Design of Surface Modifications for Nanoscale Sensor Applications

Nanoscale biosensors provide the possibility to miniaturize optic, acoustic and electric sensors to the dimensions of biomolecules. This enables approaching single-molecule detection and new sensing modalities that probe molecular conformation. Nanoscale sensors are predominantly surface-based and label-free to exploit inherent advantages of physical phenomena allowing high sensitivity without distortive labeling. There are three main criteria to be optimized in the design of surface-based and label-free biosensors: (i) the biomolecules of interest must bind with high affinity and selectively to the sensitive area; (ii) the biomolecules must be efficiently transported from the bulk solution to the sensor; and (iii) the transducer concept must be sufficiently sensitive to detect low coverage of captured biomolecules within reasonable time scales. The majority of literature on nanoscale biosensors deals with the third criterion while implicitly assuming that solutions developed for macroscale biosensors to the first two, equally important, criteria are applicable also to nanoscale sensors. We focus on providing an introduction to and perspectives on the advanced concepts for surface functionalization of biosensors with nanosized sensor elements that have been developed over the past decades (criterion (iii)). We review in detail how patterning of molecular films designed to control interactions of biomolecules with nanoscale biosensor surfaces creates new possibilities as well as new challenges

Electrochemical Readout in Lab-On-Chip Platforms: Overview of State of the Art and Future Perspectives

Lab-on-chip (LoC) platforms are disruptive technologies in analytical chemistry and bioengineering and are known to enable miniaturised and sensitive analysis of complex biological and chemical samples. Electrochemical detection became one of the most explored among several readout methods, hence its versatility, robustness, straightforward execution, sensitivity, and portability. Therefore, this brief review critically examines the principles, applications, and prospects of electrochemical detection in LoC technologies, highlighting its significance in advancing various fields, including clinical diagnostics, environmental monitoring, and drug discovery

Detection of Zn in water using novel functionalised planar microwave sensors

Metal pollution in aquatic environments has attracted global attention. Current methods are not able to monitor water quality in-situ at low-cost. This paper reports on a novel approach for detecting changes in the concentration of zinc in water using electrical and a microwave sensor method, adopting two planar sensors: one was functionalised with a screen-printed β-Bi2O3 based coating, while the other was uncoated. Results show that both electrical and the microwave sensor responses were dependent on the presence and concentration of Zn in water with R2 = 0.93-0.99. The functionalised sensor with a 60 μm thick β-Bi2O3 based film offers improved performance compared with both uncoated and functionalised sensors with 40 μm thick coating for detecting the changes of Zn concentrations in water for low levels (100 and 500 μg/L). This novel sensing system could be a cost-effective alternative to the current offline methods

Surface engineering of poly(methylmethacrylate): Effects on fluorescence immunoassay

The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer

Surface engineering of poly(methylmethacrylate): Effects on fluorescence immunoassay

The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer

Nanoscale Studies of Proteins and Thin Films Using Scanning Probe Microscopy

Nanostructures of organosilanes, thin metal films, and protein nanopatterns were prepared and analyzed with atomic force microscopy (AFM). Organosilanes with designed functional groups were used to selectively pattern green fluorescent protein at the nanoscale using protocols developed with particle lithography. Mesospheres are deposited onto a substrate to produce a surface mask. Organosilanes are deposited to form a matrix film surrounding nanopores for depositing proteins. The nanopatterns were characterized using AFM, after steps of particle lithography for directly visualizing surface changes. Studies with AFM also provide a compelling tool for teaching undergraduates to introduce concepts of nanoscience. An undergraduate laboratory was developed with particle lithography to introduce the concepts of nanoscience and surface chemistry. Nanopatterns of organosilane films are prepared using protocols of particle lithography. An organic thin film is applied to the substrate using steps of either heated vapor deposition or immersion in solution. At the molecular level, two types of sample morphology can be made depending on the step for depositing organosilanes. Experience with advanced AFM instrumentation is obtained for data acquisition, digital image processing and analysis. Skills with chemical analysis are gained with bench methods of sample preparation. Concepts such as the organization of molecules on surfaces and molecular self-assembly are demonstrated with the visualization of nanopatterns prepared by students. Experiments with particle lithography can be used as a laboratory module or for undergraduate research projects, and are suitable for students with a multidisciplinary science background. The kinetics and properties of thin gold films during dewetting were studied using AFM. Thin films of gold with varying initial thickness were first deposited onto fire polished glass slides and imaged with AFM. Next, the films were annealed for two hours, and then imaged after annealing. Gold islands with varying degrees of separation were formed. Surface plasmon spectroscopy was also used to analyze the gold films. To further this study, a kinetic study was done. Two gold thin films of 10 nm each were imaged after being annealed for 15, 30, 45, 60 and 120 minutes. It was found that after the first 15 minutes of annealing, gold islands were observed

