Recent trends in field-effect transistors-based immunosensors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Immunosensors are analytical platforms that detect specific antigen-antibody interactions and play an important role in a wide range of applications in biomedical clinical diagnosis, food safety, and monitoring contaminants in the environment. Field-effect transistors (FET) immunosensors have been developed as promising alternatives to conventional immunoassays, which require complicated processes and long-time data acquisition. The electrical signal of FET-based immunosensors is generated as a result of the antigen-antibody conjugation. FET biosensors present real-time and rapid response, require small sample volume, and exhibit higher sensitivity and selectivity. This review brings an overview on the recent literature of FET-based immunosensors, highlighting a diversity of nanomaterials modified with specific receptors as immunosensing platforms for the ultrasensitive detection of various biomolecules.Immunosensors are analytical platforms that detect specific antigen-antibody interactions and play an important role in a wide range of applications in biomedical clinical diagnosis, food safety, and monitoring contaminants in the environment. Field-effec44FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)2013/22127-2; 2016/04739-9sem informação: The authors acknowledge the financial assistance provided by The São Paulo Research Foundation (FAPESP, project #2013/22127-2 and grant #2016/04739-9) and the National Council for Scientific and Technological Development (CNPq

Nanomaterials for Healthcare Biosensing Applications

In recent years, an increasing number of nanomaterials have been explored for their applications in biomedical diagnostics, making their applications in healthcare biosensing a rapidly evolving field. Nanomaterials introduce versatility to the sensing platforms and may even allow mobility between different detection mechanisms. The prospect of a combination of different nanomaterials allows an exploitation of their synergistic additive and novel properties for sensor development. This paper covers more than 290 research works since 2015, elaborating the diverse roles played by various nanomaterials in the biosensing field. Hence, we provide a comprehensive review of the healthcare sensing applications of nanomaterials, covering carbon allotrope-based, inorganic, and organic nanomaterials. These sensing systems are able to detect a wide variety of clinically relevant molecules, like nucleic acids, viruses, bacteria, cancer antigens, pharmaceuticals and narcotic drugs, toxins, contaminants, as well as entire cells in various sensing media, ranging from buffers to more complex environments such as urine, blood or sputum. Thus, the latest advancements reviewed in this paper hold tremendous potential for the application of nanomaterials in the early screening of diseases and point-of-care testing

Stencil lithography for bridging MEMS and NEMS

The damage inflicted to silicon nanowires (Si NWs) during the HF vapor etch release poses a challenge to the monolithic integration of Si NWs with higher-order structures, such as microelectromechanical systems (MEMS). This paper reports the development of a stencil lithography-based protection technology that protects Si NWs during prolonged HF vapor release and enables their MEMS integration. Besides, a simplified fabrication flow for the stencil is presented offering ease of patterning of backside features on the nitride membrane. The entire process on Si NW can be performed in a resistless manner. HF vapor etch damage to the Si NWs is characterized, followed by the calibration of the proposed technology steps for Si NW protection. The stencil is fabricated and the developed technology is applied on a Si NW-based multiscale device architecture to protectively coat Si NWs in a localized manner. Protection of Si NW under a prolonged (>3 h) HF vapor etch process has been achieved. Moreover, selective removal of the protection layer around Si NW is demonstrated at the end of the process. The proposed technology also offers access to localized surface modifications on a multiscale device architecture for biological or chemical sensing applications

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

Microfluidic Immunoassays Based on Self-Assembled Magnetic Bead Patterns and Time-Resolved Luminescence Detection

Microfluidic bio-assays have emerged as the most privileged solutions and provide the basis for the realization of miniaturized bio-analytical systems and clinical diagnostic devices that are portable, user-friendly and cost-effective (Lab-on-a-chip). Two important steps that are implemented in a microfluidic bio-assay are: (a) the immobilization and/or patterning of target-specific bio-molecules on the surface of a microfluidic channel, for selectively capturing bio-targets like antigens or pathogens, followed by (b) sensitive detection of the bio-targets. In this thesis, we demonstrate microfluidic bio-assays based on novel methods for generating protein-patterns and on sensitive detection of the bio-targets. First, we introduce a simple and fast method for creating protein micropatterns both on a bare substrate and in-situ inside a microfluidic channel, in a matter of minutes, through electrostatic self-assembly of pre-functionalized magnetic beads. A lift-off patterned positively-charged aminosilane layer is used as the template for immobilizing the protein-coated negatively-charged beads. The number and arrangement of the beads can be well-controlled by altering the silane template design. Subsequently, we use patterned beads as assay substrates for performing on-chip bioassays. We demonstrate highly-sensitive full on-chip sandwich immunoassays for single and multi-analyte detection using beads as assay substrate. We successfully explored the possibility to lower the detection limit of immunoassays by concentrating the target antigens on a very small number of patterned beads. We also present the application of bead patterns as a platform for immuno-separation, culture and analysis of target (cancer) cells. Finally, we demonstrate a rapid on-chip immuno-histo-chemical assay on breast cancer tissues. We use luminescent lanthanide probes in place of conventional fluorescent probes, as labels for detection antibodies, for sensitive detection and quantification of biomarkers. Thanks to the time resolved microscopy and luminescent probes, the background noise due to the autofluorescence of the samples (i.e. tissue, cells) and microfluidic chips is successfully eliminated resulting in an improved signal-to-noise ratio when compared with the fluorescent microscopy results. Our assay results fully agree with the clinical analyses outcome, and this opens perspectives for a fully-integrated cancer detection platform for bedside diagnostics

