Numerical and experimental analysis of enzymatic reaction in electrochemical sensors: electrochemical enzymatic analysis

The research examines, numerically and experimentally, the identification of substrate concentration in amperometric electrochemical flow cells. Three-dimensional numerical simulations have been preformed for predicting the mass transfer processes in the vicinity of the electrochemical cell using a realistic geometrical model. The experimental procedure included the fixed enzyme /spl beta/-galactosidase and the injected substrate, para-aminophenyl /spl beta/-D-galactopyranoside. For the optimization of the inlet flow rate, simulations and experiments have been preformed using flow rates between 0.05-250 /spl mu/l/min with an identical substrate concentration. The numerical simulation results were used to evaluate the species concentration distribution in the vicinity of the electrochemical cell for predicting the electric current through the electrode. Different substrate concentrations applied and ranged between 0.05-1.125 mg/ml at a chosen flow rate of 50 /spl mu/l/min. A good agreement was found between the numerical and the experimental electric current evolution values, especially for the higher substrate concentrations. The correlation coefficient was 0.98 in the higher substrate concentrations. A linear relationship was obtained between the inlet substrate concentration and the steady-state electric current for both the numerical and the experimental results. Once this linear relationship is established, the inlet substrate concentration based on the electric current through the electrode can be established.     

Sensitive chemiluminescence immunoassay by capillary electrophoresis with gold nanoparticles

This technical note describes a new chemiluminescence immunoassay hyphenated to capillary electrophoresis (CE-based CL-IA) with gold nanoparticles (AuNPs) technique for biological molecules determination. AuNPs were used as a protein label reagent in the light of its excellent catalytic effect to the CL reaction of luminol and hydrogen peroxide. AuNPs conjugate with antibody (Ab) to form tagged antibody (Ab*), and then Ab* link to antigen (Ag) to produce an Ab*-Ag complex by a noncompetitive immunoreaction. The mixture of the excess Ab* and the Ab*-Ag complex was baseline separated and detected within 5 min under the optimized conditions. This new protocol was evaluated with human immunoglobulin G (IgG) as the target molecule. The calibration curve of IgG was in the range of 0.008-5 μg/mL with a correlation coefficient of 0.995. The detection limit (S/N = 3) of IgG was 1.14 × 10(-3) μg/mL (7.1 pmol/L, 0.39 amol). The proposed AuNPs enhanced CE-based CL-IA method was successfully applied for the quantification of IgG in human sera from patients. It proves that the present method could be developed into a new and sensitive biochemical analysis technique.     

Ultrasensitive aptamer-based multiplexed electrochemical detection by coupling distinguishable signal tags with catalytic recycling of DNase I

This work reports an aptamer-based, disposable, and multiplexed sensing platform for simultaneous electrochemical determination of small molecules, employing adenosine triphosphate (ATP) and cocaine as the model target analytes. The multiplexed sensing strategy is based on target-induced release of distinguishable redox tag-conjugated aptamers from a magnetic graphene platform. The electronic signal of the aptasensors could be further amplified by coupling DNase I with catalytic recycling of self-produced reactants. The assay was based on the change in the current at the various peak potentials in the presence of the corresponding signal tags. Experimental results revealed that the multiplexed electrochemical aptasensor enabled the simultaneous monitoring of ATP and cocaine in a single run with wide working ranges and low detection limits (LODs: 0.1 pM for ATP and 1.5 pM for cocaine). This concept offers promise for rapid, simple, and cost-effective analysis of biological samples.     

Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.     

Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation

The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.     

Peanut skin extract mediated synthesis of gold nanoparticles,silver nanoparticles and gold–silver bionanocomposites for electrochemical Sudan IV sensing

Sustainable methods are needed for rapid and efficient detection of environmental and food pollutants. The Sudan group of dyes has been used extensively as adulterants in food and also are found to be polluting the soil and water bodies. There have been several methods for detection of Sudan dyes, but most of them are not practical enough for common use. In this study, the electrochemical detection efficiency and stability of gold nanoparticle (AuNPs), silver NPs and Au–Ag bionanocomposites, synthesised by peanut skin extract, modified glassy carbon electrode has been investigated. The synthesised nanomaterial samples were characterised, for their quality and quantity, using ultra–visible spectroscopy, inductive coupled plasma mass spectrophotometer, Fourier transform infrared spectroscopy, energy‐dispersive X‐ray spectroscopy, high‐resolution transmission electron microscope and field emission scanning electron microscope. The nanomaterial hybrid electrodes showed great efficiency and stability in the detection of Sudan IV compared with the other previous electrodes. The peak current of the Sudan IV oxidation and reduction was found to be proportional to its concentration, in the range of 10–80 µM, with a detection limit of 4 µM. The hybrid electrodes showed 90% stability in detection for 20 cycles.Inspec keywords: gold, silver, nanoparticles, nanocomposites, biomedical materials, electrochemical sensors, dyes, nanofabrication, ultraviolet spectra, visible spectra, spectrophotometry, Fourier transform infrared spectra, X‐ray chemical analysis, transmission electron microscopy, scanning electron microscopy, field emission electron microscopyOther keywords: peanut skin extract mediated synthesis, gold nanoparticles, silver nanoparticles, gold–silver bionanocomposites, electrochemical Sudan IV sensing, electrochemical detection efficiency, modified glassy carbon electrode, ultra–visible spectroscopy, inductive coupled plasma mass spectrophotometer, Fourier transform infrared spectroscopy, energy‐dispersive X‐ray spectroscopy, high‐resolution transmission electron microscope, field emission scanning electron microscope, oxidation, reduction, detection limit, Au, Ag, Au‐Ag     

