Nucleic acid sensing via electrochemical oligonucleotide-templated reactions.

Short single-stranded nucleic acids as found in a variety of bodily fluids have recently emerged as minimally invasive biomarkers for a broad range of pathologies, most notably cancer. Because of their small size, low natural abundance and high sequence homology between family members they are challenging to detect using standard technologies suitable for use at the point-of-care. Herein we report the design, engineering and testing of a novel sensing strategy: electrochemically active molecular probes based on peptide nucleic acid (PNA) scaffolds for the detection of single-stranded oligonucleotides, in particular microRNAs (or miRs). As a proof-of-principle, a wide range of probes were designed and tested to detect miR-141, a known diagnostic biomarker for prostate cancer. Optimal quantitative sensing of miR-141 was achieved via the first example of an electrochemical oligonucleotide-templated reaction (EOTR), whereby two PNA probes - functionalized with an aniline and a 1,4-catechol respectively - preferentially react with each other upon simultaneous hybridization to the same RNA target strand, serving here as a template. Quantitative, electrochemical detection of the product of this bio-orthogonal reaction showed direct correlation between adduct formation and miR-141 concentration. Coupling the specificity of OTR with the speed and sensitivity of electrochemical sensing delivers EOTRs as a promising new technique for fast, low-cost, quantitative and sequence-specific detection of short nucleic acids from liquid biopsies.

A Nuclease Protection ELISA Assay for Colorimetric and Electrochemical Detection of Nucleic Acids.

Early and accurate diagnosis is crucial to monitor infection outcomes and provide timely interventions. However, gold standard polymerase chain reaction assays (PCR) are labor-intensive and require expensive reagents and instrumentation. Nuclease protection has been used for decades to detect and quantify nucleic acid but has not yet been investigated as a diagnostic tool for infectious disease. In this work, we describe a nuclease protection enzyme-linked immunosorbent assay (NP-ELISA) for accurate and sensitive detection of nucleic acid. Briefly, binding of a nucleic acid target to an oligo probe protects it from digestion of un-hybridized nucleic acid by S1 nuclease. Following the workflow of an ELISA, a horseradish peroxidase (HRP)-conjugated antibody binds the probe and oxidizes its substrate to generate signal. The assay was validated with three HRP substrates for absorbance, chemiluminescence, and electrochemical readouts, demonstrating great versatility. Electrochemical detection with 3,3',5,5'-Tetramethylbenzidine (TMB) gave the highest assay sensitivity with a limit of detection of 3.72×103 molecules mL-1. Furthermore, non-complementary targets did not generate a response, indicating a high degree of specificity. This proof of principle serves as a stepping stone towards developing miniaturized, multiplexed nuclease protection assays for point-of-care diagnosis.

Correction: Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs.

Correction for 'Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs' by Suraj Pavagada et al., Chem. Commun., 2019, 55, 12451-12454.

Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs.

Herein we demonstrate the first example of oligonucleotide-templated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth.

Multilayered Microfluidic Paper-Based Devices: Characterization, Modeling, and Perspectives.

Microfluidic paper-based analytical devices (µPADs) are simple but powerful analytical tools that are gaining significant recent attention due to their many advantages over more traditional monitoring tools. These include being inexpensive, portable, pump-free, and having the ability to store reagents. One major limitation of these devices is slow flow rates, which are controlled by capillary action in the hydrophilic pores of cellulosic paper. Recent investigations have advanced the flow rates in µPADs through the generation of a gap or channel between two closely spaced paper sheets. This multilayered format has opened up µPADs to new applications and detection schemes, where large gap sizes (>300 µm) provide at least 169× faster flow rates than single-layer µPADs, but do not conform to established mathematical models for fluid transport in porous materials, such as the classic Lucas-Washburn equation. In the present study, experimental investigations and analytical modeling are applied to elucidate the driving forces behind the rapid flow rates in these devices. We investigate a range of hypotheses for the systems fluid dynamics and establish a theoretical model to predict the flow rate in multilayered µPADs that takes into account viscous dissipation within the paper. Device orientation, sample addition method, and the gap height are found to be critical concerns when modeling the imbibition in multilayered devices.

Janus electrochemistry: Simultaneous electrochemical detection at multiple working conditions in a paper-based analytical device.