Carbon Nanomaterials based on Graphene in (Electro-)chemical Sensors: Characterization, Modification and Application

The variability of graphene with its exceptional properties gives rise to improve material chemistry in various fields of applications. The development of graphene is still in the beginning and up to now only a few niche products have reached the market and are highlighted as well as the aim of this work in chapter 1. The goal of this thesis was the investigation of graphene in electrochemical sensor applications. It holds great promise in terms of miniaturization, improving sensitivity and developing new sensor concepts. Many approaches are already described for biosensor applications, often utilizing graphene in an amperometric detection scheme. Therefore, the impact of preparation technique on the detection of the model analyte H2O2 was investigated, applying different graphene materials as electrode material. Further, graphene was studied as tunable sensor material in gas sensing applications and how it can be customized for the room temperature detection of CO2. Chapter 2 summarizes the author’s publications and patents developed in the frame of this work. The perspectives of graphene in electrochemical sensors were investigated by the means of research performed in this field and are described in chapter 3. The most prominent preparation techniques and the application in biosensor and gas sensor technologies are discussed. Every method provides graphene materials of different characteristics, scalability and further usability. It was shown that defects in the ideal sp2 carbon lattice decide on the sensor performance, but also on device fabrication and appropriate functionalization. A higher quality of graphene can lead to more sensitivity and reliable device production in electrochemical biosensor technologies. In contrast, a defective structure can enhance the sensitivity and applicability in gas sensing applications, providing additional adsorption sites. In chapter 4, the experimental work, performed during this work, is described in detail. Chapter 5 comprises the results on graphene and graphene composite materials applied in the electrochemical detection of H2O2 and as tunable recognition element in gas sensors. In a first part, graphene materials derived by different preparation techniques were studied as electrode material. The electrochemical behavior of the different materials has been investigated as well as the feasibility in device fabrication was compared. It was shown, that the quality of the graphene has an enormous impact on the reductive amperometric detection of H2O2. A defective structure like in reduced graphene oxide leads to almost no significant improvement in signal enhancement compared to a standard carbon disc electrode. In contrast, fewer defects like in graphene prepared by chemical vapor deposition, resulted in a higher sensitivity, which is 50 times better compared to reduced graphene oxide. This technique was found to be most suitable for the production of highly sensitive electrodes to be further used in amperometric detection and development of biosensors. Whereas, a material of high quality is desired in electrochemical biosensor applications, defects are beneficial using graphene as transducer in a chemiresistive setup for the detection of gaseous analytes. In the next part of the work, reduced graphene oxide was demonstrated to be an applicable candidate for gas detection at moderate (85 °C) or even room temperature. Analyte gases like NO2, CH4 and H2 were detected due to fast changes in the electrical resistance at of 85 °C. To overcome the poor selectivity, the material was further altered with octadecylamine, metal nanoparticles such as Pd and Pt, and metal oxides such as MnO2, and TiO2. This changed the sensor response towards the studied gases and the different response patterns for six different materials allowed a clear discrimination of all test gases by pattern recognition based on principal component analysis. Based on the feasibility of this concept, a graphene-based sensor for the room temperature detection of CO2 was developed. Decoration of reduced graphene oxide with CuO nanoparticles led to an improved sensing performance for the target analyte. Different levels of metal oxide doping were applied by wet chemical and electrochemical preparation methods and the resulting composite materials were characterized. It was shown that a complete coverage obtained by wet chemical functionalization leads to highest sensitivity, comparable to a commercial CO2 sensor, which was also tested in the frame of this work. An array consisting of reduced graphene oxide and the composite with CuO nanoparticles was capable to differentiate CO2 from NO2, CO, H2 and CH4. This sensor material can lead to the development of miniaturized chemical sensors comprising high and adjustable sensitivity, which can be applied for monitoring air quality and ventilation management. The presented sensor concept based on customized graphene materials can be tailored for the versatile use in appropriate applications. One of the main challenges remains the reproducible large-scale production of graphene and functionalized graphene combined with reliable transfer techniques in terms of an industrial application. This problem has not been solved completely up to now. But the extensive research going on in this field will lead to a solution in near future and will help graphene to find its way to be integrated into many electrochemical sensor devices. Further studies should preferably aim for large scale production of the material and devices. The use of high quality graphene with a distinct introduction of defects and a better controlled way of functionalization may be a route to tune the material properties in favorable directions