Shape control of gold nanoparticles by silver underpotential deposition

Four different gold nanostructures: octahedra, rhombic dodecahedra, truncated ditetragonal prisms, and concave cubes, have been synthesized using a seed-mediated growth method by strategically varying the Ag(+) concentration in the reaction solution. Using X-ray photoelectron spectroscopy and inductively coupled plasma atomic emission spectroscopy, we provide quantitative evidence that Ag underpotential deposition is responsible for stabilizing the various surface facets that enclose the above nanoparticles. Increasing concentrations of Ag(+) in the growth solution stabilize more open surface facets, and experimental values for Ag coverage on the surface of the particles fit well with a calculated monolayer coverage of Ag, as expected via underpotential deposition.     

Electrochemical detection of chloride by underpotentially deposited silver films on polycrystalline gold

This paper describes an electrochemical method for measuring dilute levels of chloride using an underpotentially deposited (UPD) Ag adlayer on polycrystalline Au substrates as a sensing agent. Specifically, chloride ions adsorb onto the Ag UPD adlayer and effect changes in the electrochemical deposition and stripping characteristics of the silver film. Cyclic voltammograms (CVs) of the native Au/Ag(UPD) electrode in 0.1 M H2SO4(aq) exhibit a primary stripping peak for the Ag UPD adlayer at 550 mV vs Ag(+/0), and chloride adsorption onto the Au/Ag(UPD) surface effects a peak shift to approximately 600 mV vs Ag(+/0), depending on the amount of adsorbed Cl-, as affected by the Cl- concentrations and contact times employed in the derivatization. The chloride-treated electrodes also exhibit a stripping peak at 275 mV that is not observed on the native substrate and increases in intensity with Cl- concentration and derivatization time. The integrated charge density for this latter stripping peak relative to that for the primary stripping peak at 550-610 mV provides a useful metric for quantifying adsorbed Cl- levels, and these values allow measurement of Cl- concentrations in dilute aqueous solutions. For Cl- concentrations between 0.5 and 100 microM, the kinetics of Cl- adsorption followed a transient Langmuir adsorption model and allowed measured surface coverages to be used for determining Cl- solution concentrations. Using contact times of 1 min for Cl- adsorption, the electrodes showed a linear response across Cl- concentrations of 0.5-20 microM.     

Biological synthesis of gold and silver nanoparticles mediated by the bacteria Bacillus subtilis

Biological synthesis of gold and silver nanoparticles was carried out using the bacteria Bacillus subtilis. The reduction processes of chloroaurate and silver ions by B. subtilis were found to be different. Gold nanoparticles were synthesized both intra- and extracellularly, while silver nanoparticles were exclusively formed extracellularly. The gold nanoparticles were formed after 1 day of addition of chloroaurate ions, while the silver nanoparticles were formed after 7 days. The nanoparticles were characterized by X-ray diffraction, UV-vis spectra and transmission electron spectroscopy. X-ray diffraction revealed the formation of face-centered cubic (fcc) crystalline gold nanoparticles in the supernatant, broth solution and bacterial pellet. Silver nanoparticles also exhibited diffraction peaks corresponding to fcc metallic silver. UV-vis spectra showed surface plasmon vibrations for gold and silver nanoparticles centered at 530 and 456 nm, respectively. TEM micrographs depicted the formation of gold nanoparticles intra- and extracellularly, which had an average size of 7.6 +/- 1.8 and 7.3 +/- 2.3 nm, respectively, while silver nanoparticles were exclusively formed extracellularly, with an average size of 6.1 +/- 1.6 nm. The bacterial proteins were analyzed by sodium dodecyl sulfonate-polyacrylamide electrophoresis (SDS-PAGE) before and after the addition of metal ion solutions. We believe that proteins of a molecular weight between 25 and 66 kDa could be responsible for chloroaurate ions reduction, while the formation of silver nanoparticles can be attributed to proteins of a molecular weight between 66 and 116 kDa. We also believe that the nanoparticles were stabilized by the surface-active molecules i.e., surfactin or other biomolecules released into the solution by B. subtilis.     

