An impedimetric micro-immunosensing assay to detect Alzheimer's disease biomarker: Aß40.

A miniaturized biosensing platform, based on monoclonal amyloid-beta antibodies (mAßab) that were immobilized on a disc-shaped platinum/iridium (Pt/Ir) microelectrode surface coupled with an impedimetric signal transducer, was developed for the label-free and sensitive detection of amyloid-beta peptide fragment 1-40 (Aß40); a reliable biomarker for early diagnosis of Alzheimer's disease (AD). A Pt/Ir microelectrode was electropolymerized with poly (ortho-phenylenediamine), a conducting free amine-containing aromatic polymer; followed by crosslinking with glutaraldehyde for subsequent coupling of mAßab on the microelectrode surface. This modification strategy efficiently improved the impedimetric detection performance of Aß40 in terms of charge transfer resistance (∼400-fold difference) and normalized impedance magnitude percentage change (∼40% increase) compared with a passive adsorption-based immobilization method. The sensitivity of the micro-immunosensing assay was found to be 1056 kΩ/(pg/mL)/cm2 and the limit of detection was found to be 4.81 pg/mL with a dynamic range of 1-104 pg/mL (R2 = 0.9932). The overall precision of the assay, as measured by relative standard deviation, ranged from 0.84 to 5.15%, demonstrating its reliability and accuracy; while in respect to assay durability and stability, the immobilized mAßab were able to maintain 80% of their binding activity to Aß40 after incubation for 48 h at ambient temperature (25 °C). To validate the practical applicability, the assay was tested using brain tissue lysates prepared from AD-induced rats. Results indicate that the proposed impedimetric micro-immunosensing platform is highly versatile and adaptable for the quantitative detection of other disease-related biomarkers.

A voltammetric immunosensor based on a nanoporous alumina millirod for detection of porcine serum albumin.

A voltammetric immunosensor was developed for detection of porcine serum albumin (PSA) to identify raw meat products adulterated with pork. A novel strategy to fabricate multiple individual nanoporous alumina (NPA) millirods (length, 5.0 mm; diameter, 1.0 mm) as the biorecognition platform is described. Each NPA millirod was covalently bioconjugated with anti-PSA capturing antibodies (α-PSAC). Following immunocapture, the PSA bound to the α-PSAC/NPA millirod bioconjugate were tagged with gold nanoparticles (AuNPs) functionalized with anti-PSA detection antibodies as the signaling probe. Subsequently, the AuNPs were voltammetrically analyzed to quantify the target PSA. The immunosensor exhibited 100 % specificity and high sensitivity to PSA with a limit of detection (LoD) of 50 (range, 0-1000) pg/mL (R2 = 0.9907). Real-world applicability was successfully validated using pork/beef adulterated mixtures with a LoD of 0.05 % (w/w). Overall, the detection performance of the proposed immunosensor was excellent and, thus, is suitable for surveillance of food safety and quality.

Electrochemical genosensor for specific detection of the food-borne pathogen, Vibrio cholerae.

A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/µl of V. cholerae genomic DNA.

Synthesis of Various Size Gold Nanoparticles by Chemical Reduction Method with Different Solvent Polarity.

Complicated and strict protocols are followed to tune the size of gold nanoparticles (GNPs) in chemical synthesis methods. In this study, we address the polarity of solvents as a tool for tailoring the size of GNPs in the chemical reduction method. The effects of varying polarity index of the reaction medium on synthesizing gold nanoparticles by chemical reduction method have been investigated. Ethanol as a polar solvent, ethanol-water mixture as reaction medium, L-ascorbic acid as reducing agent, and polyvinylpyrrolidone as stabilizer were used to synthesize GNPs. The polarity index of the reaction medium was adjusted by changing the volume ratio of ethanol to water. UV-Vis, dynamic light scattering (DLS), and transmission electron microscopy (TEM) characterizations reveal that the growth of nanoparticles was gradually increased (~ 22 to 219 nm hydrodynamic diameter) with decreasing value of polarity index of the reaction medium (~ 8.2 to 5.2). Furthermore, the high polarity index of the reaction medium produced smaller and spherical nanoparticles, whereas lower polarity index of reaction medium results in bigger size of GNPs with different shapes. These results imply that the mechanistic of the growth, assembly, and aggregation phenomena of ligand or stabilizer-capped GNPs strongly rely on the polarity of solvent molecules. Using the proposed methodology, wide size range of GNPs with different morphology sizes can be synthesized by simply modulating the volume percentage of organic solvent in the reaction medium.

A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents.

A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R2=0.992) and log10 (1-104 CFU/ml) (R2=0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens.

An Electrochemical Genosensing Assay Based on Magnetic Beads and Gold Nanoparticle-Loaded Latex Microspheres for Vibrio cholerae Detection.

An ultrasensitive electrochemical genosensing assay was developed for the sequence-specific detection of Vibrio cholerae DNA using magnetic beads as the biorecognition surface and gold nanoparticle-loaded latex microspheres (latex-AuNPs) as a signal-amplified hybridization tag. This biorecognition surface was prepared by immobilizing specific biotinylated capturing probes onto the streptavidin-coupled magnetic beads. Fabricating a hybridization tag capable of amplifying the electrochemical signal involved loading multiple AuNPs onto polyelectrolyte multilayer film-coated poly(styrene-co-acrylic acid) latex microspheres as carrier particles. The detection targets, single-stranded 224-bp asymmetric PCR amplicons of the V. cholerae lolB gene, were sandwich-hybridized to magnetic bead-functionalized capturing probes and fluorescein-labeled detection probes and tagged with latex-AuNPs. The subsequent electrochemical stripping analysis of chemically dissolved AuNPs loaded onto the latex microspheres allowed for the quantification of the target amplicons. The high-loading capacity of the AuNPs on the latex microspheres for sandwich-type dual-hybridization genosensing provided eminent signal amplification. The genosensing variables were optimized, and the assay specificity was demonstrated. The clinical applicability of the assay was evaluated using spiked stool specimens. The current signal responded linearly to the different V. cholerae concentrations spiked into stool specimens with a detection limit of 2 colony-forming units (CFU)/ml. The proposed latex-AuNP-based magnetogenosensing platform is promising, exhibits an effective amplification performance, and offers new opportunities for the ultrasensitive detection of other microbial pathogens.

A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae.

Vibrio cholerae is a human pathogen that causes mild to severe diarrheal illnesses and has major public health significance. Herein, we present a thermostabilized electrochemical genosensing assay combining the use of magnetic beads as a biorecognition platform and gold nanoparticles as a hybridization tag for the detection and quantification of V. cholerae lolB gene single-stranded asymmetric PCR amplicons as an alternative to the time-consuming classical isolation method. This thermostabilized, pre-mixed, pre-aliquoted and ready-to-use magnetogenosensing assay simplified the procedures and permitted the reaction to be conducted at room temperature. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. The real-time stability evaluation of thermostabilized assay was found to be stable for at least 180 days at room temperature (25-30°C). The analytical specificity of the assay was 100%, while its analytical sensitivity was linearly related to different concentrations of 200-mer synthetic target, purified genomic DNA, and bacterial culture with a limit of detection (LoD) of 3.9nM, 5pg/µl, and 10(3)CFU/ml, respectively. The clinical applicability of the assay was successfully validated using spiked stool samples with an average current signal-to-cut-off ratio of 10.8. Overall, the precision of the assay via relative standard deviation was <10%, demonstrating its reliability and accuracy.