大気中の物質を捕捉するためのポリマーブラシを有する機能性マイクロファイバー不織布

This study developed adsorbents that target volatile organic compounds (VOCs) and influenza viruses among the threats in the atmosphere through polymer brushes. Confirmed that VOCs could be adsorbed when introducing appropriate functional groups and metal ions through the Cambridge Crystal Graphic Data Centre data and conducted experiments accordingly. As a result, the copper ion-immobilized adsorbent at the degree of grafting (dg) 170% removed up to 50% of 4L acetone gas (50 ppm), and the amount of adsorbed acetone was 0.04mmol/g. In the case of the influenza virus, because hemagglutinin present on the surface of influenza binds to sialic acid through specific recognition, sialic acid was introduced into a polymer brush, and adsorption was attempted. However, wheat germ agglutinin was used as a substitute instead of hemagglutinin. As a result, when dg 87%, the maximum 118.2 μg in 200 μg lectin was adsorbed, and the adsorption rate was 59.1%.北九州市立大

Smart and Functional Interfaces for Sensitive SPR Biosensing Towards Biomedical Applications

The aim of this thesis is to develop Surface Plasmon Resonance (SPR) methods that improve biosensing performance, in particular the sensitivity and selectivity of the analysis and their adaptation for biomedical applications to samples with complex background. This is of significant importance in translating the biosensor technologies to deliver the same results as the conventional methods but in a more accessible, efficient, and economical manner. The first study exploited SPR as a DNA biosensor for the detection of Malaria Plasmodium falciparum parasite with the hybridization chain reaction (HCR), which resulted in the formation of self-assembled target DNA nanostructures for signal enhancement. The sensitivity was further improved by using gold nanoparticles (AuNPs) for additional signal amplification. Tests with human blood plasma indicated the results were comparable to analyses in buffer, despite noticeable non-specific binding from the plasma. The concern of non-specific binding was systematically investigated in the second study where an antifouling surface consists of supported lipid bilayer membranes (SLBs) and protein A was developed for detection of trace amount of proteins in undiluted human serum. Specifically, cholera toxin (CT) spiked into the serum was used as the target, and advanced interface was further extended to immunosensing of immunoglobulin G (IgG). In the third study, SPR biosensor was employed in combination of bright field microscopy to characterize cellular apoptosis. HeLa cells undergoing apoptosis induced by hydrogen peroxide (H2O2) were monitored by SPR and the signals were compared to microscopic analysis of the morphological changes. SPR study revealed a decreased signal as cell confluency decreases, with the rates increasing as H2O2 concentration increases. An abnormality was found at high concentrations when both apoptosis and necrosis were induced. A mathematical model was proposed to explain SPR response where a non- uniform adsorbed layer was partially responsible. The significance of this thesis is that a number of high performing biosensing approaches have been developed and demonstrated. In addition to the advantages of SPR (e.g. label-free, real-time biomolecules binding, and portability), these methods have paved the way towards realizing effective sensing in biomedical research, especially in the early detection of infectious diseases and in the treatment of cancers

Desenvolvimento de biossensor para detec??o direta de Flavivirus utilizando nanobast?es de ouro.