Chemical stabilization of gold coated by silver core-shell nanoparticles via electron transfer

Silver nanoparticles are notoriously susceptible to oxidation, yet gold nanoparticles coated in silver exhibit a unique electronic interaction that occurs at the interface of the two metals, leading to enhanced stability properties for the silver shell. In order to probe the phenomenon, the stability of gold nanoparticles coated by silver was studied in the presence of various chloride-containing electrolytes. It was found that a critical silver shell thickness of approximately 1 nm exists that cannot be oxidatively etched from the particle surface: this is in contrast to the observation of complete oxidative etching for monometallic silver nanoparticles. The results are discussed in terms of particle composition, structure and morphology before and after exposing the particles to the electrolytes. Raman analysis of the reporter molecule 3-amino-1,2,4-triazole-5-thiol adsorbed on the particle surface illustrates the feasibility of using gold coated by silver nanoparticle probes in sensing applications that require the presence of high levels of salt. The results provide insight into the manipulation of the electronic and stability properties for gold- and silver-based nanoparticles.     

Influences of gold and silver nanoparticles in loop-mediated isothermal amplification reactions

Loop-mediated isothermal amplification (LAMP) performed with protein DNA polymerase Bst and DNA chains was influenced by nanoparticles in different ways. The effects of different concentrations of gold nanoparticles (AuNPs) with diameters 10 and 20?nm and silver nanoparticles (AgNPs) 1–10?nm in diameter on the amplification of the pR72H gene of Vibrio parahaemolyticus were investigated. AuNPs with a diameter of 10?nm in 0.6–60?nM concentration accelerated initiation of the LAMP reaction, 3?nM AuNPs reduced the reaction time by about 10?min, whereas 20?nm AuNPs did not, although neither size increased the yield after 60?min. AgNPs inhibited the LAMP reaction both in speed and yield at concentrations of 0.6–60?nM; the yield of amplification was reduced by 50% and 80% for 12 and 60?nM, respectively, after reaction for 1?h. This indicated that strong bactericidal effects of silver are also observed in its nanoparticles. The molecular mechanism of AuNPs and AgNPs in LAMP needs to be explored further, although their size-related electronic, magnetic and optical properties, as well as their ability to affect protein denaturation, or hydrophilic/hydrophobic effects may be involved.     

pH-insensitive fabrication of gold nanoparticles with high concentration by ultrasound-assisted electrochemical process via aid of chitosan

In this work, we report a new pathway to prepare pure gold nanoparticles with high concentrations in acid solutions via the aid of chitosan without the addition of other stabilizers and reductants based on electrochemical methods. Interestingly, this fabrication of gold nanoparticles with high concentrations in solutions is pH-insensitive. The characteristics of prepared gold nanoparticles were examined by transmission electron microscopy (TEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (HRXPS) measurements. Experimental results indicate the concentration and the particle size of Au nanoparticles prepared in 0.1N HCl are ca. 50 ppm and 12 nm in diameter, respectively. Similar experiment performed in 0.1N NaCl with the aid of chitosan shows that the corresponding concentration of prepared Au nanoparticles is ca. 100 ppm. Further similar experiments performed in 0.1N HCl and 0.1N NaCl without the aid of chitosan show that the corresponding concentrations of prepared Au nanoparticles are ca. 1 and 60 ppm, respectively.     

Synthesis and enhanced electrochemical properties of pod-shaped gold/silica nanoparticles

Pod-shaped gold/silica nanoparticles (PGSNPs) were prepared using perfluorooctanoic acid (PFOA) and cetyltrimethylammonium bromide (CTAB) as cotemplates. The PGSNPs were utilized to explore a novel biosensor through coupling myoglobin (Mb) with chitosan (Chi). Compared with Mb-Chi-PSNPs (pod-shaped silica nanoparticles)/GC modified electrode, Mb-Chi-PGSNPs/GC electrode exhibited a pair of much stronger redox peaks at − 0.28 V (vs. Ag/AgCl). Moreover, facilitated direct electron transfer of the metalloenzymes with smaller peak-to-peak separation (ΔEp) of about 46 mV was acquired on the PGSNPs-based enzyme electrode. The PGSNPs-based biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with a wide linear range (1-540 µM) and high sensitivity (661 mA cm^− 2 M^− 1). Together, the Mb-Chi-PGSNPs film is one of ideal candidate materials for direct electrochemistry of redox proteins, and may find potential applications in biomedical, food, and environmental analysis and detection.     

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.     

Synthesis of gold and silver nanoparticles by electron irradiation at 5-15 keV energy

Thin coatings (～10?μm) made from a mixture of polyvinyl alcohol (PVA) and HAuCl(4) or PVA and AgNO(3) on quartz plates were irradiated with 5-15?keV electrons, at room temperature. The electron energy was varied from coating to coating in the range of 5-15?keV, but electron fluence was kept constant at ～10(15)?e?cm(-2). Samples were characterized by the UV-vis, XRD, SEM and TEM techniques. The plasmon absorption peaks at ～511 and ～442?nm confirmed the formation of gold and silver nanoparticles in the respective electron-irradiated coatings. The XRD, SEM and TEM measurements reveal that the average size of the particles could be tailored in the range of 130-50?nm for gold and from 150-40?nm for silver by varying the electron energy in the range of 5-15?keV. These particles of gold and silver embedded in the polymer could also be separated by dissolving the coatings in distilled water.