Microgravimetric immunosensor for direct detection of aerosolized influenza A virus particles.

The development and characterization of a quartz crystal microbalance (QCM) sensor for the direct detection of aerosolized influenza A virions is reported. Self-assembled monolayers (SAMs) of mercaptoundecanoic acid (MUA) are formed on QCM gold electrodes to provide a surface amenable for the immobilization of anti-influenza A antibodies using NHS/EDC coupling chemistry. The surface-bound antibody provides a selective and specific sensing interface for the capture of influenza virions. A nebulizer is used to create aerosolized samples and is directly connected to a chamber housing the antibody-modified crystal ("immunochip"). Upon exposure to the aerosolized virus, the interaction between the antibody and virus leads to a dampening of the oscillation frequency of the quartz crystal. The magnitude of frequency change is directly related to virus concentration. Control experiments using aerosols from chicken egg allantoic fluid and an anti-murine antibody based immunosensor confirm that the observed signal originates from specific viral binding on the chip surface. Step-by-step surface modification of MUA assembly, antibody attachment, and antibody-virus interaction are characterized by atomic force microscopy (AFM) imaging analysis. Using the S/N = 3 principle, the limit of detection is estimated to be 4 virus particles/mL. The high sensitivity and real-time sensing scheme presented here can play an important role in the public health arena by offering a new analytical tool for identifying bio-contaminated areas and assisting in timely patient diagnosis

QCM Aptasensor for Rapid and Specific Detection of Avian Influenza Virus

There has been a need for rapid detection of avian influenza virus (AIV) H5N1 due to it being a potential pandemic threat. Most of the current methods, including culture isolation and PCR, are very sensitive and specific but require specialized laboratories and trained personnel in order to complete the tests and are time-consuming. The goal of this study was to design a biosensor that would be able to rapidly detect AIV H5N1 using aptamers as biosensing material and a quartz crystal microbalance (QCM) for transducing method. Specific DNA aptamers against AIV H5N1 were immobilized, through biotin and streptavidin conjugation, onto the gold surface of QCM sensor to capture the target virus. Magnetic nanobeads (150 nm in diameter) were then added as amplifiers considering its large surface/volume ratio which allows for a higher target molecule binding rate and faster movement. The result showed that the captured AIV caused frequency change, and more change was observed when the AIV concentration increased. The nanobead amplification was effective at the lower concentrations of AIV; however, it was not significant when the AIV concentration was 1 HAU or higher. The detection limit of the aptasensor was 1 HAU with a detection time of 1 h. The capture of the target virus on to the surface of QCM sensor and the binding of magnetic nanobeads with the virus was confirmed with electron microscopy. Aptamers have unlimited shelf life and are temperature stable which allows this aptasensor to give much more consistent results specifically for in field applications

Aptamer-based SPR Biosensor for Detection of Avian Influenza Virus

Rapid and specific detection of avian influenza (AI) virus is urgently needed with the concerns over the outbreaks of highly pathogenic H5N1 avian influenza in animal and human infection. Aptamers are artificial oligonucleic acid that can specifically bind to target molecules. They show comparable affinity for target virus and better thermal stability than monoclonal antibodies. Those advantages make aptamers promising candidates in diagnostic and detection applications. The goal of this research was to use DNA&ndashaptamer as the specific recognition element in a portable surface plasmon resonance (SPR) biosensor for detection of AI H5N1 virus in poultry. A SPR biosensor was fabricated using the selected aptamers based on streptavidin&ndashbiotin method. Streptavidin was directly adsorbed onto the surface of a gold waveguide in the SPR biosensor. Then, biotinylated aptamers were immobilized on the sensor surface via streptavidin&ndashbiotin binding. The immobilized aptamers captured AI H5N1 virus in a sample solution, causing an increase in refraction index (RI). Performances of the aptamer&ndashbased SPR biosensor were studied in streptavidin modification, aptamer immobilization and virus detection. The optimal concentrations of streptavidin and aptamers were determined to improve the sensitivity of the biosensor. The response of the aptamer&ndashvirus interaction was shown to be virus titer&ndashdependent, and a linear range for the titers of AI H5N1 was found between 0.128 and 1.28 HA unit. The aptamer&ndashbased SPR biosensor could detect the H5N1 virus at a titer greater than 0.128 HA unit within 1.5 h. No significant interference was observed from non&ndashtarget subtypes such as AI H7N2, H9N2, H2N2, H1N1 and H5N2. The aptamer&ndashbased SPR biosensor was further evaluated for detection of AI virus in poultry swab samples. All of the AI viruses used in this study were killed ones to ensure biological safety

