Annual report

Informe annual de l'Institut de Biotecnologia i Biomedicina "Vicent Villar Palasí

Escherichia coli stress response systems and their reaction to terahertz radiation

In this review, we summarize the latest data concerning the reactions of Escherichia coli to nonthermal terahertz radiation and the underlying molecular mechanisms. E. coli is the most simple and convenient model object for studying the effects of terahertz radiation: both its genetics and metabolism are well studied, and it is easily amenable to genetic engineering allowing one to create biosensors using promoters of genes activated by certain stress factors and the reporter GFP protein. Transformed E. coli cells containing biosensors can be used to visualize their reactions to terahertz radiation based on the intensity of GFP fluorescence. In this review, we present data on the response of certain E. сoli stress response systems to terahertz radiation obtained by us, as well as by other authors. We discuss experimental results for E. сoli/ pKatG-GFP, E. сoli/pCopA-GFP, and E. сoli/ pEmrR-GFP biosensors that are used to detect E. сoli genetic networks responding to oxidative stress, copper ion homeostasis failures, and antiseptics, respectively. The obtained data indicate that exposure to nonthermal terahertz radiation induces E. сoli gene networks of oxidative stress and copper ion homeostasis, but does not activate those responding to antibiotics, protonophores, or superoxide anions. The fact that E. сoli/pKatG-GFP and E. сoli/pCopA-GFP biosensors have different activation and reaction periods when exposed to terahertz radiation and natural inducers suggests that reactions of oxidative stress and copper ion homeostasis systems to terahertz radiation are specific

Molecular and Biotechnological Approaches in the Diagnosis of Leprosy

Leprosy is a worldwide health problem, which needs the development of new and innovative strategies to be controlled. Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease; thus, the development of biotechnology platforms, which include the mapping of antigens with potential to be used in immunodiagnostic and molecular methods for the detection of Mycobacterium leprae, is an important tool to confirm the clinical diagnostic. Molecular biology and biotechnological methods have been used to assist in the diagnosis of this disease, each one with its advantages and drawbacks. Enzyme-linked immunosorbent assay (ELISA) is the used method for leprosy diagnosis, and it allows the detection of infection-related antigens. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. On the other hand, lateral flow assay (LFA) and optical and electrochemical biosensors are rapid and portable methods, capable of performing diagnosis in the field without sample preparation. This chapter presents such techniques, their uses in the diagnosis and detection of M. leprae, as well as the potential for the development of new techniques and strategies that can help to control and understand mycobacteriosis

Advanced Electrochemical and Opto-Electrochemical Biosensors for Quantitative Analysis of Disease Markers and Viruses

The recent global events of the SARS-CoV-2 pandemic in 2020 have alerted the world to the urgent need to develop fast, sensitive, simple, and inexpensive analytical tools that are capable of carrying out a large number of quantitative analyses, not only in centralized laboratories and core facilities but also on site and for point-of-care applications. In particular, in the case of immunological tests, the required sensitivity and specificity is often lacking when carrying out large-scale screening using decentralized methods, while a centralized laboratory with qualified personnel is required for providing quantitative and reliable responses. The advantages typical of electrochemical and optical biosensors (low cost and easy transduction) can nowadays be complemented in terms of improved sensitivity by combining electrochemistry (EC) with optical techniques such as electrochemiluminescence (ECL), EC/surface-enhanced Raman spectroscopy (SERS), and EC/surface plasmon resonance (SPR). This Special Issue addresses existing knowledge gaps and aids in exploring new approaches, solutions, and applications for opto-electrochemical biosensors in the quantitative detection of disease markers, such as cancer biomarkers proteins and allergens, and pathogenic agents such as viruses. Included are seven peer-reviewed papers that cover a range of subjects and applications related to the strategies developed for early diagnosis

A Low-Cost Genomic Sensor for Ocean-Observing Systems and Infectious Disease Detection

abstract: Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and power hungry. In this dissertation project, an inexpensive, portable, low-energy consuming, and highly quantitative microbiological genomic sensor has been developed for in situ ocean-observing systems. A novel real-time colorimetric loop-mediated isothermal amplification (LAMP) technology has been developed for quantitative detection of microbial nucleic acids. This technology was implemented on a chip-level device with an embedded inexpensive imaging device and temperature controller to achieve quantitative detection within one hour. A bubble-free liquid handling approach was introduced to avoid bubble trapping during liquid loading, a common problem in microfluidic devices. An algorithm was developed to reject the effect of bubbles generated during the reaction process, to enable more accurate nucleic acid analysis. This genomic sensor has been validated at gene and gene expression levels using Synechocystis sp. PCC 6803 genomic DNA and total RNA. Results suggest that the detection limits reached 10 copies/μL and 100 fg/μL, respectively. This approach was highly quantitative, with linear standard curves down to 103 copies/μL and 1 pg/μL, respectively. In addition to environmental microbe characterization, this genomic sensor has been employed for viral RNA quantification during an infectious disease outbreak. As the Zika fever was spreading in America, a quantitative detection of Zika virus has been performed. The results show that the genomic sensor is highly quantitative from 10 copies/μL to 105 copies/μL. This suggests that the novel nucleic acid quantification technology is sensitive, quantitative, and robust. It is a promising candidate for rapid microbe detection and quantification in routine laboratories. In the future, this genomic sensor will be implemented in in situ platforms as a core analytical module with minor modifications, and could be easily accessible by oceanographers. Deployment of this microbial genomic sensor in the field will enable new scientific advances in oceanography and provide a possible solution for infectious disease detection.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Coronaviruses Research in BRICS Countries

