A low-cost biological agglutination assay for medical diagnostic applications

Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted

Amélioration de l'évidence et des mécanismes de pro-action et de rétroaction dans le développement de nouvelles technologies de diagnostic en bio médecine

La compétition retrouvée entre les différentes compagnies oeuvrant dans le domaine pharmaceutique est très forte. En effet, dans le domaine du biodiagnostic, on retrouve toute une gamme de technologies plus ou moins complémentaires en voie de développement et en cours d'utilisation. Dans l'étude actuelle, il a été entrepris d'optimiser le processus de R&D et d'utilisation effectués sur un appareil de biodiagnostic, l'ADVIA 120 de Bayer. L'approche préconisée pour réaliser cette optimisation a été l'utilisation des technologies de communication. Ainsi, au cours de ce projet, une méthodologie de communication a été développée afin de supporter les différents échanges d'informations effectués par les participants dans un processus de transfert des technologies. Le développement de cette méthode de communication a été élaboré en se basant sur des notions théoriques issues d'un cadre d'évaluation intégré. Ce cadre conceptuel a aussi été développé au cours de ce projet. L'objectif d'un tel cadre est de décrire le processus de transfert des technologies par une approche longitudinale et transversale et d'ainsi obtenir un modèle flexible qui est adapté au contexte des technologies de biodiagnostic."--Résumé abrégé par UM

Biosensors and Nanobiosensors: Design and Applications

The goal of this chapter is to cover the full scope of biosensors. It offers a survey of the principles, design, operation, and biomedical applications of the most popular types of biosensing devices in use today. By discussing recent research and future trends based on many excellent books and reviews, it is hoped to give the readers a comprehensive view on this fast growing field

Nanotechnology in Sepsis: Diagnosis and Treatment, Review

AbstractSepsis is one of the main reasons of deaths internationally, with excessive mortality rates and a pathological complexity hindering early and correct diagnosis. Today, laboratory culture checks are the epitome of pathogen recognition in sepsis. However, their consistency stays a problem of controversy with false negative results frequently observed. Clinically used blood markers, C reactive protein (CRP) and procalcitonin (PCT) are indications of an acute-phase response and as a result lack specificity, supplying restrained diagnostic efficacy. In addition to bad diagnosis, inefficient drug delivery and the increasing prevalence of antibiotic-resistant microorganisms represent significant obstacles in antibiotic stewardship and hinder high quality therapy. These challenges have brought on the exploration for choice techniques that pursue accurate prognosis and high-quality treatment. Nanomaterials are examined for each diagnostic and therapeutic functions in sepsis. The nanoparticle (NP)-enabled seize of sepsis causative agents and/or sepsis biomarkers in biofluids can revolutionize sepsis diagnosis. From the therapeutic factor of view, presently current nanoscale drug transport structures have established to be extraordinary allies in focused therapy, whilst many different nanotherapeutic functions are envisioned. Herein, the most applicable purposes of nanomedicine for the diagnosis, prognosis, and treatment of sepsis is reviewed, imparting a quintessential evaluation of their potentiality for scientific translation