Biosensors for Rapid Detection of Avian Influenza

The scope of this chapter was to review the advancements made in the area of biosensors for rapid detection of avian influenza viruses (AIVs). It is intended to provide general background about biosensor technology and to discuss important aspects for developing biosensors, such as selection of the suitable biological recognition elements (anti-AIV bioreceptors) as well as their immobilization strategies. A major concern of this chapter is also to critically review the biosensors’ working principles and their applications in AIV detection. A table containing the types of biosensor, bioreceptors, target AIVs, methods, etc. is given in this chapter. A number of papers for the different types of biosensors give hints on the current trends in the field of biosensor research for its application on AIV detection. By discussing recent research and future trends based on many excellent publications and reviews, it is hoped to give the readers a comprehensive view on this fast-growing field

Development and evaluation of QCM sensors for the detection of influenza virus from clinical samples

The Quartz crystal microbalance (QCM) is a very sensitive mass-detecting device which is based on changes in to the vibrational frequency of quartz crystals after adsorption of substances to a modified crystal surface. In this study a QCM-based biosensor was developed for the rapid diagnosis of influenza viruses and its suitability and limitations were compared with currently available diagnostic methods on 67 clinical samples (nasal washes) received during the 2005 Australian winter. The type-specific and conserved viral M1 proteins of both A/PR/8/34 and B/Lee/40 viruses were used to prepare polyclonal antisera for the development of an ELISA. The limits of detection of ELISAs for the detection of purified A/PR/8/34 and B/Lee/40 ƒnviruses were 20ƒÝg/mL ƒnand 14 ƒÝg/mL using polyclonal antibodies, and 30 ƒÝg/mL and 20 ƒÝg/mL for monoclonal antibodies, respectively. The limit for detection of each virus was 104 pfu/mL, irrespective of whether antisera or monoclonal antibodies were used for capture. Non-purified cell culture-grown preparations of either virus could be detected at 103 pfu/mL The QCM utilised the same reagents used in ELISAs. However, a number of parameters were then further optimised to improve the sensitivity of the tests. These included blocking of non-specific binding, examination of the effects of flow-cell compression, the role of pH, flow rate, antibody concentration and the addition of protein A to the crystal surfaces of the biosensor. The lowest virus concentration that could be detected with the QCM was 104 pfu/mL for egg-grown preparations of both A/PR/8/34 and B/Lee/40, which could be detected within 30 min. However, conjugation of 13 nm gold nanoparticles to a second detector antibody resulted in a 10-fold increase in sensitivity and a detection limit of 103 pfu/mL that could be determined within 1 h. The direct detection of the influenza viruses in nasal samples was not possible by QCM because of the significant frequency fluctuation that was probably caused by the viscosity of the samples. Therefore, an additional culture step of 12 h was required, which increased the processing time to 2 days. The QCM/nanoparticle method was shown to be as sensitive as the standard cell culture method, and the QCM method as sensitive as the shell vial method. The QCM and QCM/nanoparticle methods were shown to be 81 and 87% as sensitive and both were 100% as specific as the real-time polymerase chain reaction. However, for use in rapid diagnosis, improvements are required to remove frequency fluctuation resulting from the direct use of nasal samples

