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.

Highly sensitive and fast Legionella spp. in situ detection based on a loop mediated isothermal amplification technique combined to an electrochemical transduction system.

A rapid highly sensitive genosensor has been developed for monitoring the presence of Legionella spp. in different water systems (domestic hot water, heating/cooling systems or cooling towers) in order to avoid its spreading from the source of contamination. The genosensor integrates a loop mediated isothermal amplification (LAMP) reaction with an electrochemical transduction signal, producing a very simple, rapid to perform and cost effective method, suitable for in situ analyses. This approach detects as low as 10 fg of Legionella nucleic acid, corresponding to only 2 number copies of the bacteria. The use of an electrochemical redox-active double stranded DNA (dsDNA) intercalating molecule, known as methylen blue (MB), allows the immediate electrochemical reading during the DNA polymerization. The sensor can obtain quantitative results in 20 min with a correlation between the electrochemical data and Legionella spp. copy number (at a logarithmic scale) of r = -0.97. In conclusion, a fast, easy to use, and accurate electrochemical genosensor, with high precision, sensitivity, and specificity has been developed for in situ detection of Legionella spp. enabling real time decision making and improving significantly the current detection methods for the prevention and screening of Legionella.

A novel Real Time micro PCR based Point-of-Care device for Salmonella detection in human clinical samples.

Our POC (Point of Care) device is intended to be a diagnostic tool for routine use in the clinical sector. The validation of the whole procedure, including bacterial genomic DNA isolation and the Real Time detection of Salmonella spp., was conducted on 29 clinical stool samples that had been diagnosed with Salmonella spp. by a routine culture technique. The entire process was achieved in a single microfluidic chip within 35 min. In comparison to the culture reference method that is used in the clinical laboratories, this new device performed well in regards to the analytical parameters of sensitivity, specificity and accuracy. Therefore, the POC device reported in this study proved to be very appropriate for the fully integrated analysis system. To the best of our knowledge, this is the first work to report the sample preparation and followed by Real Time PCR (Polymerase Chain Reaction) on a single 2.5 µl chamber chip for the detection of Salmonella spp. bacteria in stool samples.

The SmartBioPhone, a point of care vision under development through two European projects: OPTOLABCARD and LABONFOIL.

This paper describes how sixteen partners from eight different countries across Europe are working together in two EU projects focused on the development of a point of care system. This system uses disposable Lab on a Chips (LOCs) that carry out the complete assay from sample preparation to result interpretation of raw samples. The LOC is either embedded in a flexible motherboard with the form of a smartcard (Labcard) or in a Skinpatch. The first project, OPTOLABCARD, extended and tested the use of a thick photoresit (SU-8) as a structural material to manufacture LOCs by lamination. This project produced several examples where SU-8 microfluidic circuitry revealed itself as a viable material for several applications, such as the integration on chip of a Polymerase Chain Reaction (PCR) that includes sample concentration, PCR amplification and optical detection of Salmonella spp. using clinical samples. The ongoing project, LABONFOIL, is using two results of OPTOLABCARD: the sample concentration method and the capability to fabricate flexible and ultra thin LOCs based on sheets instead of wafers. This rupture from the limited and expensive wafer surface heritage allows the development of a platform where LOCs are big enough to include all the sample preparation subcomponents at a low price. These LOCs will be used in four point of care applications: environment, food, cancer and drug monitoring. The user will obtain the results of the tests by connecting the Labcard/Skinpatch reader to a very popular interface (a smartphone), creating a new instrument namely "The SmartBioPhone". All standard smartphone capabilities will be at the disposal of the point of care instrument by a simple click. In order to guarantee the future mass production of these LOCs, the project will develop a large dry film equipment where LOCs will be fabricated at a low cost.