SARS-CoV-2 has infected more than 105 million people worldwide. During this pandemic, researchers and clinicians have been working to understand the molecular mechanisms that underpin viral pathogenesis by studying viral–host interactions. Now, with the global rollout of various COVID-19 vaccines—based on the neutralization of the spike protein using different technologies—viral immunology and cell-based immunity are being investigated. Researchers are also studying how various SARS-CoV-2 genetic mutations will impact the efficacy of these COVID-19 vaccines. At the same time, various antiviral drugs have been identified or repurposed that have potential as anti-SARS-CoV-2 treatments. BRICS (Brazil, Russia, India, China, and South Africa) is the acronym used to associate five major emerging national economies. The BRICS countries are known for their significant influence on regional affairs, including being leaders in scientific and clinical research and innovation. This Special Issue includes researchers from BRICS countries, in particular South Africa, involved in the study of SARS-CoV-2 and COVID-19. Original articles, as well as new perspectives or reviews on the matter, were welcomed. Research in the fields of vaccine studies, pathogenesis, genetic mutations, viral immunology, and antiviral drugs were especially encouraged

<i>E. coli</i> as an Indicator of Contamination and Health Risk in Environmental Waters

Good public health depends on regular monitoring of water quality as faecal contamination is a serious problem due to the potential for contracting disease. Bacterial contamination in water is measured using indicator organisms, notably Escherichia coli and Enterococci which are used as primary indicators of contamination in fresh and marine water quality, respectively, rather than the total coliforms present. Although most E. coli and Enterococci strains cause only mild infections, their presence is indicative of the potential presence of other more pathogenic organisms which are a danger to human health. The acceptable levels of indicator organisms are defined in legislation and are set for drinking, river, well and marine water. This chapter will consider current gold standard culture methods of analysis for E. coli and compare them with molecular DNA procedures. Established culture methods use β‐D-glucuronidase to identify E. coli and β‐D-galactosidase to detect coliforms. Emphasis will be placed on newer procedures that can be used onsite supported by laboratory procedures used for confirmation. Available rapid fluorimetric procedures which have been developed for use in the field, based on the assay of β‐D-glucuronidase, will be discussed. The rapid advances in procedures using a molecular approach will be considered and compared with the more established methods for determining E. coli in water. It is essential that all these methods should be quantitative in order to comply with legal norms, and in this regard, the potential involvement of biosensor technology will be of great value in successfully transferring laboratory procedures to the field

Integrated Electrochemical Biosensors for Detection of Waterborne Pathogens in Low-Resource Settings

More than 783 million people worldwide are currently without access to clean and safe water. Approximately 1 in 5 cases of mortality due to waterborne diseases involve children, and over 1.5 million cases of waterborne disease occur every year. In the developing world, this makes waterborne diseases the second highest cause of mortality. Such cases of waterborne disease are thought to be caused by poor sanitation, water infrastructure, public knowledge, and lack of suitable water monitoring systems. Conventional laboratory-based techniques are inadequate for effective on-site water quality monitoring purposes. This is due to their need for excessive equipment, operational complexity, lack of affordability, and long sample collection to data analysis times. In this review, we discuss the conventional techniques used in modern-day water quality testing. We discuss the future challenges of water quality testing in the developing world and how conventional techniques fall short of these challenges. Finally, we discuss the development of electrochemical biosensors and current research on the integration of these devices with microfluidic components to develop truly integrated, portable, simple to use and cost-effective devices for use by local environmental agencies, NGOs, and local communities in low-resource settings

The emerging significance of nanomedicine-based approaches to fighting COVID-19 variants of concern: A perspective on the nanotechnology’s role in COVID-19 diagnosis and treatment

COVID-19, one of the worst-hit pandemics, has quickly spread like fire across nations with very high mortality rates. Researchers all around the globe are making consistent efforts to address the main challenges faced due to COVID-19 infection including prompt diagnosis and therapeutics to reduce mortality. Conventional medical technology does not effectively contain the havoc caused by deadly COVID-19. This signals a crucial mandate for innovative and novel interventions in diagnostics and therapeutics to combat this ongoing pandemic and counter its successor or disease if it were ever to arise. The expeditious solutions can spring from promising areas such as nanomedicine and nanotechnology. Nanomedicine is a dominant tool that has a huge potential to alleviate the disease burden by providing nanoparticle-based vaccines and carriers. Nanotechnology encompasses multidisciplinary aspects including artificial intelligence, chemistry, biology, material science, physical science, and medicine. Nanoparticles offer many advantages compared to larger particles, including better magnetic properties and a multiplied surface-to-volume ratio. Given this, the present review focuses on promising nanomedicine-based solutions to combat COVID-19 and their utility to control a broad range of pathogens and viruses, along with understanding their role in the therapy, diagnosis, and prevention of COVID-19. Various studies, reports, and recent research and development from the nanotechnology perspective are discussed in this article

