DNA nanotechnology for nucleic acid analysis: sensing of nucleic acids with DNA junction-probes.

DNA nanotechnology deals with the design of non-naturally occurring DNA nanostructures that can be used in biotechnology, medicine, and diagnostics. In this study, we introduced a nucleic acid five-way junction (5WJ) structure for direct electrochemical analysis of full-length biological RNAs. To the best of our knowledge, this is the first report on the interrogation of such long nucleic acid sequences by hybridization probes attached to a solid support. A hairpin-shaped electrode-bound oligonucleotide hybridizes with three adaptor strands, one of which is labeled with methylene blue (MB). The four strands are combined into a 5WJ structure only in the presence of specific DNA or RNA analytes. Upon interrogation of a full-size 16S rRNA in the total RNA sample, the electrode-bound MB-labeled 5WJ association produces a higher signal-to-noise ratio than electrochemical nucleic acid biosensors of alternative design. This advantage was attributed to the favorable geometry on the 5WJ nanostructure formed on the electrode's surface. The 5WJ biosensor is a cost-efficient alternative to the traditional electrochemical biosensors for the analysis of nucleic acids due to the universal nature of both the electrode-bound and MB-labeled DNA components.

Single-Step Integration of Poly(3-Octylthiophene) and Single-Walled Carbon Nanotubes for Highly Reproducible Paper-Based Ion-Selective Electrodes.

Calibration of ion-selective electrodes (ISEs) is cumbersome, time-consuming, and constitutes a significant limitation for the development of single-use and wearable disposable sensors. To address this problem, we have studied the effect of ion-selective membrane solvent on ISE reproducibility by comparing tetrahydrofuran (THF) (a typical solvent for membrane preparation) and cyclohexanone. In addition, a single-step integration of semiconducting/transducer polymer poly(3-octylthiophene) (POT) with single-walled carbon nanotubes (SWCNTs) into the paper-based ISEs (PBISEs) substrate was introduced. PBISEs for potassium and sodium ions were developed, and these ISEs present outstanding sensor performance and high potential reproducibility, as low as ±1.0 mV (n = 3).

Selective Determination of Isothermally Amplified Zika Virus RNA Using a Universal DNA-Hairpin Probe in Less than 1 Hour.

The recent outbreak of the Zika virus (ZIKV) in the Americas and multiple studies that linked the virus to the cases of microcephaly and neurological complications have revealed the need for cost efficient and rapid ZIKV diagnostics tests. Here, a diagnostic platform relying on a four-way junction (4WJ)-based biosensor with electrochemical readout using a Universal DNA-Hairpin (UDH) probe for the selective recognition of an isothermally amplified ZIKV RNA fragment is developed. The 4WJ structure utilizes an electrode-immobilized stem-loop (DNA-hairpin) probe and two DNA adaptor strands complementary to both the stem-loop probe and the targeted fragment of a ZIKV amplicon. One of the adaptor strands is responsible for high selectivity of the target recognition, while another helps unwinding the target secondary structure. The first adaptor strand contains a redox label methylene blue to trigger the current change in response to the target-dependent formation of the 4WJ structure on the surface of the electrode. The amplicon can be analyzed directly from the amplification sample without the need for its purification. The proposed diagnostic methodology exhibits the limit of ZIKV RNA detection of 1.11 fg/µL (∼0.3 fM) and high selectivity that allows for reliable discrimination of ZIKV from West Nile virus and four dengue virus serotypes. Overall, the analysis of ZIKV RNA can be completed in less than 1 h, including amplification and electrochemical detection.

Visible light-triggered fluorescence and pH modulation using metastable-state photoacids and BODIPY.

Small changes in the pH gradient play a critical role in numerous biological and chemical pathways. Systems capable of monitoring and regulating these changes with high sensitivity and minimum photo-fatigue are in demand. Herein, we propose a visible light-triggered molecular system that allows for reversible regulation of acidity and fluorescence. This robust bi-functional system opens a new horizon towards novel studies that rely on small changes in acid-mediated controlled processes with high sensitivity. The two photosensitive compounds employed, a metastable-state photoacid (mPAH) and a boron-dipyrromethene (BODIPY) derivative, allow for consistent modulation of both fluorescence (based on the working principle of the inner filter effect) and pH (around a magnitude) over multiple cycles.

Circumventing Traditional Conditioning Protocols in Polymer Membrane-Based Ion-Selective Electrodes.

