Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, ...glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-μl samples (concentration: 100 pg/ml-100 ng/ml) were needed to form the antigen-antibody complexes during 20-30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses.
•A sensitive paper based electrochemical sensor for label-free detection of bacteria in food samples is presented.•Ab-SWCNT conjugate was used to decrease the functionalization steps and increase the ...robustness of the immunosensors.•A detection limit of 13 CFUmL-1 in spiked milk samples was obtained with rapid detection time (∼30min).•A highly linear behavior and high specificity were observed.
The need for low-cost, sensitive, and reliable sensors for the detection of whole bacterial cells in food samples without pre-treatment has been increasing. Outbreaks of foodborne diseases can be severe, especially in developing countries; however, most bio-detection tools are unaffordable. Here, we have developed a rapid and low-cost paper-based electrochemical immunosensor for label-free detection of Staphylococcus aureus, using antibody (Ab)-single walled carbon nanotube (SWCNT) bio-conjugates. Anti-S. aureus antibodies were covalently attached onto the SWCNTs, using the N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide coupling reagent. These Ab-SWCNT bio-conjugates were then immobilized on the working electrode, and the presence of S. aureus was detected by analyzing the change in peak current following antigen-antibody complex formation. Differential pulse voltammetry was performed with a bacterial concentration ranging from 10 to 107 colony forming units (CFU) mL−1. A selectivity assay using Escherichia coli B, Bacillus subtilis, and S. epidermidis (to examine cross-reactivity) showed that the sensor was specific to S. aureus. Moreover, this immunosensor showed a good linear relationship (R2=0.976) between the increase in peak current and logarithmic S. aureus concentration, with a rapid detection time (30min) and a limit of detection of 13CFUmL−1 in spiked milk samples. This low-cost immunosensor can be used for rapid detection of pathogens in actual food samples with high sensitivity and specificity.
We present a novel vertical flow-based paper immunosensor for the rapid and sensitive electrochemical and colorimetric detection of influenza H1N1 viruses using a different pore size (DP) sample pad. ...The DP sample pad consisted of two different pore size papers: larger pores (diameter: 11 µm) facing the inlet, and smaller pores (diameter: 0.45 µm) facing the conjugate pad. This sample pad offered moderate and uniform flows, and hence concentrated horseradish peroxidase tagged antibodies (HRP-Abs)-H1N1 complexes from 40 µL of sample volumes on a conjugate pad for 2 min after sample injection, thereby providing fast detection (6 min for both detection methods) with 100 µL of flushing afterwards, high sensitivity, and the simplicity of the sensor. The filtration characteristics of the DP sample pad were evaluated using fluorescent beads, indicating that only small-sized bio-particles such as viruses can pass through the sample pad. Sandwich immunoreactions of HRP-Ab-H1N1-Ab were performed on the gold paper electrode of the immunoStrip, which was determined by electrochemical impedance spectroscopy (EIS) measurements. Simultaneously, the color signal of free HRP-Ab captured on the colorimetric zone was obtained using a scanner, and the intensity was analyzed using ImageJ. This immunosensor detected H1N1 virus concentration as low as 3.3 plaque forming units (PFU)/mL (phosphate buffer saline; PBS) and 4.7 PFU/mL (saliva) by EIS, and 1.34 PFU/mL (PBS) and 2.27 PFU/mL (saliva) by the colorimetric method. Furthermore, integrating these two detection methods can reduce false results with double assurance, and this device can provide a simple and economical on-site detection platform.
•The first paper immunosensor using a double pore size (DP) sample pad.•The first vertical flow assay-based, sandwich ELISA-type paper immunosensor for the detection of influenza viruses.•Dual (electrochemical and colorimetric) detection methods to provide double assurance.•The DP sample pad offered fast detection (~6 min), high sensitivity (the limit of detection <5 PFU/mL), and the simplicity.•The DP sample pad also acted as a filter to pass only small-sized particles.
Rapid subtyping of influenza viruses in clinical laboratories has been increasingly important because three subtypes (seasonal H1N1, H3N2, and 2009 H1N1) of influenza A virus currently disseminated ...in humans have variable susceptibilities to antiviral drug. Herein, we present DNA aptamers for selective detection of influenza A H1N1 (seasonal and 2009 pandemic H1N1) viruses by targeting recombinant influenza A mini-hemagglutinin (mini-HA) protein (the stable stem region of HA) and whole H1N1 viruses. The dissociation constants (KD) of aptamer candidates V46 and V57 were 19.2 nM and 29.6 nM, respectively, according to electrochemical characterization (differential pulse voltammetry), demonstrating strong binding to mini-HA. In comparison, the KD of the influenza virus antibodies is in the range of 1 μM–10 nM. Aptamer V46 showed higher specificity and binding affinity to the mini-HA protein and H1N1 subtypes, and it was also incorporated into an indium tin oxide-based electrochemical sensor, showing sensitive and specific detection of H1N1 viruses, with a limit of detection (LOD) of 3.7 plaque-forming units per mL. The binding affinity, specificity, and LOD achieved with the electrochemical sensor suggest that it can be used for rapid subtyping of H1N1. We also propose that this aptamer can be used for the neutralization of H1N1 subtypes, suggesting potential therapeutic and diagnostic applications.
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•An ssDNA aptamer selected for subtyping of influenza A H1N1 viruses via SELEX is reported for the first time.•Aptamers that recognize H1N1 subtypes alone were selected using mini HA protein and whole H1N1 viruses.•The dissociation constant of 19.2 nM was obtained for aptamer V46.•The electrochemical sensor based on the DNA aptamer distinguished H1N1 subtypes from other subtypes of influenza A virus.
