A highly sensitive electrochemical methodology for end-point detection of loop-mediated isothermal nucleic acid amplification reactions was developed. It is based on the oxidation process of phenol ...red (PR), commonly used as a visual indicator. The dependence of its redox process on pH, which changes during amplification, allows performing quantitative measurements. Thus, the change in the oxidation potential of PR during the amplification is used, for the first time, as the analytical signal that correlates with the number of initial DNA copies. As a proof-of-concept, the amplification of the pneumolysin gene from Streptococcus pneumoniae, one of the main pathogens causing community-acquired pneumonia, is performed. Combination of isothermal amplification with electrochemical detection, performed on small-size flexible electrodes, allows easy decentralization. Adaptation to the detection of other pathogens causing infectious diseases would be very useful in the prevention of future epidemics.
Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural ...skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE.
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•New hydrogel formulation based on native skin extracellular matrix components.•The formulation promotes cell viability and enhances in vitro wound healing.•Biofabrication of a bilayered dermal/hypodermal 3D model.•The model achieves biomimetic mechanical and biological properties similar to skin.
Quantitative polymerase chain reaction (qPCR) is considered the gold standard for pathogen detection. However, improvement is still required, especially regarding the possibilities of ...decentralization. Apart from other reasons, infectious diseases demand on‐site analysis to avoid pathogen spreading and increase treatment efficacy. In this paper, the detection of SARS‐CoV‐2 is carried out by reverse transcription loop‐mediated isothermal amplification, which has the advantage of requiring simple equipment, easily adaptable to decentralized analysis. It is proposed, for the first time, the use of palladium nanoclusters (PdNCs) as indicators of the amplification reaction at end point. The pH of the medium decreases during the reaction and, in turn, a variation in the catalytic activity of PdNCs on the oxygen reduction reaction (ORR) can be electrochemically observed. For the detection, flexible and small‐size screen‐printed electrodes can be premodified with PdNCs, which together with the use of a simple and small electrochemical equipment would greatly facilitates their integration in field‐deployable devices. This would allow a faster detection of SARS‐CoV‐2 as well as of other future microbial threats after an easy adaptation.
In this article, authors propose an innovative methodology for electrochemical COVID‐19 diagnosis using palladium nanoclusters (PdNCs)‐based voltammetry. The product of the isothermal loop‐mediated amplification (LAMP) is transferred to a card with screen‐printed electrodes, previously modified with PdNCs. The change in the pH that occurs during a LAMP reaction produces a variation in the catalytic activity of PdNCs.
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•A fast, sensitive, and simple on-site method is presented for COVID-19 diagnosis.•Electrochemical detection is combined with RT-LAMP for on-site SARS-CoV-2 detection.•Malachite green ...LAMP probe is monitored electrochemically for quantitative readout.•Electrochemical measurement of malachite green is presented for SARS-CoV-2 monitoring.•Dual visual/electrochemical LAMP detection is made using malachite green indicator.
Loop-mediated isothermal amplification (LAMP) has arisen as an outstanding molecular technique for the amplification of nucleic acids, becoming an important alternative to PCR due to its simplicity and high sensitivity. However, traditional visual readout used in LAMP only provides qualitative results and can thus not provide a precise quantification of the target DNA/RNA in the sample. To overcome this, electroactive reporters incorporated in the reaction mix can provide a subsequent quantitative result of the analysis. In this work, malachite green, a commonly used colorimetric LAMP indicator, is used as an electrochemical probe to obtain quantitative results. For this purpose, its optical and electrochemical behavior has been assessed for further use in LAMP detection. Using SARS-CoV-2 RNA as template, we have developed a quantitative detection method, proving the usefulness of this molecule as a dual optical/electrochemical indicator. Thereby, with the electrochemical detection using screen-printed electrodes on which a drop of the LAMP reaction is deposited, a calibration curve with a linear dynamic range comprised between 22 and 4296 copies/μL was achieved, with a practical limit of detection of 22 copies/µL. In this method, validated with clinical samples, malachite green is used, by the first time, as a dual visual electrochemical/visual probe that can provide both an initial visual qualitative assessment together with an electrochemical quantification.
