex DNA strand displacement networks for rapid signal amplification.

Probing and Controlling Dynamic Interactions at Biomolecule-Nanoparticle Interfaces Using Stochastic DNA Walkers.

Herein, we report a bottom-up approach to assemble a series of stochastic DNA walkers capable of probing dynamic interactions occurring at the bio-nano interface. We systematically investigated the impact of varying interfacial factors, including intramolecular interactions, orientation, cooperativity, steric effect, multivalence, and binding hindrance on enzymatic behaviors at the interfaces of spherical nucleic acids. Our mechanistic study has revealed critical roles of various interfacial factors that significantly alter molecular binding and enzymatic behaviors from bulk solutions. The improved understanding of the bio-nano interface may facilitate better design and operation of nanoparticle-based biosensors and/or functional devices. We successfully demonstrate how improved understanding of the bio-nano interface help rationalize the design of amplifiable biosensors for nucleic acids and antibodies.

Simulation-guided engineering of an enzyme-powered three dimensional DNA nanomachine for discriminating single nucleotide variants.

Single nucleotide variants (SNVs) are important both clinically and biologically because of their profound biological consequences. Herein, we engineered a nicking endonuclease-powered three dimensional (3D) DNA nanomachine for discriminating SNVs with high sensitivity and specificity. Particularly, we performed a simulation-guided tuning of sequence designs to achieve the optimal trade-off between device efficiency and specificity. We also introduced an auxiliary probe, a molecular fuel capable of tuning the device in solution via noncovalent catalysis. Collectively, our device produced discrimination factors comparable with commonly used molecular probes but improved the assay sensitivity by ∼100 times. Our results also demonstrate that rationally designed DNA probes through computer simulation can be used to quantitatively improve the design and operation of complexed molecular devices and sensors.

Enzyme-Powered Three-Dimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing.

Herein, we report a DNA nanomachine, built from a DNA-functionalized gold nanoparticle (DNA-AuNP), which moves a DNA walker along a three-dimensional (3-D) DNA-AuNP track and executes the task of releasing payloads. The movement of the DNA walker is powered by a nicking endonuclease that cleaves specific DNA substrates on the track. During the movement, each DNA walker cleaves multiple substrates, resulting in the rapid release of payloads (predesigned DNA sequences and their conjugates). The 3-D DNA nanomachine is highly efficient due to the high local effective concentrations of all DNA components that have been co-conjugated on the same AuNP. Moreover, the activity of the 3-D DNA nanomachine can be controlled by introducing a protecting DNA probe that can hybridize to or dehybridize from the DNA walker in a target-specific manner. This property allows us to tailor the DNA nanomachine into a DNA nanosensor that is able to achieve rapid, isothermal, and homogeneous signal amplification for specific nucleic acids in both buffer and a complicated biomatrix.

Chemical speciation of Zn, Cd, Cu, and Pb in pore waters of agricultural and contaminated soils using Donnan dialysis.

Knowledge of trace metal speciation in soil pore waters is important in addressing metal bioavailability and risk assessment of contaminated soils. Numerous analytical methods have been utilized for determining trace metal speciation in aqueous environmental matrixes; however, most of these methods suffer from significant interferences. The Donnan dialysis membrane technique minimizes these interferences and has been used in this study to determine free Zn2+, Cd2+, Cu2+, and Pb2+ activities in pore waters from 15 agricultural and 12 long-term contaminated soils. The soils vary widely in their origin, pH, organic carbon content, and total metal concentrations. Pore water pM2+ activities also covered a wide range and were controlled by soil pH and total metal concentrations. For the agricultural soils, most of the free metal activities were below detection limit, apart from Zn2+ for which the fraction of free Zn2+ in soluble Zn ranged from 2.3 to 87% (mean 43%). Five of the agricultural soils had detectable free Cd2+ with fractions of free metal ranging from 59 to 102% (mean 75%). For the contaminated soils with detectable free metal concentrations, the fraction of free metal as a percentage of soluble metal varied from 9.9 to 97% (mean 50%) for Zn2+, from 22 to 86% (mean 49%) for Cd2+, from 0.4 to 32.1% (mean 5%) for Cu2+, and from 2.9 to 48.8% (mean 20.1%) for Pb2+. For the contaminated soils, the equilibrium speciation programs GEOCHEM and WHAM Model VI provided reasonable estimates of free Zn2+ fractions in comparison to the measured fractions (R2 approximately 0.7), while estimates of free Cd2+ fractions were less agreeable (R2 approximately 0.5). The models generally predicted stronger binding of Cu2+ to DOC and hence lower fractions of free Cu2+ as compared with the observed fractions. The binding of Cu2+ and Pb2+ to DOC predicted by WHAM Model VI was much strongerthan that predicted by GEOCHEM.