169 research outputs found
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
Electrochemical microfluidic multiplexed biosensor platform for point-of-care testing
Early and accurate diagnosis of a specific disease plays a decisive role for its effective treatment. However, in many cases the clinical findings, based on a single biomarker detection alone, are not sufficient for the appropriate diagnosis as well as monitoring of its treatment. Furthermore, it is highly desirable to screen multi-analytes (e.g. various diseases and drugs) at the same time enabling a low-cost, quick and reliable quantification. Thus, multiplexing, simultaneous detection of different analytes from a single sample, has become in recent years essential for diagnostics, especially for point-of-care testing (POCT). This thesis focuses on the scientific issue regarding the sensitivity enhancement of microfluidic biosensor platforms. Simulations, design studies and experiments are employed to investigate the interplay between the immobilization area and the resulting sensitivity. Thereby, a novel concept comprising design rules for microfluidic biosensors using the stop-flow technique has been introduced. In combination with different technical measures it allows the realization of an electrochemical lab-on-a-chip (LOC) platform for the fast, sensitive and simultaneous POCT in clinically relevant samples. This system employs a universally applicable, bioaffinity based biomolecule immobilization along with an amperometric readout. By means of the dry film photoresist technology, the fabrication of disposable microfluidic biosensors is enabled with high yield on wafer-level. The presented LOC platform offers three different biosensors with a microfluidic channel network of two, four or eight discrete immobilization sections, each with a volume of 680 nl. They can be actuated by individual channel inlets allowing a high flexibility in the assay design with respect to its format (e.g. competitive) and its technology (e.g. genomics). The feasibility for multiplexing is successfully demonstrated with DNA-based antibiotic assays for tetracycline and streptogramin, both important growth promoters in livestock breeding. The extensive usage of antibiotics is one of the major causes of the multi-drug-resistant bacteria and so, it has to be kept under surveillance. This platform allows the simultaneous POCT of different antibiotics from human plasma along with a limit of detection of less than 10 ng ml⁻¹, a wide working range up to 1,600 ng ml⁻¹ and inter-assay precisions of about 10 %. Moreover, the microfluidic LOC system provides a low consumption of reagent and sample, reduces the total assay time drastically with a sample-to-result time of only 10 min. The shelf-life of the biosensors is proven to be at least 3 months at +4 °C. The introduced design concept with specific technical measures facilitates the implementation of microfluidic multiplexed biosensors in a low-cost, compact, and at the same time sensitive manner. This platform targets the POCT in the first place, yet, owing to its multiplexing approach it can be expanded for in vitro diagnostics
Point-of-Care Devices for Viral Detection: COVID-19 Pandemic and Beyond
The pandemic of COVID-19 and its widespread transmission have made us realize the importance of early, quick diagnostic tests for facilitating effective cure and management. The primary obstacles encountered were accurately distinguishing COVID-19 from other illnesses including the flu, common cold, etc. While the polymerase chain reaction technique is a robust technique for the determination of SARS-CoV-2 in patients of COVID-19, there arises a high demand for affordable, quick, user-friendly, and precise point-of-care (POC) diagnostic in therapeutic settings. The necessity for available tests with rapid outcomes spurred the advancement of POC tests that are characterized by speed, automation, and high precision and accuracy. Paper-based POC devices have gained increasing interest in recent years because of rapid, low-cost detection without requiring external instruments. At present, microfluidic paper-based analysis devices have garnered public attention and accelerated the development of such POCT for efficient multistep assays. In the current review, our focus will be on the fabrication of detection modules for SARS-CoV-2. Here, we have included a discussion on various strategies for the detection of viral moieties. The compilation of these strategies would offer comprehensive insight into the detection of the causative agent preparedness for future pandemics. We also provide a descriptive outline for paper-based diagnostic platforms, involving the determination mechanisms, as well as a commercial kit for COVID-19 as well as their outlook
NANOTECHNOLOGY FOR DETECTION OF DISEASES CAUSED BY VIRUSES-CURRENT OVERVIEW
Nanotechnology is having a high impact on the development of a novel class of biosensors called nanobiosensors. This technology has utilized some extremely exciting elements for sensing phenomenon improvement. The utilization of nano-materials, nano-rods, nano-particles, nano-tubes have aided rapid, reliable reproducibility and its detection in a much better way. The unique properties of nanobiosensors and its varied applications have influenced biosensing research. Since longtime, nanobiosensors have been utilized worldwide for the diagnosis of diseases co-related with molecular detection of biomarkers. This paper highlights the use of such nanobiosensors for the detection of the virus, infections, fungal pathogens, Human Immunodeficiency Virus (HIV) related diseases such as Cardiovascular diseases (CDVs), Renal Arthritis (RA) through different techniques including electrochemical biosensing, optical biosensing, point of care-diagnostics etc
Electrochemical Sensors for Flow-Enhanced Immunoassays and Flow Monitoring of Porous Materials
