Cardiac troponin T (cTnT) is considered a clinical standard for its high specificity and sensitivity when diagnosing acute myocardial infarction; however, most studies on the electrical sensors of ...cardiac troponin biomarkers have focused on cTnI rather than cTnT. This study presents label-free, low-cost, transparent, and flexible aptamer-based immunosensors for the electrical detection of cTnT using reduced graphene oxide (rGO) sheets. GO was first deposited by AC dielectrophoresis between two predefined source and drain electrodes on a 3-aminopropyltriethoxylsilane-modified polyethylene terephthalate substrate. The GO was then reduced using hydrazine vapour without damaging the substrate, resulting in uniform, controlled, and stable deposition of rGO sheets, and demonstrating more stability than those directly deposited by dielectrophoresis. Amine-modified single-strand DNA aptamers against cTnT were immobilized onto the rGO channels. The relative resistance change of this sensor owing to the attachment of cTnT was quantified as the cTnT concentration decreased from 10 ng/mL to 1 pg/mL in phosphate buffered saline (PBS) and 10-fold diluted human serum in PBS, with the limits of detection being 1.2 pg/mL and 1.7 pg/mL, respectively, which is sufficiently sensitive for clinical applications. High-yield and rapid fabrication of the present rGO sensors will have significant influences on scaled-up fabrication of graphene-based sensors.
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.
Display omitted
•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.
Electrostatic samplers have been increasingly studied for sampling of viral and bacterial aerosols, and bioaerosol samplers are required to provide concentrated liquid samples with high physical ...collection and biological recovery, which would be critical for rapid detection. Here, the effects of sampling media and protocols on the physical collection and biological recovery of two airborne bacteria (Pseudomonas fluorescens and Micrococcus luteus) under electrostatic field were investigated using a personal electrostatic particle concentrator (EPC). Deionized (DI) water with/without sodium dodecyl sulfate (SDS) and phosphate buffered saline were tested as sampling media. A polystyrene container was mounted onto the collection electrode of the EPC for stable storage and vortexing after capture. Many bacterial cells were found to be deposited on the bottom surface of the container submerged in the media via electrophoresis, and among the tested sampling protocols, wet sampling with a container and subsequent vortexing offered the most bacteria in the collection suspension. Experiments with several sampling media showed that 0.001-0.01% SDS-DI water demonstrated the highest recovery rate in the EPC. These findings would be valuable in the field of sampling and subsequent rapid detection of bioaerosols.
Display omitted
•A transparent CNT immunosensor is presented for detection of a prostate cancer biomarker osteopontin.•This immunosensor showed a highly linear and reproducible behavior from 1pgmL−1 ...to 1μgmL−1.•The limit of detection of the immunosensor was 0.3pgmL−1.•This immunosensor demonstrated high selectivity against bovine serum albumin and human serum.
Osteopontin (OPN) is involved in almost all steps of cancer development, and it is being investigated as a potential biomarker for a diagnosis and prognosis of prostate cancer. Here, we report a label-free, highly sensitive and transparent immunosensor based on single-walled carbon nanotubes (SWCNTs) for detection of OPN. A high density of COOH functionalized SWCNTs was deposited between two gold/indium tin oxide electrodes on a glass substrate by dielectrophoresis. Monoclonal antibodies specific to OPN were covalently immobilized on the SWCNTs. Relative resistance change of the immunosensors was measured as the concentration of OPN in phosphate buffer saline (PBS) and human serum was varied from 1pgmL−1 to 1μgmL−1 for different channel lengths of 2, 5, and 10μm, showing a highly linear and reproducible behavior (R2>97%). These immunosensors were also specific to OPN against another test protein, bovine serum albumin, PBS and human serum, showing that a limit of detection for OPN was 0.3pgmL−1. This highly sensitive and transparent immunosensor has a great potential as a simple point-of-care test kit for various protein biomarkers.
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.
Fine particulate matter (PM 2.5 ) is a major environmental health risk. Several instruments based on the quartz crystal microbalance (QCM) have been developed for PM 2.5 measurement because of their ...accurate, sensitive, real-time, and low-cost mass measurements. However, prolonged or non-uniform deposition on the quartz crystal can cause nonlinear responses between frequency shifts and mass deposition, and its frequent manual cleaning with wet sponges is required. These disable long-term measurements of the instruments, thus limiting their applications in remote areas. Herein, we present a new PM 2.5 instrument called qEPC-Snow. This instrument consists of a QCM crystal embedded in an electrostatic particle concentrator (EPC) for collection and sensing of PM 2.5 and a carbon dioxide aerosol (snow) jet unit for residue-free, rapid, effective, and non-destructive cleaning of the crystal. Laboratory tests were conducted with aerosolized 100-nm and 2-<inline-formula> <tex-math notation="LaTeX">\mu \text{m} </tex-math></inline-formula> polystyrene latex microspheres as PM 2.5 representatives to evaluate (i) frequency responses and (ii) mass sensitiveness of qEPC-Snow, (iii) particle removal efficiencies, and (iv) reuse of the used crystals. Experimental results demonstrated high removal efficiencies (approximately 99.9% for both particle sizes) and statistical similarity between the initial and cleaned QCM crystals in the frequency shift-mass deposition relationship, thereby enabling measurement for more than one month without demounting the crystals. The mass sensitivity was 57.34 (Hz/<inline-formula> <tex-math notation="LaTeX">\mu \text{g} </tex-math></inline-formula>) with R 2 = 0.9904, corresponding to 0.05667 (Hz/min)/(<inline-formula> <tex-math notation="LaTeX">\mu \text{g}/\text{m}^{3}) </tex-math></inline-formula> in mass concentration sensitivity for the PM 2.5 representatives. The influence of particle sizes on qEPC-Snow's frequency behaviors will also be discussed in detail.
PDF