Nuevas estrategias miniaturizadas basadas en inmunoanálisis electroquímico soportado sobre partículas magnéticas para la detección y el control de la micotoxina Zearalenona

El trabajo de tesis se ha centrado en el desarrollo de nuevas estrategias analíticas miniaturizadas basadas en inmunoanálisis electroquímico, para llevar a cabo la detección y el control de la micotoxina Zearalenona en alimentos infantiles, con el fin de desarrollar rutas alternativas y competitivas a las técnicas clásicas, las cuales emplean instrumentación analítica cara y sofisticada. El analito elegido, la zearalenona (ZEA), es una micotoxina no esteroidea con actividad estrogénica. Ésta micotoxina está siendo objeto de estudio por parte de la comunidad científica debido a que está presente en un gran número de alimentos básicos de la dieta (aparece fundamentalmente en cereales) y debido al riesgo que supone sobre la salud humana y animal. La cantidad máxima permitida de ZEA, varía de unos países a otros siendo 20 [my]g Kg-1 (ppb) el límite más restrictivo aplicado a alimentos infantiles. Debido a la toxicidad, su detección se ha convertido en uno de los campos más importantes dentro del análisis de alimentos. Además, teniendo en cuenta los bajos niveles de concentración a los que se encuentran y con objeto de mejorar su regulación y control en alimentos, ha surgido la necesidad de desarrollar métodos cada vez más sensibles y específicos que permitan su determinación por debajo de los límites exigidos por la legislación. Asimismo, los métodos inmunoanalíticos han llegado a ser la metodología de elección para la determinación de micotoxinas en general, y de ZEA en particular, gracias a su gran sensibilidad y especificidad. Asimismo, la detección electroquímica (ED) ha demostrado ser una valiosa herramienta en inmunoanálisis debido a su buena sensibilidad, sencillez, bajo coste, gran cantidad de marcadores existentes y debido al buen comportamiento de las reacciones enzimáticas acopladas a las reacciones de transferencia de carga. Siendo en el campo de la miniaturización y de los sistemas microfluídicos, donde esta detección alcanza una mayor importancia, debido a su miniaturización inherente sin pérdida de sensibilidad y a su compatibilidad con las técnicas de microfabricación. Por todo ello, el diseño de esta tesis tiene como punto de partida el desarrollo de un método ELISA convencional con detección electroquímica, a partir del cual se evoluciona hacia dos estrategias miniaturizadas siguiendo las tendencias más actuales de la Química Analítica, que persiguen el desarrollo de sistemas miniaturizados, simplificados y automatizados. Estas nuevas estrategias incluyen el desarrollo de un inmunosensor electroquímico sobre electrodos serigrafiados de carbono desechables y la integración total del ELISA electroquímico en una plataforma microfluídica. En todas las estrategias desarrolladas, los resultados obtenidos pueden considerarse excelentes, demostrando su aplicabilidad para la determinación de ZEA en muestras alimentarias infantiles, con excelente fiabilidad y a concentraciones muy por debajo de la exigida por la legislación vigente

