A Single Electrochemical Biosensor Designed to Detect Any Virus.

Viruses are the primary cause of many infectious diseases in both humans and animals. Various testing methods require an amplification step of the viral RNA sample before detection, with quantitative reverse transcription polymerase chain reaction (RT-qPCR) being one of the most widely used along with lesser-known methods like Nucleic Acid Sequence-Based Amplification (NASBA). NASBA offers several advantages, such as isothermal amplification and high selectivity for specific sequences, making it an attractive option for low-income facilities. In this research, we employed a single electrochemical biosensor (E-Biosensor) designed for potentially detecting any virus by modifying the NASBA protocol. In this modified protocol, a reverse primer is designed with an additional 22-nucleotide sequence (tag region) at the 5'-end, which is added to the NASBA process. This tag region becomes part of the final amplicon generated by NASBA. It can hybridize with a single specific E-Biosensor probe set, enabling subsequent virus detection. Using this approach, we successfully detected three different viruses with a single E-Biosensor design, demonstrating the platform's potential for virus detection.

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.

Highly reproducible electrochemical biosensor for Influenza A virus towards low-resource settings.

A highly reproducible electrochemical biosensor, employing a five-stranded four-way junction (5S-4WJ) system through square wave voltammetry, has been successfully validated for the detection of Influenza A virus (InfA). A comprehensive assessment of its linearity, precision, accuracy, and robustness has demonstrated its compliance with FDA standards. Integration with Nucleic Acid-Based Amplification (NASBA) has showcased its selectivity for InfA, enabling the detection of InfA RNA with a standard heater set at 41 °C. This platform offers a straightforward setup well-suited for use at low-resource facilities.

Cost-Effective Modular Biosensor for SARS-CoV-2 and Influenza A Detection.

A modular, multi-purpose, and cost-effective electrochemical biosensor based on a five-stranded four-way junction (5S-4WJ) system was developed for SARS-CoV-2 (genes S and N) and Influenza A virus (gene M) detection. The 5S-4WJ structure consists of an electrode-immobilized universal stem-loop (USL) strand, two auxiliary DNA strands, and a universal methylene blue redox strand (UMeB). This design allows for the detection of specific nucleic acid sequences using square wave voltammetry (SWV). The sequence-specific auxiliary DNA strands (m and f) ensure selectivity of the biosensor for target recognition utilizing the same USL and UMeB components. An important feature of this biosensor is the ability to reuse the USL-modified electrodes to detect the same or alternative targets in new samples. This is accomplished by a simple procedure involving rinsing the electrodes with water to disrupt the 5S-4WJ structure and subsequent re-hybridization of the USL strand with the appropriate set of strands for a new analysis. The biosensor exhibited minimal loss in signal after rehybridization, demonstrating its potential as a viable multiplex assay for both current and future pathogens, with a low limit of quantification (LOQ) of as low as 17 pM.

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.

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).

Enhanced Fouling Resistance and Antimicrobial Property of Ultrafiltration Membranes Via Polyelectrolyte-Assisted Silver Phosphate Nanoparticle Immobilization.

Ultrafiltration (UF) is a low-pressure membrane that yields higher permeate flux and saves significant operating costs compared to high-pressure membranes; however, studies addressing the combined improvement of anti-organic and biofouling properties of UF membranes are lacking. This study investigated the fouling resistance and antimicrobial property of a UF membrane via silver phosphate nanoparticle (AgPNP) embedded polyelectrolyte (PE) functionalization. Negatively charged polyacrylic acid (PAA) and positively charged polyallylamine hydrochloride (PAH) were deposited on the membrane using a fluidic layer-by-layer assembly technique. AgPNPs were immobilized within the crosslinked "bilayers" (BL) of PAH/PAA. The effectiveness of AgPNP immobilization was confirmed by microprofile measurements on membrane surfaces using a solid contact Ag micro-ion-selective electrode. Upon stable and uniform BL formation on the membrane surface, the permeate flux was governed by a combined effect of PAH/PAA-derived hydrophilicity and surface/pore coverage by the BLs "tightening" of the membrane. When fouled by a model organic foulant (humic acid), the functionalized membrane exhibited a lower flux decline and a greater flux recovery due to the electrostatic repulsion imparted by PAA when compared to the unmodified membrane. The functionalization rendered antimicrobial property, as indicated by fewer attachments of bacteria that initiate the formation of biofilms leading to biofouling.

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.

An electrochemical biosensor based on Hairpin-DNA modified gold electrode for detection of DNA damage by a hybrid cancer drug intercalation.

An efficient and new electrochemical biosensor for detection of DNA damage, induced by the interaction of the hybrid anti-cancer compound (7ESTAC01) with DNA, was studied by differential pulse voltammetry (DPV). The biosensor consists of a Stem-Loop DNA (SL-DNA) probe covalently attached to the gold electrode (GE) surface that hybridizes to a complementary DNA strand (cDNA) to form a double-stranded DNA (dsDNA). The interaction and DNA damage induced by 7ESTAC01 was electrochemically studied based on the oxidation signals of the electroactive nucleic acids on the surface of the GE by DPV. As a result, the SL-DNA/GE and dsDNA/GE were tested with the reduced 7ESTAC01, showing the voltammetric signal of guanine and adenine, increase in the presence of 7ESTAC01. Under optimum conditions, the dsDNA/GE biosensor exhibited excellent DPV response in the presence of 7ESTAC01. The bonding interaction between 7ESTAC01 and calf thymus DNA (ctDNA) was confirmed by UV-Vis absorption spectroscopy, dynamic simulations (performed to investigate the DNA structure under physiological conditions), and molecular docking. Theoretical results showed the presence of hydrogen bonding and intercalation in the minor groove of DNA, involving hydrophobic interactions.