Designing Paper-Based Immunoassays for Biomedical Applications

Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture

Plasmonic Nanostars for Sensitive SERS-based Immunodetection

Malaria remains a global health problem and detection is essential to combat this disease. Rapid diagnostic tests in a lateral flow assay (LFA) format using Plasmodium falciparum histidine-rich pro- tein II (PfHRPII) as biomarker is the most common malaria detection method. These LFAs are usually low-cost and rapid but suffer from low sensitivity. Surface-enhanced Raman scattering (SERS) is a technique with high sensitivity and combined with LFA, is possible to detect PfHRPII through colori- metric and SERS assays, increasing the limits of detection. The recombinant antigen PfHRPII was expressed using a vector containing a His-tag in Escherichia coli and purified by a Ni-NTA column. The SDS-PAGE and western blot confirmed the presence of PfHRPII with a molecular weight of 67 kDa (at 0.12 ± 0.03 mg∙mL-1 ). The SERS tags were developed with star-shaped gold nanoparticles functionalised with a Raman reporter, 4-mercaptobenzoic acid, and covalently conjugated with an antibody. A proof-of-concept was made with Peroxidase/anti-Peroxidase complex with bovine serum albumin to block unspecific interactions and the SERS tags formation and activity were fully charac- terised through UV-Vis spectra, agarose gel electrophoresis, enzymatic activity assay, and dynamic light scattering. Each component of LFA was evaluated by the pixel intensity difference between the background and test line by ImageJ. The LFA optimisations led to the selection of a nitrocellulose membrane (CNPF8) not blocked, a sample pad (GFB-R7L), and an absorbent pad (AP-045). Anti- Peroxidase (0.9 mg∙mL-1 ) and anti-IgG (0.5 mg∙mL-1 ) were immobilised on the test and control lines, respectively. The sample was mixed into the SERS tags at 0.2 nM and deposited in the sample pad. The LOD and LOQ were determined as 5.24 and 7.28 μg∙mL-1 , respectively. Lastly, an LFA was in- cubated with recombinant PfHRPII at 50 ng∙mL-1 and the SERS performance was compared with a negative control (no PfHRPII). The samples had a small but significant difference nevertheless further studies are needed to reduce the non-specific interactions from the control sample.A malária continua a ser um problema de saúde global e a sua deteção é essencial para combater a doença. O método de deteção de malária mais comum é o teste de diagnóstico rápido em formato de teste de fluxo lateral (conhecido pelo acrónimo de LFA) usando como biomarcador a proteína rica em histidina II de Plasmodium falciparum (PfHRPII). Estes LFAs são de baixo custo e rápidos, oferecem pouca sensibilidade. Espectroscopia de Raman aumentada pela superfície (conhecida pelo acrónimo de SERS) é uma técnica com grande sensibilidade e quando combinada com LFA, pode detetar PfHRPII por análise colorimétrica e por SERS, aumentando os limites de deteção. O antigénio recom- binante PfHRPII foi expresso em Escherichia coli usando um vetor e purificado por uma coluna de níquel (Ni-NTA) através da cauda de histidinas. As técnicas SDS-PAGE e western blot confirmaram a presença de PfHRPII com um peso molecular de 67 kDa (a 0,12 ± 0,03 mg∙mL-1 ). As sondas de SERS foram produzidas com nanopartículas de ouro em formato de estrela, funcionalizadas com um repórter de Raman, o ácido 4-mercaptobenzóico, e conjugado covalentemente com um anticorpo. Através do complexo da enzima peroxidase/anti-peroxidase, e com albumina de soro bovino para bloquear intera- ções inespecíficas, foi feito uma prova de conceito e a formação e a atividade das sondas de SERS foram caracterizadas através de espectro de UV-Vis, eletroforese em gel de agarose, ensaio de ativida- de enzimática e dispersão dinâmica de luz. Todos os componentes de LFA foram avaliados pela dife- rença de intensidade de pixel entre o fundo e a linha de teste por ImageJ. As otimizações de LFA per- mitiram a seleção da membrana de nitrocelulose (CNPF8) não bloqueada, do bloco de amostra (GFB- R7L), e do bloco absorvente (AP-045). O anticorpo anti-peroxidase (0,9 mg∙mL-1 ) e o anti-IgG (0,5 mg∙mL-1 ) foram imobilizados nas linhas de teste e controlo, respetivamente. A amostra foi misturada nas sondas de SERS a 0,2 nM e depositadas no bloco de amostra. O LOD e o LOQ foram determina- dos como 5,24 e 7,28 μg∙mL-1 , respetivamente. Por último, um LFA foi incubado com PfHRPII re- combinante a 50 ng∙mL-1 e o desempenho de SERS foi comparado com o controlo negativo (sem PfHRPII). As amostras tiveram uma pequena, mas significativa diferença, no entanto, são necessários mais estudos para reduzir as interações não específicas na amostra de controlo

Micro/nanofluidic and lab-on-a-chip devices for biomedical applications

Micro/Nanofluidic and lab-on-a-chip devices have been increasingly used in biomedical research [1]. Because of their adaptability, feasibility, and cost-efficiency, these devices can revolutionize the future of preclinical technologies. Furthermore, they allow insights into the performance and toxic effects of responsive drug delivery nanocarriers to be obtained, which consequently allow the shortcomings of two/three-dimensional static cultures and animal testing to be overcome and help to reduce drug development costs and time [2–4]. With the constant advancements in biomedical technology, the development of enhanced microfluidic devices has accelerated, and numerous models have been reported. Given the multidisciplinary of this Special Issue (SI), papers on different subjects were published making a total of 14 contributions, 10 original research papers, and 4 review papers. The review paper of Ko et al. [1] provides a comprehensive overview of the significant advancements in engineered organ-on-a-chip research in a general way while in the review presented by Kanabekova and colleagues [2], a thorough analysis of microphysiological platforms used for modeling liver diseases can be found. To get a summary of the numerical models of microfluidic organ-on-a-chip devices developed in recent years, the review presented by Carvalho et al. [5] can be read. On the other hand, Maia et al. [6] report a systematic review of the diagnosis methods developed for COVID-19, providing an overview of the advancements made since the start of the pandemic. In the following, a brief summary of the research papers published in this SI will be presented, with organs-on-a-chip, microfluidic devices for detection, and device optimization having been identified as the main topics.info:eu-repo/semantics/publishedVersio

