Automated Paper-Based Femtogram Sensing Device for Competitive Enzyme-Linked Immunosorbent Assay of Aflatoxin B1 Using Submicroliter Samples.

Microfluidic paper-based analytical devices (µPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A µPAD for automating the competitive enzyme-linked immunosorbent assay (ELISA) of small-sized target detection at the femtogram level using submicroliter samples is reported in this study. The proposed µPAD was integrated with a sucrose valve to automate the sequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location at the zones on the device that had been prepared with an antibody-conjugated enzyme before immersion in a running buffer allowed minimization of sample volume to 0.6 µL, while eliminating the possible loss of a target molecule by adsorption on the membrane, thus improving detection sensitivity. Furthermore, the proposed device was successfully applied to the automation of competitive ELISA for the detection of aflatoxin B1 (AFB1), a potent carcinogen that causes substantial health risks to humans worldwide, with a detection limit of 60 femtograms or 0.1 ng/mL. The method developed in this study provides high sensitivity, small sample volume, on-site and equipment-free measurements, low-cost operation, and user-friendliness. This approach could be used to analyze small-sized molecules in the fields of food safety and quality control, environmental monitoring, and clinical diagnostics.

Black tea polyphenol theaflavin as promising antioxidant and potential copper chelator.

BACKGROUND: In this work, we investigated the antioxidant and copper chelating abilities of theaflavin, a polyphenol responsible for astringency, color, and sensation in black tea. Using voltammetric techniques, the analyses were conducted with disposable electrochemical printed carbon chips in conjunction with a portable hand-held potentiostat. RESULTS: Voltammograms of theaflavin showed five separate oxidation peaks, corresponding to the oxidation of five individual functional groups. Electroanalytical data indicated that, after interaction with copper, theaflavin had higher antioxidant potential and was a better copper chelator than epigallocatechin gallate, a major polyphenol present in green tea and a well-known antioxidant. This could be attributed to the extra fused ring and larger number of OH groups in theaflavin. CONCLUSIONS: Our findings introduce another natural compound as a potential nutraceutical in oxidation- and copper-modulated illnesses. This simple and fast approach would also be highly pertinent to the inspection of the health benefits of natural food products. To the best of our knowledge, this is the first report of the electrochemical analysis of Cu (II) chelation with theaflavin. © 2020 Society of Chemical Industry.

Design of dual working electrodes for concentration process in metalloimmunoassay.

Electrochemical immunosensing, particularly through a metalloimmunoassay, is a promising approach for development of point-of-care (POC) diagnostics devices. This study investigated the structure of dual working electrodes (W1 and W2), used in a silver nanoparticles-labeled sandwich-type immunoassay and silver concentration process, paying special attention to the position of W1 relative to W2. The new structures of the dual working electrodes were fabricated for efficient silver concentration and evaluated experimentally, which showed that the duration of prereduction before current measurement decreased from 480 s to 300 s by transforming the position of W1 from 1 line to 2 lines or 6 parts. The experimental results were also compared with numerical simulations based on three-dimensional diffusion, and the prereduction step almost followed the three-dimensional diffusion equation. Using numerical simulations, the ideal structures of dual working electrodes were designed based on relationships between the structures and duration of prereduction or the LOD. In the case of 36 lines at an area ratio of W1 to W1 + W2 of 1 to 10, the prereduction duration decreased to 96 s. The dual working electrodes designed in this study promise to shorten the total analysis time and lower the LOD for POC diagnostics.

Sensing technique of silver nanoparticles as labels for immunoassay using liquid electrode plasma atomic emission spectrometry.

We report the use of liquid electrode plasma-atomic emission spectrometry (LEP-AES) in protein sensing studies employing Ag nanoparticle labeling. LEP-AES requires no plasma gas and no high-power source and is suitable for onsite portable analysis. Human chorionic gonadotropin (hCG) was used as a model target protein, and the immunoreaction in which hCG is sandwiched between two antibodies, one of which is immobilized on the microwell and the second is labeled with Ag nanoparticles, was performed. Sensing occurs at the narrow pass in the center of a quartz chip following oxidative dissolution of the Ag nanoparticles by nitric acid. hCG was analyzed in the range from 10 pg/mL to 1 ng/mL, and the detection limit for hCG was estimated at 1.3 pg/mL (22.8 fM). The proposed detection method has a wide variety of promising applications in metal-nanoparticle-labeled biomolecule detection.

