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

brightening the future of bioanalysis

This work was fnanced by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects LA/P/0037/2020, UIDP/50025/2020 and UIDB/50025/2020 of the Associate Laboratory Institute of Nanostructures, Nanomodelling and Nanofabrication—i3N and by the FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the following Projects: UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO; LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB; grant PTDC/NAN-MAT/30589/2017; and fellowship SFRH/BD/132057/2017 also from MIT Portugal PhD Program (to M.J.O.). Funding also from the European Community H2020 program under grant agreement No. 716510 (ERC-2016-STG TREND), No. 640598 (ERC-StG-2014, NEWFUN), and No. 685758 (1D-Neon).A new avenue has opened up for applications of surface-enhanced Raman spectroscopy (SERS) in the biomedical field, mainly due to the striking advantages offered by SERS tags. SERS tags provide indirect identification of analytes with rich and highly specific spectral fingerprint information, high sensitivity, and outstanding multiplexing potential, making them very useful in in vitro and in vivo assays. The recent and innovative advances in nanomaterial science, novel Raman reporters, and emerging bioconjugation protocols have helped develop ultra-bright SERS tags as powerful tools for multiplex SERS-based detection and diagnosis applications. Nevertheless, to translate SERS platforms to real-world problems, some challenges, especially for clinical applications, must be addressed. This review presents the current understanding of the factors influencing the quality of SERS tags and the strategies commonly employed to improve not only spectral quality but the specificity and reproducibility of the interaction of the analyte with the target ligand. It further explores some of the most common approaches which have emerged for coupling SERS with microfluidic technologies, for biomedical applications. The importance of understanding microfluidic production and characterisation to yield excellent device quality while ensuring high throughput production are emphasised and explored, after which, the challenges and approaches developed to fulfil the potential that SERS-based microfluidics have to offer are described.publishersversionpublishe