The simultaneous detection of multiple analytes from a single sample is a critical tool for the analysis of real world samples. However, this is challenging to accomplish in the field by current electroanalytical techniques, where tuning assay conditions towards a target analyte often results in poor selectivity and sensitivity for other species in the mixture. In this work, an electrochemical paper-based analytical device (ePAD) capable of performing simultaneous electrochemical experiments in different solution conditions on a single sample was developed for the first time. We refer to the system as a Janus-ePAD after the two-faced Greek god because of the ability of the device to perform electrochemistry on the same sample under differing solution conditions at the same time with a single potentiostat. In a Janus-ePAD, a sample wicks down two channels from a single inlet towards two discreet reagent zones that adjust solution conditions, such as pH, before flow termination in two electrochemical detection zones. These zones feature independent working electrodes and shared reference and counter electrodes, facilitating simultaneous detection of multiple species at each species' optimal solution condition. The device utility and applicability are demonstrated through the simultaneous detection of two biologically relevant species (norepinephrine and serotonin) and a common enzymatic assay product (p-aminophenol) at two different solution pH conditions. Janus-ePADs show great promise as an inexpensive and broadly applicable platform which can reduce the complexity and/or number of steps required in multiplexed analysis, while also operating under the optimized conditions of each species present in a mixture.

Thermoplastic Electrode Arrays in Electrochemical Paper-Based Analytical Devices.

Electrochemical paper-based analytical devices (ePADs) have garnered significant interest as an alternative to traditional benchtop methods due to their low cost and simple fabrication. Historically, ePADs have relied almost exclusively on single electrode detection, limiting potential gains in sensitivity and selectivity achievable with multiple electrodes. Herein we describe incorporation of thermoplastic electrode (TPE) arrays into flow ePADs. Quasi-steady flow was solely generated by capillary action through a fan-shaped paper device. The electrode arrays were fabricated using a simple solvent-assisted method with inexpensive materials (i.e., graphite and thermoplastic binder). These electrodes can be employed as an array of individually addressable detectors or connected as an interdigitated electrode array. The TPEs were characterized through SEM, optical profilometry and cyclic voltammetry. Chronoamperometry was used to characterize the flow-based TPE-ePADs. Trace detection of a ferrocene complex (FcTMA+) was demonstrated through generation-collection experiments, achieving a limit of detection of 0.32 pmol. These TPE arrays containing ePADs show great promise as a rapid, sensitive, and low-cost sensor for point-of-need (PON) applications.

Development of an Electrochemical Paper-Based Analytical Device for Trace Detection of Virus Particles.

Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 µL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.

Rapid flow in multilayer microfluidic paper-based analytical devices.

Microfluidic paper-based analytical devices (µPADs) are a versatile and inexpensive point-of-care (POC) technology, but their widespread adoption has been limited by slow flow rates and the inability to carry out complex in field analytical measurements. In the present work, we investigate multilayer µPADs as a means to generate enhanced flow rates within self-pumping paper devices. Through optical and electrochemical measurements, the fluid dynamics are investigated and compared to established flow theories within µPADs. We demonstrate a ∼145-fold increase in flow rate (velocity = 1.56 cm s-1, volumetric flow rate = 1.65 mL min-1, over 5.5 cm) through precise control of the channel height in a 2 layer paper device, as compared to archetypical 1 layer µPAD designs. These design considerations are then applied to a self-pumping sequential injection device format, known as a three-dimensional paper network (3DPN). These 3DPN devices are characterized through flow injection analysis of a ferrocene complex and anodic stripping detection of cadmium, exhibiting a 5× enhancement in signal compared to stationary measurements.

A paper-based colorimetric spot test for the identification of adulterated whiskeys.

A colorimetric point-of-care paper-based analytical device (PAD) is developed for detecting adulterated beverages using whiskey falsified with caramel color as a model. Combining principal component analysis and calibration curves facilitated identification of adulteration in samples seized by the Brazilian Federal Police, at only ∼$0.02 per sample.

Boron Doped Diamond Paste Electrodes for Microfluidic Paper-Based Analytical Devices.

