RESUMEN
In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: â¼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.
RESUMEN
Clinical assessment based on a single biomarker is in many circumstances not sufficient for adequate diagnosis of a disease or for monitoring its therapy. Multiplexing, the measurement of multiple analytes from one sample and/or of the same target from different samples simultaneously, could enhance the accuracy of the diagnosis of diseases and their therapy success. Thus, there is a great and urgent demand for multiplexed biosensors allowing a low-cost, easy-to-use, and rapid on-site testing. In this work, we present a simple, flexible, and highly scalable strategy for implementing microfluidic multiplexed electrochemical biosensors (BiosensorX). Our technology is able to detect 4, 6, or 8 (different) analytes or samples simultaneously using a sequential design concept: multiple immobilization areas, where the assay components are adsorbed, followed by their individual electrochemical cells, where the amperometric signal readout takes place, within a single microfluidic channel. Here, first we compare vertical and horizontal designs of BiosensorX chips using a model assay. Owing to its easier handling and superior fluidic behavior, the vertical format is chosen as the final multiplexed chip design. Consequently, the feasibility of the BiosensorX for multiplexed on-site testing is successfully demonstrated by measuring meropenem antibiotics via an antibody-free ß-lactam assay. The multiplexed biosensor platform introduced can be further extended for the simultaneous detection of other anti-infective agents and/or biomarkers (such as renal or inflammation biomarkers) as well as different (invasive and non-invasive) sample types, which would be a major step towards sepsis management and beyond.
Asunto(s)
Técnicas Biosensibles , Microfluídica , Biomarcadores , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Synthetic biology applies concepts from electrical engineering and information processing to endow cells with computational functionality. Transferring the underlying molecular components into materials and wiring them according to topologies inspired by electronic circuit boards has yielded materials systems that perform selected computational operations. However, the limited functionality of available building blocks is restricting the implementation of advanced information-processing circuits into materials. Here, a set of protease-based biohybrid modules the bioactivity of which can either be induced or inhibited is engineered. Guided by a quantitative mathematical model and following a design-build-test-learn (DBTL) cycle, the modules are wired according to circuit topologies inspired by electronic signal decoders, a fundamental motif in information processing. A 2-input/4-output binary decoder for the detection of two small molecules in a material framework that can perform regulated outputs in form of distinct protease activities is designed. The here demonstrated smart material system is strongly modular and can be used for biomolecular information processing for example in advanced biosensing or drug delivery applications.
Asunto(s)
Modelos Teóricos , Biología Sintética , Sistemas de Liberación de Medicamentos , Péptido HidrolasasRESUMEN
Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy.
Asunto(s)
Antibacterianos , Técnicas Biosensibles , Animales , Monitoreo de Drogas , Pruebas en el Punto de AtenciónRESUMEN
Synthetic biology is strongly inspired by concepts of engineering science and aims at the design and generation of artificial biological systems in different fields of research such as diagnostics, analytics, biomedicine, or chemistry. To this aim, synthetic biology uses an engineering approach relying on a toolbox of molecular sensors and switches that endows cellular hosts with non-natural computing functions and circuits. Importantly, this concept is not only limited to cellular approaches. Synthetic biological building blocks have also conferred sensing and switching capability to otherwise inactive materials. This principle has attracted high interest for the development of biohybrid materials capable of sensing and responding to specific molecular stimuli, such as disease biomarkers, antibiotics, or heavy metals. Moreover, the interconnection of individual sense-and-respond materials to complex materials systems has enabled the processing of, for example, multiple inputs or the amplification of signals using feedback topologies. Such systems holding high potential for applications in the analytical and diagnostic sectors will be described in this chapter.