Development of an Electrochemical Sensor for SARS-CoV-2 Detection Based on Loop-Mediated Isothermal Amplification.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused more than 6 million deaths all over the world, demonstrating the need for a simple, fast and cost-effective point-of-care (POC) test for the detection of the virus. In this work, we developed an electrochemical sensor for SARS-CoV-2 virus detection on clinical samples based on loop-mediated isothermal amplification (LAMP). With the development of this novel sensor, the time of each measurement is significantly reduced by avoiding the DNA extraction step and replacing it with inactivation of the sample by heating it at 95 °C for 10 min. To make the reaction compatible with the sample pre-treatment, an RNase inhibitor was added directly to the premix. The LAMP product was measured in a novel, easy-to-use manufactured sensor containing a custom-made screen-printed carbon electrode. Electrochemical detection was performed with a portable potentiostat, and methylene blue was used as the redox-transducing molecule. The developed sensor achieved a limit of detection of 62 viral copies and was 100% specific for the detection of the SARS-CoV-2 virus. The performance of the electrochemical sensor was validated with nasopharyngeal samples, obtaining a sensibility and specificity of 100% compared to the gold standard RT-PCR method.

Potential use of heat shock protein 90 as a biomarker for the diagnosis of human diseases.

INTRODUCTION: The heat shock protein 90 (Hsp90) is a protein involved in many different biological processes and especially in cell survival. Some of these functions require the participation of other biological molecules, so Hsp90 is a chaperone that takes part in many protein-protein interactions working as a critical signaling hub protein. As a member of the heat shock protein family, Hsp90 expression is regulated under certain environmental and/or stressful situations, therefore Hsp90 concentration can be monitored and linked to these effects. AREAS COVERED: This review discusses the Hsp90 expression in samples from individuals affected by different diseases (from infectious to cancer origin), and the biological consequences of these disorders, including the potential use of Hsp90 as a biomarker for the diagnosis of human diseases. EXPERT OPINION: The potential of Hsp90 as a biomarker disease has been demonstrated in several studies in relation to infectious diseases and especially cancer. However, further research in this field is still needed, mainly to validate in statistically significant clinical studies that the detection of Hsp90 protein allows the diagnosis of some cancers at an early stage and also that it can act as a biomarker for monitoring the efficacy of their therapies.

Extraction-Free Colorimetric RT-LAMP Detection of SARS-CoV-2 in Saliva.

The pandemic situation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need of fast, simple, and cost-effective tests for the diagnosis of emerging pathogens. RT-qPCR has been established as the reference technique for the diagnosis of SARS-CoV-2 infections. This method requires a time-consuming protocol for the extraction of the nucleic acids present in the sample. A colorimetric reverse transcription loop-mediated isothermal amplification using the calcein molecule combined with a simple extraction-free method for saliva samples (calcein RT-LAMP) has been developed. Samples are heated 95 °C for 10 min before amplification at 63 °C for 40 min. The results can be observed by fluorescence or by the naked eye with a color change from orange to green. The method was compared with commercialized available colorimetric and fluorescent RT-LAMP kits. The developed method shows better sensitivity and specificity than the colorimetric commercial RT-LAMP and the same as the fluorescent RT-LAMP, without the need of a fluorescent reader. Moreover, the calcein RT-LAMP has, compared to RT-qPCR, a sensitivity of 90% and a specificity of 100% for saliva samples with a Ct ≤ 34, without the need for expensive RT-qPCR instruments, demonstrating the potential of this method for population screening.

Design and 3D printing of an electrochemical sensor for Listeria monocytogenes detection based on loop mediated isothermal amplification.

The aim of this work is the design and 3D printing of a new electrochemical sensor for the detection of Listeria monocytogenes based on loop mediated isothermal amplification (LAMP). The food related diseases involve a serious health issue all over the world. Listeria monocytogenes is one of the major problems of contaminated food, this pathogen causes a disease called listeriosis with a high rate of hospitalization and mortality. Having a fast, sensitive and specific detection method for food quality control is a must in the food industry to avoid the presence of this pathogen in the food chain (raw materials, facilities and products). A point-of-care biosensor based in LAMP and electrochemical detection is one of the best options to detect the bacteria on site and in a very short period of time. With the numerical analysis of different geometries and flow rates during sample injection in order to avoid bubbles, an optimized design of the microfluidic biosensor chamber was selected for 3D-printing and experimental analysis. For the electrochemical detection, a novel custom gold concentric-3-electrode consisting in a working electrode, reference electrode and a counter electrode was designed and placed in the bottom of the chamber. The LAMP reaction was optimized specifically for a primers set with a limit of detection of 1.25 pg of genomic DNA per reaction and 100% specific for detecting all 12 Listeria monocytogenes serotypes and no other Listeria species or food-related bacteria. The methylene blue redox-active molecule was tested as the electrochemical transducer and shown to be compatible with the LAMP reaction and very clearly distinguished negative from positive food samples when the reaction is measured at the end-point inside the biosensor.