Estimation of maturity of compost from food wastes and agro-residues by multiple regression analysis.

The composting process of food wastes and tree cuttings was examined on four composting types composed from two kinds of systems and added mixture of microorganisms. The time courses of 32 parameters in each composting type were observed. The efficient composting system was found to be the static aerated reactor system in comparison with the turning pile one. Using the multiple regression analysis of all the data (159 samples) obtained from this study, some parameters were selected to predict the germination index (GI) value, which was adopted as a marker of compost maturity. For example, using the regression model generated from pH, NH(4)(+) concentration, acid phosphatase activity, and esterase activity of water extracts of the compost, GI value was expressed by the multi-linear regression equation (p<0.0001). High correlations between the measured GI value and the predicted one were made in each type of compost. As a result of these observations, the compost maturity might be predicted by only sensing of the water extract at the composting site without any requirements for a large-size equipment and skill, and this prediction system could contribute to the production of a stable compost in wide-spread use for the recycling market.

PEP-on-DEP: A competitive peptide-based disposable electrochemical aptasensor for renin diagnostics.

Antibody-based immunosensors are relatively less accessible to a wide variety of unreachable targets, such as low-molecular-weight biomarkers that represent a rich untapped source of disease-specific diagnostic information. Here, we present a peptide aptamer-based electrochemical sensor technology called 'PEP-on-DEP' to detect less accessible target molecules, such as renin, and to improve the quality of life. Peptide-based aptamers represent a relatively smart class of affinity binders and show great promise in biosensor development. Renin is involved in the regulation of arterial blood pressure and is an emerging biomarker protein for predicting cardiovascular risk and prognosis. To our knowledge, no studies have described aptamer molecules that can be used as new potent probes for renin. Here, we describe a portable electrochemical biosensor platform based on the newly identified peptide aptamer molecules for renin. We constructed a randomized octapeptide library pool with diversified sequences and selected renin specific peptide aptamers using cDNA display technology. We identified a few peptide aptamer sequences with a KD in the µM binding affinity range for renin. Next, we grafted the selected peptide aptamers onto gold nanoparticles and detected renin in a one-step competitive assay using our originally developed DEP (Disposable Electrochemical Printed) chip and a USB powered portable potentiostat system. We successfully detected renin in as little as 300ngmL(-1) using the PEP-on-DEP method. Thus, the generation and characterization of novel probes for unreachable target molecules by merging a newly identified peptide aptamer with electrochemical transduction allowed for the development of a more practical biosensor that, in principle, can be adapted to develop a portable, low-cost and mass-producible biosensor for point-of-care applications.

Effects of tamoxifen, 17α-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female Japanese medaka (Oryzias latipes).

The effects of oral administration of tamoxifen, 17α-ethynylestradiol (EE2), flutamide, and methyltestosterone (MT), on plasma vitellogenin levels of male and female medaka were investigated. Medaka were fed diets containing different concentrations of these chemicals for 7 days, and these plasma vitellogenin levels were measured. Tamoxifen increased significantly the vitellogenin levels in male, but inhibited the normal vitellogenin induction in female in the high concentration groups. EE2 increased significantly vitellogenin levels in both sexes. Flutamide increased significantly the vitellogenin levels in female, but gave no effects on male. MT inhibited the normal vitellogenin induction in female, but increased slightly vitellogenin levels in male without a clear tendency. Administration of tamoxifen, EE2, flutamide, and MT showed the different pattern in vitellogenin levels in both sexes.

Effect of alkylphenols on adult male medaka: plasma vitellogenin goes up to the level of estrous female.

The effect that oral administration of four alkylphenols, (1) bisphenol A (BPA), (2) p-t-octylphenol (OP), (3) p-nonylphenol (NP) and (4) p-n-nonylphenol (n-NP), as well as 17α-ethynylestradiol (EE2) had on male medaka fish vitellogenin was investigated. The male medaka was fed diets containing different concentrations of these chemicals for 7 days, after which their plasma vitellogenin levels were measured. Vitellogenin levels up to ≈10(7) ng/ml were found. This value is close to that of the normal estrous female medaka. The median effective concentration (EC(50)) values resulting from BPA, OP, NP and EE2 in the diet were calculated as 1600, 2600, 940 and 0.37 µg/g diet, respectively.

Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka (Oryzias latipes).

Effects of two widely found chemical pollutants, bis(2-ethylhexyl) phthalate (DEHP) and benzo[a]pyrene (BaP), on the embryos of Japanese medaka were investigated. The embryos were exposed to different concentrations (0.01, 0.1, 1, and 10µg/l) of DEHP and BaP. The following were investigated: (1) hatching time and hatching rate in embryos, (2) mortality, sex ratio, body weight and gonadosomatic index (GSI) in adulthood. These two chemicals delayed the hatching time without dose-dependence, but these chemicals had no effect on hatching rate. Mortality was raised and body weight was reduced by DEHP and BaP-treatment; distortion of sex ratio appeared at the lowest concentration of DEHP tested. GSI was decreased because of the BaP-treatment. DEHP and BaP negatively affected Japanease medaka embryos, and the influences of the effects continued into adult stage. Moreover, the effects did not appear to be necessarily dose-dependent.

Effects of bis(2-ethylhexyl) phthalate, γ-hexachlorocyclohexane, and 17ß-estradiol on the fry stage of medaka (Oryzias latipes).

The effects of bis(2-ethylhexyl) phthalate (DEHP), γ-hexachlorocyclohexane (γ-HCH), and 17ß-estradiol (E2) on the fry stage of medaka were investigated. The medaka fry were exposed to different concentrations (0.01, 0.1, 1, and 10µg/L) of these chemicals for 3 weeks after hatching. Then, mortality, body weight, sex ratio, and gonadosomatic index (GSI) of the matured fish (after 5 months) were measured. Mortality was increased significantly in the 10µg/L E2 group. Distortion of sex ratio was found in 1 and 10µg/L E2 groups. DEHP treated groups showed the GSI reduction only in male fish. All the γ-HCH and parts of the E2 treated groups showed the GSI reduction in both sexes. Exposure of DEHP, γ-HCH, and E2 during the fry stage affected normal maturation of medaka at the concentrations which had no impact on mortality or sex ratio.

Development of automated paper-based devices for sequential multistep sandwich enzyme-linked immunosorbent assays using inkjet printing.

To the best of our knowledge, this is the first report on paper-based devices for automating the sequential multistep procedures of a sandwich-type enzyme-linked immunosorbent assay (ELISA) that require only a single-step application of the sample solution. The device was based on a piece of nitrocellulose (NC) membrane with specially designed channels, where all the reagents are applied at different locations in order to control the fluid travel to the detection region. The inkjet printing method, a simple and low-cost process, was used to create the flow channel and device barrier patterns. The fabricated barrier was found to be an efficient boundary for the liquid along the printed design in the NC membrane, enabling direct control of the reagent flow time. ELISA results were obtained with a single-step sample application. The developed devices (so-called automated paper-based devices) provided a simple procedure for the sandwich ELISA, while reducing assay time and reagent consumption. Colorimetric results were measured using digital camera imaging with software processing. The capability of the method developed herein was successfully used to determine the levels of human chorionic gonadotropin (hCG) by ELISA.

Gold-linked electrochemical immunoassay on single-walled carbon nanotube for highly sensitive detection of human chorionic gonadotropin hormone.

A new sensitive gold-linked electrochemical immunoassay (GLEIA) for the detection of the pregnancy marker human chorionic gonadotropin (hCG) has been developed using the direct electrochemical detection of Au nanoparticles. We utilized single-walled carbon nanotube (SWCNT) microelectrodes; 24 SWCNT microelectrodes were arrayed on a single Si substrate 25×30 mm² in size, for the development of a new GLEIA (SWCNT-GLEIA). This SWCNT-GLEIA provided convenient and cost-effective tests with the required antibody and antigen sample volumes as small as 2.0 µL for a group of 4 SWCNT microelectrodes. In addition, this assay also exhibited properties of high sensitivity and selectivity benefitting from the intrinsic extraordinary features of SWCNTs. Using scanning electron microscopy, we also observed Au nanoparticle-labeled antigen-antibody complexes immobilized on the surface of the SWCNT microelectrodes. The concentration of the pregnancy marker (hCG) showed a linear relationship with the current intensity obtained from differential pulse voltammetry measurements with a limit of detection (LOD) of 2.4 pg/mL (0.024 mIU/mL) hCG. This LOD is 15 times more sensitive than a previous GLEIA, which used screen-printed carbon electrodes.