Development of a low-cost graphene-based impedance biosensor

PhD ThesisThe current applicability and accuracy of point-of-care devices is limited, with the need of future technologies to simultaneously target multiple analytes in complex human samples. Graphene’s discovery has provided a valuable opportunity towards the development of high performance biosensors. The quality and surface properties of graphene devices are critical for biosensing applications with a preferred low contact resistance interface between metal and graphene. However, each graphene production method currently results in inconsistent properties, quality and defects thus limiting its application towards mass production. Also, post-production processing, patterning and conventional lithography-based contact deposition negatively impact graphene properties due to chemical contamination. The work of this thesis focuses on the development of fully-functional, label-free graphene-based biosensors and a proof-of-concept was established for the detection of prostate specific antigen (PSA) in aqueous solution using graphene platforms. Extensive work was carried out to characterize different graphene family nanomaterials in order to understand their potential for biosensing applications. Two graphene materials, obtained via a laser reduction process, were selected for further investigations: reduced graphene oxide (rGO) and laser induced graphene from polyimide (LIG). Electrically conductive, porous and chemically active to an extent, these materials offer the advantage of simultaneous production and patterning as capacitive biosensing structures, i.e. interdigitated electrode arrays (IDE). Aiming to enhance the sensitivity of these biosensors, a novel, radio-frequency (RF) detection method was investigated and compared with conventional electrochemical impedance spectroscopy (EIS) on a well-known biocompatible material: gold (standard). It was shown that the RF detection methods require careful design and testing setup, with conventional EIS performing better in the given conditions. The method was further used on rGO and LIG IDE devices for the electrochemical impedance detection of PSA to assess the feasibility of the graphene based materials as biosensors. The graphene-based materials were successfully functionalized via the available carboxylic groups, using the EDC-NHS chemistry. Despite the difficulty of producing reproducible graphene-based electrodes, highly required for biosensor development, extensive testing was carried out to understand their feasibility. The calibration curves obtained via successive PSA addition showed a moderate-to-high ii sensitivity of both rGO and LIG IDE. However, further adsorption and drift testing underlined some major limitations in the case of LIG, due to its complex morphology and large porosity. To enable low contact resistance to these biosensors, the electroless nickel coating process is shown to be compatible with various graphene-based materials. This was demonstrated by tuning the chemical nickel bath and method conditions for pristine graphene and rGO for nickel contacts deposition

Capillary Microfluidic Chips for Point-of-Care Testing:from Research Tools to Decentralized Medical Diagnostics

Research on microfluidic devices for biological analysis has progressed sufficiently to be developed into point-of-care diagnostics products. The goal of this thesis is to improve multiple aspects of capillary-driven microfluidic devices. In particular, the objective is to provide devices with a fast time to result, that are simple to use (one-step), that can be portable, that accept a variety of samples, that operate reliably, that provide a range of detection signals, that are mass manufacturable at lost cost, and that are able to detect medically relevant biological molecules. First, we survey the evolution of microfluidic research into portable medical diagnostic devices. By looking at several gaps and opportunities in current medical diagnostics, we provide an overview of research topics that have the potential to shape the next generation of point-of-care diagnostics. Specifically we explain technologies in the order of sample interacting with different components of a device. We investigate the materials, surface treatments, sample processing, microfluidic elements (such as valves, pumps and mixers), receptors and analytes and the integration of these components into a device that might conceivably leave the laboratory for the hands of consumers. The knowledge of what is important in a point-of-care diagnostics device was used to develop a proof of concept. One of the main challenges is to make microfluidics easy to use by incorporating reagents and microfluidic elements. We integrated a number of functional elements on a chip such as a sample collector, delay valves, flow resistors, a deposition zone for detection antibodies (dAbs), a reaction chamber sealed with a polydimethylsiloxane (PDMS) substrate, and a capillary pump and vents. We further incorporated capture antibodies (cAbs), detection antibodies (dAbs) and analyte molecules for making one-step immunoassays. The integrated microfluidic chip requires only the addition of sample to trigger a sequence of events controlled by capillary forces to detect C-reactive protein (CRP), a general inflammation and cardiac marker, at a concentration of 1 ng mL-1 within 14 min using only 5 µL of human serum. The proof-of-concept is extended to easily modify several assay parameters such as the flow rates and the volumes of samples for tests, and the type of reagents and receptors for analytes. The multiparametric microfluidic chip is capable of analyzing 20 µL of human serum in 6 parallel flow paths in a range of flow rates with filling times from 10 minutes to 72 minutes. The asymmetric release of dAbs in a stream of human serum is compensated by a Dean flow mixer. Sample is equally split into 6 reaction chambers connected to flow resistances that vary flow rates, and the kinetics of capture of analyte-dAb complexes. The increased incubation time leads to a fourfold increase in detection signal in the reaction chamber with the longer incubation time. Furthermore, integrating reagents and controlling their release is essential for simple and accurate point-of-care diagnostic devices. We developed reagent integrators (RIs) to release small amounts of dried reagents (ng quantities and less) into microliters of sample. Typical RIs are composed of an inlet splitting into a central reagent channel, with a high hydraulic resistance, and two diluter channels. Reagents spotted in the central channel reconstitute in sample during filling and merge at the end of the RI with a dilution factor corresponding to the relative hydraulic resistance of the channels forming the RI. RIs are simple to integrate in lateral flow assays and provide a great degree of control over reagent integration and dissolution. Finally, the one-step capillary-driven microfluidic chips have the ability to not only detect a variety of proteins, but also to detect nucleic acids for molecular diagnostics. These devices, especially if manufactured in low cost plastic and used with portable fluorescence readers, have the potential to identify a wide variety of health conditions and to enable truly decentralized medical diagnostics