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.

A universal and label-free impedimetric biosensing platform for discrimination of single nucleotide substitutions in long nucleic acid strands.

We report a label-free universal biosensing platform for highly selective detection of long nucleic acid strands. The sensor consists of an electrode-immobilized universal stem-loop (USL) probe and two adaptor strands that form a 4J structure in the presence of a specific DNA/RNA analyte. The sensor was characterized by electrochemical impedance spectroscopy (EIS) using K3[Fe(CN)6]/K4[Fe(CN)6] redox couple in solution. An increase in charge transfer resistance (RCT) was observed upon 4J structure formation, the value of which depends on the analyte length. Cyclic voltammetry (CV) was used to further characterize the sensor and monitor the electrochemical reaction in conjunction with thickness measurements of the mixed DNA monolayer obtained using spectroscopic ellipsometry. In addition, the electron transfer was calculated at the electrode/electrolyte interface using a rotating disk electrode. Limits of detection in the femtomolar range were achieved for nucleic acid targets of different lengths (22 nt, 60 nt, 200 nt). The sensor produced only a background signal in the presence of single base mismatched analytes, even in hundred times excess in concentration. This label-free and highly selective biosensing platform is versatile and can be used for universal detection of nucleic acids of varied lengths which could revolutionize point of care diagnostics for applications such as bacterial or cancer screening.

In Situ Monitoring of Pb2+ Leaching from the Galvanic Joint Surface in a Prepared Chlorinated Drinking Water.

A novel method using a micro-ion-selective electrode (micro-ISE) technique was developed for in situ lead monitoring at the water-metal interface of a brass-leaded solder galvanic joint in a prepared chlorinated drinking water environment. The developed lead micro-ISE (100 µm tip diameter) showed excellent performance toward soluble lead (Pb2+) with sensitivity of 22.2 ± 0.5 mV decade-1 and limit of detection (LOD) of 1.22 × 10-6 M (0.25 mg L-1). The response time was less than 10 s with a working pH range of 2.0-7.0. Using the lead micro-ISE, lead concentration microprofiles were measured from the bulk to the metal surface (within 50 µm) over time. Combined with two-dimensional (2D) pH mapping, this work clearly demonstrated that Pb2+ ions build-up across the lead anode surface was substantial, nonuniform, and dependent on local surface pH. A large pH gradient (ΔpH = 6.0) developed across the brass and leaded-tin solder joint coupon. Local pH decreases were observed above the leaded solder to a pH as low as 4.0, indicating it was anodic relative to the brass. The low pH above the leaded solder supported elevated lead levels where even small local pH differences of 0.6 units (ΔpH = 0.6) resulted in about four times higher surface lead concentrations (42.9 vs 11.6 mg L-1) and 5 times higher fluxes (18.5 × 10-6 vs 3.5 × 10-6 mg cm-2 s-1). Continuous surface lead leaching monitoring was also conducted for 16 h.

A Single Electrochemical Probe Used for Analysis of Multiple Nucleic Acid Sequences.

Electrochemical hybridization sensors have been explored extensively for analysis of specific nucleic acids. However, commercialization of the platform is hindered by the need for attachment of separate oligonucleotide probes complementary to a RNA or DNA target to an electrode's surface. Here we demonstrate that a single probe can be used to analyze several nucleic acid targets with high selectivity and low cost. The universal electrochemical four-way junction (4J)-forming (UE4J) sensor consists of a universal DNA stem-loop (USL) probe attached to the electrode's surface and two adaptor strands (m and f) which hybridize to the USL probe and the analyte to form a 4J associate. The m adaptor strand was conjugated with a methylene blue redox marker for signal ON sensing and monitored using square wave voltammetry. We demonstrated that a single sensor can be used for detection of several different DNA/RNA sequences and can be regenerated in 30 seconds by a simple water rinse. The UE4J sensor enables a high selectivity by recognition of a single base substitution, even at room temperature. The UE4J sensor opens a venue for a re-useable universal platform that can be adopted at low cost for the analysis of DNA or RNA targets.

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.

Adsorption of Proteins to Thin-Films of PDMS and Its Effect on the Adhesion of Human Endothelial Cells.

This paper describes a simple and inexpensive procedure to produce thin-films of poly(dimethylsiloxane). Such films were characterized by a variety of techniques (ellipsometry, nuclear magnetic resonance, atomic force microscopy, and goniometry) and used to investigate the adsorption kinetics of three model proteins (fibrinogen, collagen type-I, and bovine serum albumin) under different conditions. The information collected from the protein adsorption studies was then used to investigate the adhesion of human dermal microvascular endothelial cells. The results of these studies suggest that these films can be used to model the surface properties of microdevices fabricated with commercial PDMS. Moreover, the paper provides guidelines to efficiently attach cells in BioMEMS devices.

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.