Fabrication of plasmonic nanosubstrate for SERS-based nucleic acid detection

Dissertação de mestrado em Biofísica e BionanossistemasUm rápido diagnóstico nos primeiros estadios de doenças é um passo crucial para melhorar a eficácia dos tratamentos de cancro e outras doenças. O estudo de alterações genéticas é particularmente essencial na análise da presença de mutações e alterações transcriptómicas no cancro, sendo crucial no desenvolvimento de estratégias de tratamento personalizado e no avanço da eficácia da terapia. O mesmo conceito é aplicado ao estudo de ácidos nucleicos (NA) de origem viral, o que pode ajudar na diminuição da taxa de infeção de doenças como a COVID-19. Atualmente, testes de amplificação de Nas de pacientes são o método principal no diagnóstico da COVID-19 e na identificação de mutações no cancro. Contudo, este método requer mão de obra especializada nos processos de extração e amplificação, e a obtenção dos resultados demora entre 2 e 3 horas o que limita o potencial ‘point-of care’ (POC) da sua aplicação em zonas com recursos limitados. Este estudo foi realizado com o intuito de desenvolver dispositivos médicos à base de espectroscopia Raman amplificada por superfície (SERS) para a identificação rápida de NAs sem processos de amplificação, o que pode não melhorar a eficácia de deteção, mas simplificará o diagnóstico POC e alargar a sua implementação. Duas estratégias à base de SERS foram desenvolvidas: um ‘nanoassembly’ de dímeros de nanopartículas de ouro (AuNP) para a deteção de miR-223 (marcador oncogénico) e um ‘hairpin design’ num substrato de ouro@prata ‘core shell nanorods’ (Au@Ag NRs) para a deteção de NAs derivados de SARS-CoV-2. A estratégia de ‘nanoassembly’ consiste na formação de nanoestruturas diméricas na presença da sequência-alvo miRNA (miR-223), o que resulta em zonas electromagneticamente ativas que aumentam a intensidade do sinal Raman. O princípio básico da estratégia ‘hairpin design’ depende no uso de sondas moleculares que reconhecem sequências-alvo específicas ao vírus SARS-CoV-2, monitorizando este processo de hibridização com espetros Raman. Três sequências virais, regularmente usadas em estudos da COVID-19, foram selecionadas como sequências-alvo. As sondas moleculares foram desenvolvidas e integradas num ‘chip’ de SERS para a identificação simultânea destes oligonucleótidos. Passos de otimização do protocolo é ainda necessário, seguidos de ensaios de deteção com RNA extraído de vírus inativado e com amostras clínicas de pacientes.Rapid diagnosis at an early stage is a critical step for successful outcome in cancer and in other diseases. In particular, the study of genetic alterations is paramount to analyze the presence of mutations and transcriptomic alterations in cancer which is crucial to design personalized treatment strategies and advance therapeutic efficacy. The same concept applies to the detection of viral-specific nucleic acid (NA), which can aid on the early diagnosis and prevention of disease spreading, like in the case of COVID-19. Currently, NA amplification technologies constitute the standard for diagnosis of SARS-CoV-2-infected individuals and for the identification of tumor-related genetic alterations in cancer. However, these techniques require trained personnel to deal with the extraction and amplification steps and it takes up to 3 hours to obtain the results, which limits point-of-care (POC) applications in resource-limited settings. The scope of this project is to develop new strategies based on surface enhanced Raman scattering spectroscopy (SERS) for fast screening of specific NAs without the need of amplification, which might not improve the efficiency and sensitivity of detection but will simplify the POC diagnosis and widen its implementation. Herein, two different SERS strategies were developed: a gold nanoparticle (AuNP) dimer nanoassembly for miR-223 detection (oncogenic marker) and a hairpin design on gold@silver core-shell nanorods (Au@Ag NRs) substrate for SARS-CoV-2 nucleic acid detection The AuNP dimer nanoassembly, strategy consisted of the formation of structurally reproducible dimeric nanostructures in the presence of the target miRNA (miR-223), where the newly generated electromagnetic hot spots provide significantly enhanced Raman scattering. The basic principle of the hairpin-based strategy is to employ molecular probes to recognize a set of target sequences specific for the SARS-CoV-2 virus, while monitoring this hybridization process with SERS spectrum. Three viral NA sequences, which have been commonly used for COVID-19 diagnosis sequences have been selected as the detection targets. The molecular probes have been designed and integrated into a SERS chip for simultaneous identification of these oligonucleotdies. Further optimizations will still need to be conducted for optimal results, followed by assays involving RNA extracted from inactivated virus samples and further validation with clinical samples

Plasmonic nanoparticle sensors: current progress, challenges, and future prospects

Plasmonic nanoparticles (NPs) have played a significant role in the evolution of modern nanoscience and nanotechnology in terms of colloidal synthesis, general understanding of nanocrystal growth mechanisms, and their impact in a wide range of applications. They exhibit strong visible colors due to localized surface plasmon resonance (LSPR) that depends on their size, shape, composition, and the surrounding dielectric environment. Under resonant excitation, the LSPR of plasmonic NPs leads to a strong field enhancement near their surfaces and thus enhances various light–matter interactions. These unique optical properties of plasmonic NPs have been used to design chemical and biological sensors. Over the last few decades, colloidal plasmonic NPs have been greatly exploited in sensing applications through LSPR shifts (colorimetry), surface-enhanced Raman scattering, surface-enhanced fluorescence, and chiroptical activity. Although colloidal plasmonic NPs have emerged at the forefront of nanobiosensors, there are still several important challenges to be addressed for the realization of plasmonic NP-based sensor kits for routine use in daily life. In this comprehensive review, researchers of different disciplines (colloidal and analytical chemistry, biology, physics, and medicine) have joined together to summarize the past, present, and future of plasmonic NP-based sensors in terms of different sensing platforms, understanding of the sensing mechanisms, different chemical and biological analytes, and the expected future technologies. This review is expected to guide the researchers currently working in this field and inspire future generations of scientists to join this compelling research field and its branches.This article is published as Kant, Krishna, Reshma Beeram, Lara González Cabaleiro, Yi Cao, Daniel Quesada-González, Heng Guo, Sergio Gomez-Grana et al. "Roadmap for Plasmonic Nanoparticle Sensors: Current Progress, Challenges and Future Prospects." Nanoscale Horizons (2024). doi: https://doi.org/10.1039/D4NH00226A