Programa de P?s-Gradua??o em Biotecnologia. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.A co-circula??o de Dengue virus, Zika virus, Yellow fever virus, Chikungunia virus e Mayaro virus tem causado grande impacto na sa?de p?blica brasileira e mundial. Os programas de controle e preven??o de arboviroses e estudos epidemiol?gicos poderiam ser muito beneficiados e aperfei?oados com a utiliza??o de metodologias de detec??o direta dos v?rus em mosquitos, pois possibilitaria uma vigil?ncia mais precisa nas popula??es de vetores ao longo do tempo. Por?m, os m?todos de diagn?sticos hoje dispon?veis, apesar de possu?rem alta sensibilidade e especificidade s?o, em sua maioria, onerosos, demandam treinamento espec?fico para aplica??o e interpreta??o e n?o provem um diagn?stico r?pido. Neste contexto, o desenvolvimento de novas tecnologias de biossensores que apresentem benef?cios quanto ao tempo de an?lise, sensibilidade e simplicidade de manipula??o t?m sido objeto de pesquisa nas esferas p?blica e privada. A partir deste fato, este trabalho prop?e desenvolver e analisar a efic?cia de metodologias de funcionaliza??o de nanobast?es de ouro (NBs) com anticorpos espec?ficos anti-DENV2 e anti-flavivirus para testar a capacidade de detec??o de liga??o anticorpo-part?cula viral. A detec??o de biomol?culas ligadas ?s nanopart?culas de ouro (AuNPs), tais como prote?nas e anticorpos, pode ser feita a partir da an?lise do seu espectro de absor??o. Para isso, nanobast?es de ouro foram sintetizados, funcionalizados a 4 mM de polietilenoimina, conjugados a 0,4 ?g/mL de anticorpos e incubados com 103 UFP/mL de v?rus. Os dados foram obtidos atrav?s da leitura da resson?ncia plasm?nica de superf?cie localizada em espectr?metro de varredura UV-Vis. A s?ntese dos nanobast?es, que utilizou o m?todo de crescimento com sementes com a utiliza??o de diferentes agentes redutores, mostrou-se reprodut?vel. Al?m disso, um deslocamento muito expressivo no pico de absor??o foi observado quando biossensores contendo anti-flavivirus foram incubados em solu??o com DENV2 e ZIKV, mas n?o foi observado ap?s incuba??o com MAYV, um Alphavirus utilizado como controle negativo. Como esperado, nos biossensores contendo anti-DENV2 o deslocamento foi observado apenas na presen?a de DENV2, demonstrando a especificidade dos anticorpos utilizados. Na presen?a de soro humano dilu?do 1:3200 e macerado de mosquito dilu?do 8x, contendo DENV2 ou ZIKV, o limite de detec??o estimado foi de 100 UFP/mL. Assim, sugere-se que os nanobast?es de ouro apresentam potencial para o desenvolvimento de novas metodologias de diagn?stico mais r?pidos, precisos e pr?ticos que as t?cnicas j? existentes e viabilizar estudos de vigil?ncia da virologia em mosquitos.The co-circulation of Dengue virus, Zika virus, Yellow fever virus, Chikungunia virus and Mayaro virus has a major impact on Brazilian and world?s public health. Arbovirus prevention and control programs and epidemiological studies can be greatly benefited and improved by the use of direct virus detection methodologies in mosquitoes, as would enable more accurate monitoring of vector populations over time. The diagnostic methods available today, despite having high sensitivity and specificity, are mostly costly, require specific training for application and interpretation and do not provide a rapid diagnosis. In this context, the development of new biosensor technologies that present benefits regarding the time of analysis, sensitivity and simplicity of manipulation have been the object of research in the public and private spheres. From this fact, this work proposes to develop and analyze the effectiveness of functionalization methodologies of gold nanorods with specific anti-DENV and anti-flavivirus antibodies to test the ability to detect binding antibodies-viral particle. The detection of biomolecules bound to gold nanoparticles (AuNPs), such as proteins and antibodies, can be done from analysis of the absorption spectrum. For this, gold nanorods (NRs) were synthesized, functionalized to 4 mM polyethyleneimine, conjugated to 0.4 ?g/mL of antibodies and incubated with 103 PFU/mL virus. The data were included by reading the surface plasmon resonance localized in the UV-Vis scanning spectrometer. The synthesis of nanorods, which use the seed-mediated method using different reducing agents, was reproducible. In addition, one of the most expressive peaks occurred when anti-flaviviruscontaining biosensors were incubated in solution with DENV2 and ZIKV but were not incubated with MAYV, an Alphavirus use as a negative control. As expected, in the biosensors containing anti-DENV2 the displacement was only in the presence of DENV2, demonstrating a specificity of the antibodies used. In the presence of a 1:3200 diluted human serum and 8x diluted mosquito maceration containing DENV2 or ZIKV, the estimated detection limit was 100 PFU/mL for both. Thus, the gold nanorods have the potential to develop new diagnostic methods, faster and more precious, than existing techniques and enable studies of virology vigilance in mosquitoes

Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications

Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application