Preparation of ion-selective electrodes (ISEs) often requires long and complicated conditioning protocols limiting their application as tools for in-field measurements. Herein, we eliminated the need for electrode conditioning by loading the membrane cocktail directly with primary ion solution. This proof of concept experiment was performed with iodide, silver, and sodium selective electrodes. The proposed methodology significantly shortened the preparation time of ISEs, yielding functional electrodes with submicromolar detection limits. Moreover, it is anticipated that this approach may form the basis for the development of miniaturized all-solid-state ion-selective electrodes for in situ measurements.

Visible light-induced ion-selective optodes based on a metastable photoacid for cation detection.

A new platform of ion-selective optodes is presented here to detect cations under thermodynamic equilibrium via ratiometric analysis. This novel platform utilizes a 'one of a kind' visible light-induced metastable photoacid as a reference ion indicator to achieve activatable and controllable sensors. These ion-selective optodes were studied in terms of their stability, sensitivity, selectivity, and theoretical aspects.

Nanomolar detection limits of Cd²âº, Ag⁺, and K⁺ using paper-strip ion-selective electrodes.

Paper-based ion-selective electrodes (ISEs) are simple, flexible, and cost-efficient in comparison to conventional solid-contact ISEs. Yet, paper-based ISEs have poor limits of detection (in the micromolar range) relative to conventional solid-contact ISEs. Here we describe the construction and optimization of ISEs based on commercially available filter paper modified with single-walled carbon nanotubes (SWCNTs), sputtered gold, and conductive polymer poly(3-octylthiophene) to support an ion-selective membrane. The ion-selective membrane presented here is based on the copolymer methyl methacrylate-decyl methacrylate (MMA-DMA). The copolymer MMA-DMA is highly water-repellent and has a low coefficient of diffusion, which makes it particularly suitable for the creation of sensors with high performance in reaching low limits of detection. Three different configurations of the electrodes have been characterized by using contact angle surface analysis, oxygen influence, and testing for the presence of a water layer. Paper-strip ISEs for cadmium, silver, and potassium ions were developed with groundbreaking limits of detection of 1.2, 25.1, and 11.0 nM, respectively. In addition to such low limits of detection, paper-strip ISEs display high selectivity for their ion of interest and high reproducibility.

Visible light activated ion sensing using a photoacid polymer for calcium detection.

Presented here is a sensing membrane consisting of a modified merocyanine photoacid polymer and a calcium ionophore in plasticized poly(vinyl chloride). This membrane is shown to actively exchange protons with calcium ions when switched ON after illumination at 470 nm, and the exchange can be followed by UV-vis spectroscopy. The sensing membrane shows no response in the ON state when calcium ions are absent. The limit of detection of the sensor is 5.0 × 10(-4) M with an upper detection limit of 1.0 M. Thus, we demonstrate for the first time the use of a visible light activated, lipophilic photoacid polymer in an ion-sensing membrane for calcium ions, which highly discriminates potassium, sodium, and magnesium ions.

Open-Form Configurational Isomers of a Tricyanofuran-Type Metastable-State Photoacid.

We determine the presence of four open-form configurational isomers for an unsubstituted metastable-state photoacid (mPAH) of the tricyanofuran (TCF) type in solution, at room temperature, via 2D NMR experiments. Electronic structure calculations are carried out to predict the relative stability of the isomers found experimentally and their isomerization barriers. According to the calculated rate constants for isomerization, the molecule can freely interconvert between the open-form isomers, thereby providing a thermal pathway between the isomers that might be better suited to access the cyclized closed-form configuration and those that are not. In establishing the open form isomeric makeup of the TCF mPAH under study, this work establishes the need to consider the four isomers in further studies on the thermal and excited-state isomerization processes and substituent effect thereon.

Amplified potentiometric transduction of DNA hybridization using ion-loaded liposomes.

Amplified potentiometric transduction of DNA hybridization based on using liposome 'nanocarriers' loaded with the signaling ions is reported. The liposome-amplified potentiometric bioassay involved the duplex formation, followed by the capture of calcium-loaded liposomes, a surfactant-induced release and highly-sensitive measurements of the calcium signaling ions using a Ca(2+) ion-selective electrode (ISE). The high loading yield of nearly one million signaling ions per liposome leads to sub-fmol DNA detection limits. Factors affecting the ion encapsulation efficiency and signal amplification are evaluated and discussed. The influence of the surfactant lysing agent is also examined. Such use of 'green' calcium signaling ions addresses the inherent toxicity of Ag and CdS nanoparticle tags used in previous potentiometric bioassays. The new strategy was applied for the detection of low levels of E. coli bacteria. It could be readily extended to trace measurements of other important biomolecules in connection to different biorecognition events. The attractive analytical performance makes liposomes a useful addition to the armory of potentiometric bioassays.