Surface Plasmon Resonance for Biosensing

The rise of photonics technologies has driven an extremely fast evolution in biosensing applications. Such rapid progress has created a gap of understanding and insight capability in the general public about advanced sensing systems that have been made progressively available by these new technologies. Thus, there is currently a clear need for moving the meaning of some keywords, such as plasmonic, into the daily vocabulary of a general audience with a reasonable degree of education. The selection of the scientific works reported in this book is carefully balanced between reviews and research papers and has the purpose of presenting a set of applications and case studies sufficiently broad enough to enlighten the reader attention toward the great potential of plasmonic biosensing and the great impact that can be expected in the near future for supporting disease screening and stratification

Multicomponent magnetic nanoparticle engineering: the role of structure-property relationship in advanced applications

Combining magnetic nanomaterials with materials of other classes can produce multicomponent nanoparticles with an entire ensemble of new structures and unique, enhanced, synergetic, and/or complementary functionalities. Here we discuss the most recent developments in the synthesis of multicomponent magnetic nanoparticles, describe the resulting structures and their novel properties, and explore their application in a variety of fields, including multimodal imaging, nanomedicine, sensing, surface-enhanced Raman scattering, and heterogeneous catalysis. The current synthetic methods (usu-ally bottom-up approaches) of multicomponent nanoparticles can produce a number of tailored mor-phologies (core@shell, yolk-shell, core-satellite, Janus, nanochains, anisotropic, etc.), making them invaluable for applications in biology, medicine, chemistry, physics, and engineering. But like any new technology, their synthesis methods need to be optimized to be simple, scalable, and as environmentally friendly as possible before they can be widely adopted. In particular, the use of life cycle assessment (LCA) to guide future works toward environmental sustainability is highlighted. Overall, this review not only presents a critical and timely summary of the state-of-the-art of this burgeoning field in both fundamental and applied nanotechnology, but also addresses the challenges associated with under-standing the particular structure-property relationships of multicomponent magnetic nanoparticles.The authors thank funding from the Spanish State Research Agency (AEI) through the project PID2019-106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively

Magnetic-plasmonic nanoparticles for multimodal bioimaging and hyperthermia.

257 p.El término "teranóstica" hace referencia a la integración inteligente de diagnósticos y terapias. Esta capacidad de obtener imágenes y tratar tumores simultáneamente con nanopartículas puede resultar ventajosa frente a las técnicas convencionales de diagnóstico y terapia. Así, una ventaja adicional tanto para la obtención de imágenes como para el tratamiento es poder estudiar las enfermedades in vitro utilizando diversos métodos de obtención de imágenes y combinándolos con tratamientos novedosos.La síntesis y optimización de nanopartículas híbridas que combinan propiedades magnéticas y plasmónicas se ha estudiado durante esta tesis. Además, estas nanoestructuras pueden funcionalizarse con moléculas adicionales para aplicaciones en imagen e hipertermia. La utilización de estas nanopartículas híbridas se ha estudiado para su uso específico como agentes de contraste para resonancia magnética, dispersión Raman mejorada en la superficie y microscopía de fluorescencia en modelos celulares 2D y 3D y en modelos ex vivo. Además, se ha evaluado la aplicación de los híbridos para calentamiento fototérmico en modelos celular 2D y 3D y etiquetado específico de células en modelos celulares 2D