Department of Chemical EngineeringMicrofluidics enables a miniaturization of laboratory-scale biochemical analysis into a single chip. It has been actively developed and utilized in various biomedical diagnostic devices. Especially, lab-on-a-disc (LOD) and paper microfluidic system can serve as excellent candidates for point-of-care testing (POCT) because of several advantages including relatively simple fluid transfer mechanism, portable size of the device, fast analysis time, reduced cost, automated analysis steps and low consumption of test samples and reagents. In this thesis, electrochemical detection method has been implemented in two kinds of diagnostic devices. First, lab-on-a-disc integrated with screen-printed carbon electrodes (SPCEs) was developed for electrochemical detection of protein biomarkers, C-reactive protein. Compared to conventional optical detection, the electrochemical detection could provide enhanced sensitivity as well as significant cost reduction because it is not necessary to use optical grade plastic materials for the fabrication of the disc. In addition, we have developed electrochemical sensors for the measurement of the local fluid velocity based upon the fact that the electrochemical signal is proportional to the flow rate. As a proof of the concept experiment, we have used the electrochemical sensors for real time monitoring of the flow through porous materials, which can provide a practical tool to quantify the fluid velocity on the paper-based microfluidics. Based on chronoamperometric signals measurement using SPCEs, fluid transfer phenomena through various kinds of porous materials, geometries, absorption pads, and flow modifiers were evaluated. In conclusion, we have developed a cost-effective SPCEs-based electrochemical detection method and utilized not only for the immunoassays fully integrated on a disc but also to characterize fluid transfer behavior through the porous materials. The SPCEs can be utilized as a cost-effective sensor not only for the highly sensitive electrochemical detection for bioassays but also for the flow measurements in porous materials.ope
Development of Smartphone dual-laser waveguide based fluorescent microscopy system using 3D printing
Nowadays cellphones are present everywhere, and along with the worldwide network of devices, the concept of mobile health monitoring is changing to reshape the biosensor market. The smartphone’s camera is a proven reliable candidate as a detector for the studies performed by various research groups. This study is a proof of concept of the Smartphone detection of two fluorescent dyes which can be used as biomarkers for point-of-care diagnostics through image processing techniques. A smartphone Xiaomi Redmi Note 4 along with two fluorescent dyes DyLight™ 405 NHS Ester and DyLight™ 633 NHS Ester are used in conjunction with two lasers Thorlabs 405 nm and 638nm. The captured pictures were analyzed using Image J. The limit of detection and dynamic range values were calculated for both dyes, 28.39 nM and 20-800 nM for DyLight™ 405 NHS Ester dye and 15.85 nM and 10-600 nM for DyLight™ 633 NHS Ester dye. Then this concept is realized by developing a cheap 3D printed POC device which uses the optical microscopy technology along with a PDMS chip. Hence, this integrated novel innovation which prioritizes accuracy and the ease of usage, can be a game changer for patients who live in countries of limited resources and can moreover aid to the impoverished people who are in dire need of medical help
Scanning-fluorescence Reader Based on Embedded System
To measure the concentration of C-reactive protein (CRP) in serum, a portable, scanning-fluorescence reader based on time-resolved fluoroimmunoassays was developed. The scanning-fluorescence reader integrates with the AD7707 converter, which performs at a high accuracy. The photosensitive diode acts as the photoelectric conversion device, an optical module based on optical fibers, which is able to concentrate the excitation light from an LED into a line-shape beam, was designed to sendand receive the optical signal. The device subsequently addresses waveform data using a gradient, smoothing, and binarization method. When the device measures the CRP fluorescence test strip, the results exhibited a good linearity (0.99998) and the CVs (coefficient of variation) were below 5%, which indicate high accuracy. At the same time the system is low cost and small size
Rapid Vertical Flow Assay On Aunp Plasmonic Paper For Sers-Based Point Of Need Diagnostics
SERS based immunoassays for point-of-care diagnostics is a promising tool to facilitate biomarker detection for early disease diagnosis and control. The technique is based on a sandwiched system in which antigen is first captured by a selective substrate and then labeled by an extrinsic Raman label (ERL). Here, we report on the use of gold nanoparticle modified filter paper as a novel capture membrane in a vertical flow format. This vertical flow configuration affords reproducible flow of sample and label through the capture substrate to overcome diffusion limited kinetics and significantly reduced assay time. The filter paper was selected due to its affordability and availability, while the embedded AuNPs maximized plasmonic coupling and SERS enhancement. Additionally, the embedded AuNP served as a scaffold to immobilize capture antibody to specifically bind antigen. In this work, a SERS-based rapid vertical flow (SERS-RVF) immunoassay for detection of mouse IgG was developed to establish proof of principle. Optimization of assay conditions led to a limit of detection of 3 ng/mL, which is comparable to more traditional formats carried out in multi-well plates and significantly reduced assay time to less than 2 minutes. Additionally, IgG was accurately quantified in normal serum to validate the SERS-RVF assay for application to the analysis of biological samples. These results highlight the potential advantages of the SERS-RVF platform for point-of-need testing.
KEYWORDS: Immunoassay; surface-enhanced Raman scattering (SERS); plasmonic paper; vertical flow assay; point-of-need (PON) testing;point-of-care (POC) diagnostics
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