Fabrication of new single-walled carbon nanotubes microelectrode for electrochemical sensors application.

In this paper, we describe two simple different ways to fabricate an array of single-walled carbon nanotubes (SWCNT) microelectrodes from SWCNT network, grown on Si substrate, through micro-fabrication process. Two kinds of material, photoresist - organic compound and sputtered SiO(2), were used as an insulator layer for these arrays of SWCNT microelectrodes. The SWCNT microelectrodes were characterized by scanning electron microscopy (SEM), Raman spectroscopy, and electrochemical measurements. The SWCNT microelectrodes with sputtered SiO(2) as an insulator exhibit some prior advances to these used photoresist layer as insulator such as much stable in harsh condition (high active organic solvents) and high current density (24.94 µA mm(-2) compared to 2.69 µA mm(-2), respectively). In addition, the well-defined geometry of SWCNT microelectrodes is not only useful for investigating kinetics of electron transfer, but also promising candidate in electrochemical sensors application.

Labelless impedance immunosensor based on polypyrrole-pyrolecarboxylic acid copolymer for hCG detection.

In this work, a sensitive label-free impedimetric hCG-immunosensor was constructed by using a commercial screen-printing carbon ink electrode (namely disposable electrochemical printed chip) as the basis. The carbon ink electrode of DEP chip is modified first by deposition of polypyrrole-pyrole-2-carboxylic acid copolymer and thence hCG antibody immobilization via the COOH groups of pyrrole-2-carboxylic acid, which can serve as a linker for covalent biomolecular immobilization. The experimental results exposed that the designed immunosensor is more sensitive than other previously reported immunosensors, in the case of detection limit and linear range for antigen detection. With optimal fabrication parameters, the detection limit for α-hCG was 2.3 pg/mL in 10mM phosphate buffer saline (PBS) solution containing 1% bovine serum albumine (BSA). Moreover, the use of inexpensive DEP chip as a basis for these immunosensors will allow for simple instrumentation, disposable and portable at low cost. This work also demonstrates a new approach to develop a sensitive and label-free impedimetric immunosensor based on screen-printed electrode for applications in clinical diagnosis.

Amyloid-beta detection with saccharide immobilized gold nanoparticle on carbon electrode.

The electrochemical sensing of saccharide-protein interactions using a couple of sialic acid derivatives and Alzheimer's amyloid-beta (Abeta) is described. The densely-packed saccharide area for recognition of protein was fabricated onto a carbon electrode by three steps, which were electrochemical deposition of Au nanoparticles on a screen printed strip, self-assembled monolayer (SAM) formation of the acetylenyl group on Au nanoparticles, and the cycloaddition reaction of an azide-terminated sialic acid to the acetylenyl group. The attachment of Abeta peptides to the sialic acid layer was confirmed by electrochemistry and atomic force microscopy imaging. The intrinsic oxidation signal of the captured Abeta(1-40) and (1-42) peptides, containing a single tyrosine (Tyr) residues, was monitored at a peak potential of 0.6 V (vs Ag/AgCl within this sensor) in connection with differential pulse voltammetry. The peak current intensities were concentration dependent. The proposed process provides new routes for analysis of saccharide-protein interactions and electrochemical biosensor development.

Label-free detection of melittin binding to a membrane using electrochemical-localized surface plasmon resonance.

Localized surface plasmon resonance (LSPR) and electrochemistry measurements connecting to core-shell structure nanoparticle are successfully exploited in a simultaneous detectable scheme. In this work, the surface plasmon band characterizations of this nanostructure type are initially examined by controlling the core size of the silica nanoparticle and shell thickness of the deposited gold. These results clearly show that when the shell thickness is increased, keeping the core size constant, the peak wavelength of the LSPR spectra is shifted to a shorter wavelength and the maximum of peak intensity is achieved at a particular shell thickness. On the basis of this structure, we present a membrane-based nanosensor for optically detecting the binding of peptide toxin melittin to hybrid bilayer membrane (HBM) and electrochemically assessing its membrane-disturbing properties as a function of concentrations. It will open up the way to detect functionally similar protein toxins and other membrane-targeting peptides with the intension of integrating this chip into a microfluid and expanding it into multiarray format.

