Versatile DNA Balances via Adjacent Base Stacking for Homogeneous Assay of Energy Parameters, Small Molecules, And Ribonuclease.

Homogeneous assays often obviate any separation and washing steps, thus minimizing the risks of contamination and false positive. DNA toehold exchange is a homogeneous, reversible process whose thermodynamic properties can be finely tuned for various assay applications. However, the developed probes often rely on direct interactions of analytes with DNA strands involved in toehold exchange, limiting the versatility of probe design. Here, the coaxial adjacent stacking between one auxiliary strand and another invading strand offers a favorable ΔG to shift one DNA balance, while the auxiliary strand is independent of the DNA balance itself. Therefore, such a DNA balance allowed fine tuning of the equilibrium via adjustment of the auxiliary strand alone. The energy contribution of base stacking can be quantified in a homogeneous solution based on the difference in the equilibrium constant. Besides, the proof of concept for DNA balance allows effective assay of a small molecule or ribonuclease in a homogeneous solution. This novel DNA balance via adjacent base stacking provides an interesting alternative to homogeneously assay various analytes.

Simulation-Guided Rational Design of DNA Probe for Accurate Discrimination of Single-Nucleotide Variants Based on "Hill-Type" Cooperativity.

The accurate discrimination of single-nucleotide variants is of great interest for disease diagnosis and clinical treatments. In this work, a unique DNA probe with "Hill-type" cooperativity was first developed based on toehold-mediated strand displacement processes. Under simulation, this probe owns great thermodynamics advantage for specificity due to two mismatch bubbles formed in the presence of single-nucleotide variants. Besides, the strategies of ΔG' = 0 and more competitive strands are also beneficial to discriminate single-nucleotide variants. The feasibility of this probe was successfully demonstrated in consistent with simulation results. Due to "Hill-type" cooperativity, the probe allows a steeper dynamic range compared with previous probes. With simulation-guided rational design, the resulting probe can accurately discriminate single-nucleotide variants including nucleotide insertions, mutation, and deletions, which are arbitrarily distributed in target sequence. Two specificity parameters were calculated to quantitatively evaluate its good discrimination ability. Hence, "Hill-type" cooperativity can serve as a novel strategy in DNA probe's design for accurate discrimination of single-nucleotide variants.

Target-triggered double fluorescent biosensors for rapid and sensitive detection of long-chain perfluorinated compounds using DNA probe and lysozyme fiber.

Perfluorinated compounds (PFCs) are useful man-made chemicals and serve as new emerging organic pollutants due to their environmental and health concerns. Chromatography-mass detection methods often need complex procedure and are also too expensive, so there is a critical demand to develop rapid, inexpensive, easy-to-operate and sensitive methods for PFCs detection. In this work, double fluorescent biosensors ('DT sensor' and 'FT sensor') have been designed to quantitatively detect long-chain perfluorinated compounds (PFCs), due to their strong hydrophobic interaction with DNA probe or lysozyme fiber. The ratio and rapid fluorescence responses offered more obvious signal changes, and high sensitivity with a limit of detection (LOD) of 0.16 µM (98.2 ppb) for perfluorododecanoic acid (PFDoA). For three PFCs with longer perfluoroalkyl chain (CF2), increased detection sensitivity was achieved due to a stronger hydrophobicity. The fluorescent biosensors showed a good selectivity for long-chain PFCs and served as cross-reactive sensors to differentiate three different long-chain PFCs. The biosensors also had robust signal response in tap water or serum samples, and the LOD can be further lowered to pM (ppt) level after sample preconcentration.

Ovalbumin-coated gold nanoparticles with interesting colloidal stability for colorimetric detection of carbaryl in complex media.

Pesticide carbaryl can cause serious environmental pollution and its sensitive detection is of increasing interest. Gold nanoparticles (AuNPs) are classically colorimetric probes for detection of many analytes, but the instability in complex media limits their application. Here, Au@Ova NPs have been developed as a stable, effective, sensitive, and selective sensing system for colorimetric detection of carbaryl. Au@Ova NPs present unique and proper colloidal stability in various medias containing salt, small molecules, organic solvent (DMSO), and seawater, which are distinct from previous ones including citrate (or rhodamine B) capped AuNPs. Compared with Au@BSA NPs, Au@Ova NPs showed efficient responses to carbaryl by inhibiting acetylcholinesterase, with a linear concentration range between 0 and 25 µg/L and a detection limit of 0.25 µg/L. In addition, this nanoprobe also has good selectivity and can be applied in different real samples analysis, including fruit juice (tomato and apple) and real water samples (artificial urine and seawater).

A green photocatalytic-biosensor for colorimetric detection of pesticide (carbaryl) based on inhibition of acetylcholinesterase.

Carbaryl is a widely-used carbamate pesticide and the detection of its residues in environmental, food and clinical samples is of great importance. In this sturdy, we developed a green photocatalytic-biosensor based on double strand DNA-SYBR green I complex for sensitively colorimetric detection of carbaryl. This green photocatalytic-biosensor can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into blue ox-TMB. Meanwhile thiocholine is catalytically produced by acetylcholinesterase (AChE) to directly reduce blue ox-TMB into colorless TMB. But the activity of AChE will be suppressed by carbaryl, thus generating less thiocholine and resulting in more ox-TMB for colorimetric analysis. After the careful optimization of sensing conditions (2 µM for DNA concentration, 50 × concentration for SYBR Green I, 10 min for illumination time), the lowest detectable concentration for carbaryl is 0.008 ng/mL with a linear response in the range of 0.01-0.25 ng/mL. In addition, this photocatalytic-biosensor has good selectivity over non-target chemicals (acetamiprid, atrazine, carbendazim, melamine, bisphenol A, estradiol). It also allows detection of pesticides in real samples verified by a standard HPLC method.