Advanced biosensors for detection of pathogens related to livestock and poultry

Infectious animal diseases caused by pathogenic microorganisms such as bacteria and viruses threaten the health and well-being of wildlife, livestock, and human populations, limit productivity and increase significantly economic losses to each sector. The pathogen detection is an important step for the diagnostics, successful treatment of animal infection diseases and control management in farms and field conditions. Current techniques employed to diagnose pathogens in livestock and poultry include classical plate-based methods and conventional biochemical methods as enzyme-linked immunosorbent assays (ELISA). These methods are time-consuming and frequently incapable to distinguish between low and highly pathogenic strains. Molecular techniques such as polymerase chain reaction (PCR) and real time PCR (RT-PCR) have also been proposed to be used to diagnose and identify relevant infectious disease in animals. However these DNA-based methodologies need isolated genetic materials and sophisticated instruments, being not suitable for in field analysis. Consequently, there is strong interest for developing new swift point-of-care biosensing systems for early detection of animal diseases with high sensitivity and specificity. In this review, we provide an overview of the innovative biosensing systems that can be applied for livestock pathogen detection. Different sensing strategies based on DNA receptors, glycan, aptamers and antibodies are presented. Besides devices still at development level some are validated according to standards of the World Organization for Animal Health and are commercially available. Especially, paper-based platforms proposed as an affordable, rapid and easy to perform sensing systems for implementation in field condition are included in this review

Impedance Biosensors for the Rapid Detection of Viral and Bacterial Pathogens Using Avian Influenza Virus Subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as Model Targets

This research investigated impedance biosensors for the rapid detection of viral and bacterial pathogens using avian influenza virus (AIV) subtypes H5N1 and H7N2 and Escherichia coli O157:H7 as the model targets, which were chosen due to their impact on the agricultural and food industries. For the detection of AIV H7N2, a single stranded DNA aptamer was selected using systematic evolution of ligands by exponential enrichment (SELEX). The selected aptamer and a previously selected aptamer against AIV H5N1 were used in a microfluidics chip with an embedded interdigitated array microelectrode to fabricate an impedance biosensor for specific detection of AIV H7N2 and H5N1. The developed label-free biosensor was capable of detecting AIV H7N2 and H5N1 at a concentration down to 27×10-4 hemagglutinination units (HAU) in 30 min without sample pre-treatment, comparable to previously designed biosensors though with the advantage of DNA aptamers. Two impedance biosensors based on the use of screen-printed interdigitated electrodes were developed for the detection of E. coli O157:H7. The first was a label-free biosensor based on magnetic separation and concentration of target bacteria using antibody-labelled magnetic nanobeads and Faradic impedance measurement. It was capable of detecting 1400 cells or more of E. coli O157:H7 in a total detection time of 1 h. COMSOL Multiphysics software was used to analyze the biosensor using a simplified model and determine the role of the magnetic nanobeads in the impedance measurement. The second biosensor for detection of E. coli O157:H7 was based on aptamer-labeled magnetic nanobeads and glucose oxidase/Concanavalin A-coated gold nanoparticle labels. This biosensor was capable of detecting 8 cells or more of E. coli O157:H7 in 1.5 h. The lower detection limit of the developed impedance biosensor was comparable to the most sensitive biosensors published for the detection of E. coli O157:H7 and was also more rapid and more practical for in-field tests. Multiple impedance biosensor designs were developed in this research. The developed biosensor for AIV could conceivably be adapted for detection of other AIV subtypes and the developed E. coli O157:H7 biosensors could easily be adapted to detect different bacterial pathogens

Biosensors: A Fast-Growing Technology for Pathogen Detection in Agriculture and Food Sector

Agriculture and food have a greater role to play in order to achieve sustainable development goals. Therefore, there is a need to put an end to the effect of pathogens on food quality and safety. Pathogens have been recognized as one of the major factors causing a reduction in profitable food production. The conventional methods of detecting pathogens are time-consuming and expensive for the farmers in rural areas. In view of this, this chapter reviews the biosensors that have been developed for the detection of biological hazards in food and agricultural sectors. This chapter also lays emphasis on the impact of nanotechnology on building a fast, reliable, more sensitive, accessible, user-friendly and easily adaptable technology for illiterate farmers in the rural communities. On the whole, we have addressed the past and most recent biosensors that could ensure the quick delivery of vision 2030 which aims to end hunger and poverty