NanoSERS Microfluidics platform for rapid screening for infectious diseases

Early and accurate disease detection is critical for clinical diagnosis and ultimately determining patient outcomes. Point-of-care testing (POCT) platforms are needed in low- resource settings and also to help the decentralisation of healthcare centres. Immunoas- says using Surface-Enhanced Raman Spectroscopy (SERS) are especially interesting for their increased sensitivity and specificity. Additionally, SERS can be easily translated into POCT formats with microfluidics. In this work, a sensitive, selective, capable of multiplexing, and reusable SERS-based biosensor was developed. The SERS immunoas- say relies on a sandwich format, whereby a capture platform and SERS immunotags can capture and detect a specific antigen, respectively. The SERS immunotags consisted of gold nanostars, allowing exceptionally intense SERS signals from attached Raman re- porters, and the covalent attachment of antibodies provided a stable antigen-antibody binding activity. As a capture platform, a regenerated cellulose-based hydrogel provided a robust design and the added advantage of environmental friendliness. Besides being a transparent material with low background fluorescence and Raman signal, its high-water retention capacity was particularly suited for preserving the high activity of covalently bound antibodies, improving the assay time-stability. This SERS-based immunoassay was then integrated into a microfluidic device, allowing high-throughput sample screening allied with the high sensitivity and multiplexing features of the developed assay. The de- vice was fabricated in less than 30 minutes by exploring direct patterning on shrinkable polystyrene sheets for the construction of adaptable complex three-dimensional microflu- idic chips. Finally, to validate the microfluidic system, Plasmodium falciparum infected red blood cell culture samples were tested for malaria biomarker detection. The discrimi- nation of SERS immunotags signals from the background was made through the direct classical least squares method. As a result, better data fitting was achieved, compared to the commonly used peak integral method. Considering these features, the proposed SERS-based immunoassay notably improved the detection limits of traditional enzyme- linked immunosorbent assay approaches. Its performance was better or comparable to existing SERS-based immunosensors. Moreover, this approach successfully overcame the main challenges for application at POCT, including increasing reproducibility, sensitivity, and specificity. Hence, the microfluidic SERS system represents a powerful technology which can contribute to early diagnosis of infectious diseases, a decisive step towards lowering their still substantial burden on health systems worldwide.A detecção precoce e precisa de doenças é fundamental para o diagnóstico clínico de- terminando frequentemente o prognóstico do paciente. Desta forma, plataformas de teste de rastreio (conhecidos pelo acrónimo de POCT) são extremamente necessárias, não só em locais com poucos recursos, mas também para ajudar à descentralização dos cuidados de saúde. Os ensaios imunológicos que utilizam a espectroscopia de Raman aumentada pela superfície (conhecida pelo acrónimo de SERS) são particularmente interessantes pela sua elevada sensibilidade. Além disso, os ensaios em SERS podem ser facilmente convertidos para formatos POCT quando combinados com microfluídica. Este trabalho consistiu no desenvolvimento de um biosensor sensível, selectivo, capaz de múltipla detecção e reuti- lizável baseado no fenómeno de SERS. O ensaio imunológico em SERS foi realizado num formato em sanduíche onde um antigénio específico é apreendido por uma plataforma de captura e reconhecido por imunosondas activas em SERS. Estas sondas consistem em nanopartículas de ouro em forma de estrela, que proporcionam um sinal de SERS intenso proveniente das moléculas repórter de Raman ligadas às nanopartículas. As sondas ad- quirem a especificidade necessária para o antigénio de anticorpos a elas ligados de forma covalente, e, por conseguinte, permitem uma ligação estável antigénio-anticorpo. O hidro- gel regenerado à base de celulose forneceu uma plataforma de captura de design robusto e ecológico. Além de ser um material transparente com baixa fluorescência e, portanto, de baixa interferência no sinal de Raman, é um material com uma elevada capacidade de retenção de água tornando-o particularmente adequado para preservar a actividade dos anticorpos ligados covalentemente. Deste modo, o hidrogel proporciona uma plataforma de captura estável ao longo do tempo. O immunoensaio baseado em SERS desenvolvido foi posteriormente integrado num dispositivo de microfluídica, permitindo analisar um grande número de amostras sendo simultaneamente sensível e passível para aplicações de análise de múltiplos antigénios. O dispositivo foi fabricado em menos de 30 minu- tos devido à padronização directa em folhas de poliestireno contrácteis possibilitando a construção tridimensional de um dispositivo de microfluídica. Finalmente, para validar o sistema de microfluídica, amostras de cultura de eritrócitos infectados com Plasmodium falciparum foram testadas para detecção de biomarcadores de malária. A discriminação dos sinais das immunosondas activas em SERS, relativamente a sinais interferentes, foi feita através do método clássico de quadrados mínimos. Como resultado, foi conseguido um melhor ajuste de dados em comparação com o método de cálculo do integral das áreas das bandas habitualmente utilizado. Assim, o ensaio imunológico baseado em SERS proposto neste trabalho permitiu obter um limite de detecção mais baixo do que o obtido pelas abordagens tradicionais como o ensaio de imunoabsorção enzimática (conhecido pelo acrónimo de ELISA), além de exibir um desempenho melhor ou comparável a ou- tros sensores baseados em SERS já existentes na literatura. Adicionalmente, o sistema desenvolvido neste trabalho permite ultrapassar desafios que impedem a utilização deste tipo de sensores em locais de poucos recursos, apresentando valores elevados de repro- dutibilidade, sensibilidade e especificidade. Por conseguinte, um sistema que combina SERS e microfluídica representa uma tecnologia potencialmente importante na detecção precoce, na esperança de que, num futuro próximo, as consequências das doenças infecci- osas que ainda impõem um fardo substancial ao sistema de saúde a nível mundial, sejam minoradas