Boron doped diamond (BDD) electrodes have exemplary electrochemical properties; however, widespread use of high-quality BDD has previously been limited by material cost and availability. In the present article, we report the use of a BDD paste electrode (BDDPE) coupled with microfluidic paper-based analytical devices (µPADs) to create a low-cost, high-performance electrochemical sensor. The BDDPEs are easy to prepare from a mixture of BDD powder and mineral oil and can be easily stencil-printed into a variety of electrode geometries. We demonstrate the utility and applicability of BDDPEs through measurements of biological species (norepinephrine and serotonin) and heavy metals (Pb and Cd) using µPADs. Compared to traditional carbon paste electrodes (CPE), BDDPEs exhibit a wider potential window, lower capacitive current, and are able to circumvent the fouling of serotonin. These results demonstrate the capability of BDDPEs as point-of-care sensors when coupled with µPADs.

Electrochemical flow injection analysis of hydrazine in an excess of an active pharmaceutical ingredient: achieving pharmaceutical detection limits electrochemically.

The quantification of genotoxic impurities (GIs) such as hydrazine (HZ) is of critical importance in the pharmaceutical industry in order to uphold drug safety. HZ is a particularly intractable GI and its detection represents a significant technical challenge. Here, we present, for the first time, the use of electrochemical analysis to achieve the required detection limits by the pharmaceutical industry for the detection of HZ in the presence of a large excess of a common active pharmaceutical ingredient (API), acetaminophen (ACM) which itself is redox active, typical of many APIs. A flow injection analysis approach with electrochemical detection (FIA-EC) is utilized, in conjunction with a coplanar boron doped diamond (BDD) microband electrode, insulated in an insulating diamond platform for durability and integrated into a two piece flow cell. In order to separate the electrochemical signature for HZ such that it is not obscured by that of the ACM (present in excess), the BDD electrode is functionalized with Pt nanoparticles (NPs) to significantly shift the half wave potential for HZ oxidation to less positive potentials. Microstereolithography was used to fabricate flow cells with defined hydrodynamics which minimize dispersion of the analyte and optimize detection sensitivity. Importantly, the Pt NPs were shown to be stable under flow, and a limit of detection of 64.5 nM or 0.274 ppm for HZ with respect to the ACM, present in excess, was achieved. This represents the first electrochemical approach which surpasses the required detection limits set by the pharmaceutical industry for HZ detection in the presence of an API and paves the wave for online analysis and application to other GI and API systems.

Examination of the factors affecting the electrochemical performance of oxygen-terminated polycrystalline boron-doped diamond electrodes.

In order to produce polycrystalline oxygen-terminated boron-doped diamond (BDD) electrodes suitable for electroanalysis (i.e., widest solvent window, lowest capacitive currents, stable and reproducible current responses, and capable of demonstrating fast electron transfer) for outer sphere redox couples, the following factors must be considered. The material must contain enough boron that the electrode shows metal-like conductivity; electrical measurements demonstrate that this is achieved at [B] > 10(20) B atoms cm(-3). Even though BDD contains a lower density of states than a metal, it is not necessary to use extreme doping levels to achieve fast heterogeneous electron transfer (HET). An average [B] ~ 3 × 10(20) B atoms cm(-3) was found to be optimal; increasing [B] results in higher capacitive values and increases the likelihood of nondiamond carbon (NDC) incorporation. Hydrogen-termination causes a semiconducting BDD electrode to behave metal-like due to the additional surface conductivity hydrogen termination brings. Thus, unless [B] of the material is known, the electrical properties of the electrode may be incorrectly interpreted. Note, this layer (formed on a lapped electrode) is electrochemically unstable, an effect which is exacerbated at increased potentials. It is essential during growth that NDC is minimized as it acts to increase capacitive currents and decrease the solvent window. We found complete removal of NDC after growth using aggressive acid cleans, acid cycling, and diamond polishing impossible. Although hydrogen termination can mask the NDC signature in the solvent window and lower capacitive currents, this is not a practical procedure for improving sensitivity in electroanalysis. Finally, alumina polishing of lapped, NDC free, freestanding, BDD electrodes was found to be an effective way to produce well-defined, stable, and reproducible surfaces, which support fast (reversible) HET for Fe(CN)6(4-) electrolysis, the first time this has been reported at an oxygen-terminated surface.