Development of electrochemical platforms for DNA sensing

[eng] The present doctoral thesis is framed in the research and development (R & D) project between a private biotechnology company of molecular diagnostics Genomica SAU, the Institute for Bioengineering of Catalonia (IBEC), the University of Barcelona, and the Microfluidics ChipShop Company. The main objective of the project is making, implementation and marketing of a diagnostic device for early detection of DNA sequences involved with cancer. The multi device, or lab-on-chip (LOC), consists of a central automation unit (CAU), a system in miniature of DNA amplification or chain reaction polymerase (mini-PCR), and a biosensing platform (DNA chip) that consisting of a matrix or electrochemical array. The three elements are integrated by a microfluidic system in sandwich format cartridge. For this purpose, the aim of this thesis was the creation, characterization and optimization of the biochemical recognition platform between two single strands of DNA of dissimilar lengths but with some complementary sequences for the subsequent electrochemical detection of a hybridization event between them. Then, the integration into the cartridge of above platform was done. For the creation of this platform, we chose to use a self-assembled monolayer (SAM) of thiols as biorecognition interface of the 14 DNA sequences that are part of the project. During optimization of the interface chips individual gold and various molecules were used being chosen the molecule with two arms disulfide of polyethylene glycol (PEG) and a malaimida group at the end of one of them. This linker (or MalPEG linker) reacts with the gold surface due to the dative interaction between the sulfur atoms of the disulfide and the gold atoms from the surface of the chips. At the same time, the malaimida group reacts with the thiol group of the capture probes, joining. The PEG groups function as anti-adhesion molecules. Surface plasmon resonance (SPR) and cyclic voltammetry (CV) were techniques used to characterize the substrate and the hybridization event. For the manufacture of the cartridge, this was divided into two main blocks, the biosensing or electrochemical block and PCR block. The electrochemical block is composed of 4 layers, one of 64 working electrodes and gold paths for contact with the potentiostat, another layer that defines the area of the sensors must be functionalized gold and isolating the gold surface of the tracks. The third layer is a double-sided adhesive that has a hexagonal hole working as hybridization chamber, and the last layer is a screen printing layer with the reference electrode (RE) and counter electrodes. The above layers form an electrochemical cell wherein the hybridization will occurs. Regarding the PCR block, this is a system of two layers with a type microfluidic channel kind loop and its function is to contain the solutions during the process of DNA amplification by the mini-PCR. During the integration of the optimized SAM into an electrochemical cartridge a manual and automated ways were used to immobilize the capture probes. Several tests were performed in order to obtain the best conditions and ratios between the molecules to maximize the hybridization signal during the electrochemical detection.[spa] El presente trabajo de tesis está enmarcado en un proyecto de investigación y desarrollo (I+D) entre la empresa privada Genomica S.A.U., el Instituto de Bioingeniería de Cataluña (IBEC), la Universidad de Barcelona y la empresa alemana ChipShop Microfluidics. El objetivo principal es el desarrollo, puesta a punto y comercialización de un dispositivo electroquímico de diagnóstico médico para etapas tempranas de cáncer. El objetivo de la tesis es la creación, optimización y posterior integración de una interfaz de biosensado de ADN en el dispositivo de diagnóstico, siendo pieza fundamental en el desarrollo de éste. La interfaz escogida fue una monocapa autoensamblada (SAM) que hace las veces de biosensor y que es capaz de anclar secuencias de ADN como sondas de captura y así poder detectar, selectivamente, las secuencias objetivo complementarias. El dispositivo también cuenta con un sistema microfluídico y un sistema de amplificación de ADN de reacción en cadena de la polimerasa en miniatura. La SAM esta inmovilizada en un array electroquímico que consta de 64 electrodos de trabajo que funcionan como elemento transductor de la señal electroquímica redox de los eventos de hibridación que ocurren sobre ellos. La funcionalización y puesta a punto del dispositivo se llevó a cabo inmovilizando múltiples sondas de captura después de una optimización de las concentraciones entre las diferentes partes constituyentes de la monocapa. Técnicas ópticas y electroquímicas fueron utilizadas para la caracterización de cada etapa y técnicas de fotolitografiado y de impresión por pantalla fueron utilizadas para la fabricación de los componentes del dispositivo. Finalmente, y después de algunos cambios surgidos durante el desarrollo del dispositivo, se llega a un diseño final y a las pruebas con muestras reales, proceso que aún está en etapa experimental