An electrochemical on-field sensor system for the detection of compost maturity.

A maturity sensor system was developed, based on the combination of three electrically measured parameters, pH, NH(4)(+) concentration, and phosphatase activity in the water extracts of compost samples. One of these parameters, the apparent phosphatase activity in crude test solutions was determined using screen-printed carbon strips (SPCSs) coated with alpha-naphthyl phosphate (alpha-NP) in Nafion film. The phosphatase activity was monitored in connection with differential pulse voltammetry (DPV) with an aliquot (30 microL) of the test solution on SPCS. The phosphatase activity sensor was validated using alkaline phosphatase (ALP) in Tris-HCl buffer (pH 8.0) and acid phosphatase (ACP) in citric acid buffer (pH 5.0). The activity of the spiked enzymes in the water extract of the compost sample could be confirmed with the change of corresponding oxidation peak current signal of the product, alpha-naphthol. The water extracts of compost samples (n=24) collected in various composting days were applied to our compost maturity sensor system, and the conventional germination tests. Using multiple regression analysis, the germination index (GI) was expressed by the multi-linear regression equation consisting of pH, NH(4)(+) concentration, and the phosphatase activity. The calculated GI from the regression equation had a good correlation with the measured GI of the corresponding composts (r=0.873). As a result, we have determined an equation for the determination of the compost stability using our portable sensor system rapidly at the composting site.

Gold nanoparticle-based electrochemical detection of protein phosphorylation.

In this report, we demonstrate the application of Au nanoparticles in the electrochemical detection of protein phosphorylation. The method is based on the labeling of a specific phosphorylation event with Au nanoparticles, followed by electrochemical detection. The phosphorylation reaction is coupled with the biotinylation of the kinase substrate using a biotin-modified adenosine 5'-triphosphate [gamma]-biotinyl-3,6,9-trioxaundecanediamine (ATP) as the co-substrate. When the phosphorylated and biotinylated kinase substrate is exposed to streptavidin-coated Au nanoparticles, the high affinity between the streptavidin and biotin resulted in the attachment of Au nanoparticles on the kinase substrate. The electrochemical response obtained from Au nanoparticles enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. We determined the activity of Src non-receptor protein tyrosine kinase, p60(c-Src) and protein kinase A in combination with their highly specific substrate peptides Raytide EL and Kemptide, respectively. The detection limits for Raytide EL and Kemptide were determined as 5 and 10 microM, (S/N=3), and the detection limits for the kinase activity of p60(c-Src) and protein kinase A (PKA) were determined as 5 and 10 U mL(-1), (S/N=3), respectively. Tyrosine kinase reactions were also performed in the presence of a well-defined inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), and its negative control molecule, 4-amino-7-phenylpyrazol[3,4-d] pyrimidine (PP3), which had no inhibition effect. Based on the dependency of Au nanoparticle signal on inhibitor concentration, IC(50) value, half-maximal inhibition of the inhibitors was estimated. IC(50) values of PP2, genistein and herbimycin A to p60(c-Src) were detected as 5 nM, 25 microM and 900 nM, respectively. The inhibition of PKA activity on Kemptide using ellagic acid was monitored with an IC(50) of 3.5 microM. The performance of the biosensor was optimized including the kinase reaction, incubation with streptavidin-coated Au nanoparticles, and the small molecule inhibitors. Kinase peptide-modified electrochemical biosensors are promising candidates for cost-effective kinase activity and inhibitor screening assays.

Electrochemical DNA biosensor using a disposable electrochemical printed (DEP) chip for the detection of SNPs from unpurified PCR amplicons.

In this study, we are reporting for the first time the elucidation of single nucleotide polymorphisms (SNPs) of clinically important alleles from consenting human subjects using a disposable electrochemical printed (DEP) chip in connection with differential pulse voltammetry (DPV) and a redox active molecule Hoechst 33258 [H33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi(1H-benzimidazole)]. Post-PCR products were analyzed directly without any purification process. The aggregation of the DNA-H33258 complex causes a significant drop in the peak current intensity of H33258 oxidation. The phenomenon of DNA aggregation induced by H33258 in addition to changes in anodic current peak are used to detect SNPs. Since laborious probe immobilization was not required, our biosensor offers several benefits due to its simplicity and rapid response as a promising device for genetic analysis.

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy.

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.