Although many studies concerning the detection of influenza virus have been published, a paper-based, label-free electrochemical immunosensor has never been reported. Here, we present a ...cost-effective, handmade paper-based immunosensor for label-free electrochemical detection of influenza virus H1N1. This immunosensor was prepared by modifying paper with a spray of hydrophobic silica nanoparticles, and using stencil-printed electrodes. We used a glass vaporizer to spray the hydrophobic silica nanoparticles onto the paper, rendering it super-hydrophobic. The super-hydrophobicity, which is essential for this paper-based biosensor, was achieved via 30-40 spray coatings, corresponding to a 0.39-0.41 mg cm
coating of nanoparticles on the paper and yielding a water contact angle of 150° ± 1°. Stencil-printed carbon electrodes modified with single-walled carbon nanotubes and chitosan were employed to increase the sensitivity of the sensor, and the antibodies were immobilized via glutaraldehyde cross-linking. Differential pulse voltammetry was used to assess the sensitivity of the sensors at various virus concentrations, ranging from 10 to 10⁴ PFU mL
, and the selectivity was assessed against MS2 bacteriophages and the influenza B viruses. These immunosensors showed good linear behaviors, improved detection times (30 min), and selectivity for the H1N1 virus with a limit of detection of 113 PFU mL
, which is sufficiently sensitive for rapid on-site diagnosis. The simple and inexpensive methodologies developed in this study have great potential to be used for the development of a low-cost and disposable immunosensor for detection of pathogenic microorganisms, especially in developing countries.
Acute myocardial infarction (AMI), also recognized as a “heart attack,” is one leading cause of death globally, and cardiac myoglobin (cMb), an important cardiac biomarker, is used for the early ...assessment of AMI. This paper presents an ultrasensitive, label-free electrochemical aptamer-based sensor (aptasensor) for cMb detection using polyethylenimine (PEI)-functionalized reduced graphene oxide (PEI–rGO) thin films. PEI, a cationic polymer, was used as a reducing agent for graphene oxide (GO), providing highly positive charges on the rGO surface and allowing direct immobilization of negatively charged single-strand DNA aptamers against cMb via electrostatic interaction without any linker or coupling chemistry. The presence of cMb was detected on Mb aptamer-modified electrodes using differential pulse voltammetry via measuring the current change due to the direct electron transfer between the electrodes and cMb proteins (Fe3+/Fe2+). The limits of detection were 0.97 pg mL–1 (phosphate-buffered saline) and 2.1 pg mL–1 (10-fold-diluted human serum), with a linear behavior with logarithmic cMb concentration. The specificity and reproducibility of the aptasensors were also examined. This electrochemical aptasensor using polymer-modified rGO shows potential for the early assessment of cMb in point-of-care testing applications.
Small RNAs (sRNAs) are 18-30 nt non-coding regulatory elements found in diverse organisms, which were initially identified as small double-stranded RNAs in Caenorhabditis elegans. With the ...development of new and improved technologies, sRNAs have also been identified and characterized in plant systems. Among them, micro RNAs (miRNAs) and small interfering RNAs (siRNAs) are found to be very important riboregulators in plants. Various types of sRNAs differ in their mode of biogenesis and in their function of gene regulation. sRNAs are involved in gene regulation at both transcriptional and post-transcriptional levels. They are known to regulate growth and development of plants. Furthermore, sRNAs especially plant miRNAs have been found to be involved in various stress responses, such as oxidative, mineral nutrient deficiency, dehydration, and even mechanical stimulus. Therefore, in the present review, we focus on the current understanding of biogenesis and regulatory mechanisms of plant sRNAs and their responses to various abiotic stresses.
Measurements of airborne viruses via sampling have been critical issues. Most electrostatic samplers have been assessed for bacterial aerosols or micrometer-sized viral particles; however, sampling ...of submicrometer-sized airborne viruses is necessary, especially because of the high probability of their staying airborne and their deposition in the lower respiratory tract. Here, we present a novel personal electrostatic particle concentrator (EPC) for gentle sampling of submicrometer airborne virus particles. Owing to the enhanced electric field designed in this EPC, the collection efficiencies reached values as high as 99.3-99.8% for 0.05-2 μm diameter polystyrene particles at a flow rate of 1.2 L/min. Submicrometer-sized MS2 and T3 virus particles were also collected in the EPC, and the concentrations relative to their respective initial suspensions were more than 10 times higher than those in the SKC BioSampler. Moreover, the recovery rate of T3 was 982 times higher in the EPC (-2 kV) than in the BioSampler at 12.5 L/min because of the gentle sampling of the EPC. Gentle sampling is desirable because many bioaerosols suffer from significant viability losses during sampling. The influence of ozone generated, applied electrostatic field, and the flow rate on the viability of the viruses will also be discussed.
Malaria is one of the most life-threatening infectious diseases worldwide. Immunity to malaria is slow and short-lived despite the repeated parasite exposure in endemic areas. Malaria parasites have ...evolved refined machinery to evade the immune system based on a range of genetic changes that include allelic variation, biomolecular exposure of proteins, and intracellular replication. All of these features increase the probability of survival in both mosquitoes and the vertebrate host.
species escape from the first immunological trap in its invertebrate vector host, the
mosquitoes. The parasites have to pass through various immunological barriers within the mosquito such as anti-microbial molecules and the mosquito microbiota in order to achieve successful transmission to the vertebrate host. Within these hosts,
species employ various immune evasion strategies during different life cycle stages. Parasite persistence against the vertebrate immune response depends on the balance among virulence factors, pathology, metabolic cost of the host immune response, and the parasites ability to evade the immune response. In this review we discuss the strategies that
parasites use to avoid the vertebrate host immune system and how they promote successful infection and transmission.
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