Hybrid point-of-care devices for visual detection of biomarkers and drugs

Early diagnostics is a crucial part of clinical practice offering a rapid and convenient way to investigate and quantify the presence of key biomarkers related to specific pathologies and increasing the chance of successful treatments. In this regard, point-of-care testing (POCT) shows several advantages enabling simple and rapid analyses, allowing for real-time results, and permitting home testing. Metallic nanoparticles (NPs), like gold NPs (AuNPs), can be beneficially integrated into POC devices thanks to their tunable plasmonic properties which provide a naked-eye read-out. Moreover, the high sensitivity of NPs enables the detection of biomarkers in non-invasive fluids where the concentrations are typically low. These biofluids, like saliva and urine, are functionally equivalent to serum in reflecting the physiological state of the body, whilst they are easier to handle, collect, and store. In this thesis, I first reported the design and development of a colorimetric strategy based on the morphological change of multibranched plasmonic AuNPs, aimed at detecting glucose in saliva. The sensing approach relied on a target-induced reshaping process which involves the oxidation of the NP tips and the transformation into a spherical shape, characterized by a naked-eye detectable blue-to-pink color change. The platform proved to be beneficial in the early and non-invasive diagnosis of hyperglycemia. The successful technological transfer on a solid substrate paved the way for the realization of a dipstick prototype for home testing. Then, the strategy was adapted to other biomarkers, leading to the development of a multiplexing test for the simultaneous detection of three salivary analytes (cholesterol, glucose, and lactate). This multiplexing assay enabled to save reagents, costs, and time, whilst increasing the overall clinical value of the test. Exploiting the microfluidics applied on a paper sheet, I realized a monolithic and fully integrated POC device, through a low-cost and fast CO2 laser cutter. The platform showed excellent selectivity and multiplexing ability, with negligible interferences. The second part of my thesis was focused on the development of POC devices for the detection of anticancer drug contaminations in water solutions and urine samples. Antiblastic agents have revealed high toxicity for the exposed healthcare workers who prepare and administer these drugs in occupational environments. Hence, continuous monitoring is highly required, and POCT shows tremendous potential in this context. With this aim, I realized a lateral-flow (LF) device for the assessment of doxorubicin contamination, using the fluorescent properties of the drug for naked-eye detection. The pharmacological recognition of the DNA probe was exploited to overcome the lack of anti-doxorubicin antibodies. The highly sensitive strategy was successfully adapted to a real urine sample, without resorting to complex pretreatment procedures. Then, I developed a competitive LF device for the detection of methotrexate (MTX). AuNPs were employed as the label molecules and the pharmacological competition of folic acid and MTX for the capture enzyme was exploited as the recognition mechanism, instead of costly antibodies. Despite the sensitivity requires further improvements, the strategy showed fast and reliable results, demonstrating a high potential for workers’ safety control

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

Application of SERS for Nanomaterials

This Special Issue of “Applications of SERS” for Nanomaterials is a collection of articles which is representative of much of the current research being undertaken in the field of Surface-Enhanced Raman Scattering (SERS) spectroscopy. SERS is a fascinating, multidisciplinary field of scientific study which combines elements from chemistry, physics, material science, and engineering. Essentially, SERS is a molecular spectroscopy technique by which a measurable Raman signal for molecules on metal and semiconductor surfaces is generated through the interaction of laser light, absorbed molecules, and structured nanomaterial surfaces. This Special Issue contains an article regarding the fabrication of metal nanostructured Ag-Cu chips for SERS chemical analysis and ] the electromagnetic properties of Ag, Au, and Al nano-tips for use in the SERS imaging technique for tip-enhanced Raman Scattering (TERS). In another article, SERS spectra were simulated using density Functional Theory (DFT/TD-DFT) for the N3 dye molecule on a TiO2 nanocluster, which can be compared to experimental SERS spectra found in studies of Dye-Sensitized Solar Cells (DSSCs). In two other articles, the SERS photoinduced charge-transfer mechanism was studied experimentally in wide-bandgap semiconductors regarding molecules on ZrO2 and a composite system with molecules that linked Au nanorods and a CuO2 shell. An example of the use of SERS in solid-state physics is shown in an article which examined the effect of oxygen vacancy defects in MO3 on the SERS mechanism. Finally, this Special Issue contains two noteworthy examples of SERS applications for biochemical and chemical analysis. One paper addresses the detection of the COVID-19 coronavirus SARS-CoV-2 using SERS, and the other examines a SERS assay of the notorious herbicide glyphosate. In these papers, nanomaterials all served as the enhancing substrate, while some acted as the physical–chemical system which was being investigated