Quantitative turbidity assay for lipolytic enzymes in microtiter plates

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Design of sugar–oligonucleotide conjugates installed gold nanoparticle for effective delivery to hepatic parenchymal cells

A novel oligonucleotide delivery system that is based on oligonucleotide–nanoparticle conjugates has been described. Installed oligonucleotides were modified with the carbohydrate at the 3′ terminus, accordingly, constructed nanoparticles display clustered carbohydrates on their outer layer for the targeted delivery of oligonucleotides. The method for the construction of ligand-functionalized nanoparticle was simple and reproducible. The stability of the nanoparticles displaying clustered carbohydrates greatly increased in serum compared to nanoparticles without carbohydrates. In order to investigate the targetability of oligonucleotide–nanoparticle conjugates into primary hepatic parenchymal cells, freshly isolated rat hepatocytes were incubated with nanoparticles and the amount of internalized gold nanoparticles was evaluated by an inductively coupled plasma mass spectroscopy analysis. Nanoparticles displaying clustered carbohydrates internalized more efficiently than nanoparticles without carbohydrate modifications. In particular, the cellular uptakes of oligonucleotide-conjugated gold nanoparticle increased 1.7 ～2.0-fold by galactose modification. Competition assay revealed that clustered galactose enhanced the internalization of the nanoparticle into primary hepatic parenchymal cells by a receptor-mediated process.

A surface plasmon resonance study on the optical properties of gold nanoparticles on thin gold films

We report on an investigation of the optical properties of gold nanoparticles assembled as thin films of different thickness. The nanoparticles were linked to the surface of a gold chip by dithiol reagents and studied by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. There is good correlation between the experimental findings and theoretical simulation, and the respective data reveal the presence of ordered nanostructures in the assemblies. The shift in the SPR angle is linearly dependent on the particle size and the ratio of the different particles. SPR spectroscopy also reveals important information in terms of the optical constants of such films. This shall be further applied to in-situ quality control in the fabrication of optoelectronic, solar cell and semiconductor devices.

Enhancement of the colorimetric sensitivity of gold nanoparticles with triethanolamine to minimize interparticle repulsion

Cysteine and thioglycolic acid were immobilized on gold nanoparticles via established thiolgold surface chemistry. It is found that calcium ions rapidly induce the aggregation of the functional gold nanoparticles due to the complexation of Ca(II) by immobilized cysteine. It was also found that triethanolamine enhances the effect of calcium ions by decreasing the electrostatic repulsion between the gold nanoparticles. Transmission electron microscopy, electrophoresis, zeta potential measurements and absorptiometry were used to investigate the mechanism. Under the optimum experimental condition, the cysteine/thioglycolate/triethanolamine-modified nanoparticles were highly sensitive (the detection limit being 0.3 ??M) and selective towards calcium and magnesium ions, with a linear detection range between 1.0 ??M and 14 ??M. Based on these findings, a rapid and selective colorimetric method was developed for assaying Ca(II) ions in serum.

Sensitive detection of tuberculosis using nanoparticle-enhanced surface plasmon resonance

We show that the antigen CFP-10 (found in tissue fluids of tuberculosis patients) can be used as a marker protein in a surface-plasmon resonance (SPR) based method for early and simplified diagnosis of tuberculosis. A sandwich SPR immunosensor was constructed by immobilizing the CFP-10 antibody on a self-assembled monolayer on a gold surface, this followed by blocking it with bovine serum albumin. Following exposure of the sensor surface to a sample containing CFP-10, secondary antibody immobilized on nickel oxide nanoparticles are injected which causes a large SPR signal change. The method has a dynamic range from 0.1 to around 150 ng per mL of CFP-10, and a detection limit as low as 0.1 ng per mL. This is assumed to be due to the high amplification power of the NiO nanoparticles.

Chronocoulometric DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles, poly(dopamine) and carbon nanotubes

We describe a sensitive chronocoulometric biosensor for the sequence-specific detection of DNA. It is based on a glassy carbon electrode modified with multi-walled carbon nanotubes, polydopamine, and gold nanoparticles. The ruthenium(III)hexammine complex acts as the electrochemical indicator. Electrochemical impedance spectra and scanning electron microscopy are employed to investigate the assembly of the electrode surface. The signals of the ruthenium complex electrostatically bound to the anionic phospho groups of the DNA strands are measured by chronocoulometry before and after hybridization. The difference in signal intensity is linearly related to the logarithm of the concentration of the target DNA in the range of 1.0 nM to 10 fM with a detection limit of 3.5fM (S/N?=?3) under optimal conditions. This biosensor exhibits excellent sensitivity and selectivity and has been used for an assay of complementary target DNA in human serum sample with satisfactory results.

Continuous flow synthesis and characterization of tailor-made bare gold nanoparticles for use in SERS

We describe a method for the synthesis of gold nanoparticles in a stainless steel continuous flow tubular reactor using tetrachloroauric acid as a precursor but without using a classical reducing agent. Gold(III) ion is reduced by stainless steel to form gold nanoparticles which are collected at the end of the coil. A single-phase system is introduced that generates dispersed nanoparticles in the absence of reducing agents on their surface. By controlling flow rates and temperature, the size of the nanoparticles can be tuned in the range from 24 nm to 36 nm. The reproducibility of the preparation was investigated, relative standard deviation of both the wavelength of the peak and the intensity of the plasmonic absorption band were determined and found to vary by 0.15 % and 6.5 %, respectively. Flow synthesis is found to be an excellent alternative to chemical methods to produce stable gold nanoparticles of varying size in an efficiently way. The particles obtained also perform very well when used as a substrate in surface enhanced Raman scattering as shown by the characterization of carboxylated single walled carbon nanotubes.

An indium tin oxide electrode modified with gold nanorods for use in potential-controlled surface plasmon resonance studies

The modification of electrodes with gold nanoparticles results in an increased electrode surface area, enhanced mass transport, and improved catalytic properties. We have extended this approach to indium tin oxide (ITO) electrodes to obtain optically transparent gold nanorod-modified electrodes which display enhanced electrochemical capabilities and have the additional advantage of showing a tunable surface plasmon resonance. The procedures for attaining high surface coverage (15 gold nanorods per square µm) of such electrodes were optimized, and the potential-dependent surface plasmon resonance was studied under controlled electrical potential. In an exemplary sensor application, we demonstrate the detection of mercury via potential-dependent formation of an Au-Hg amalgam.

Comparative SPR study on the effect of nanomaterials on the biological activity of adsorbed proteins

Bioactivity of proteins is evaluated to test the adverse effects of nanoparticles interjected into biological systems. Surface plasmon resonance (SPR) spectroscopy detects binding affinity that is normally related to biological activity. Utilizing SPR spectroscopy, a concise testing matrix is established by investigating the adsorption level of bovine serum albumin (BSA) and anti-BSA on the surface covered with 11-mercaptoundecanoic acid (MUA); magnetic nanoparticles (MNPs) and single-walled carbon nanotubes (SWCNTs), respectively. The immunoactivity of BSA on MNPs and SWCNT decreased by 18?% and 5?%, respectively, compared to that on the gold film modified with MUA. This indicates that MNPs cause a considerable loss of biological activity of adsorbed protein. This effect can be utilized for practical applications on detailed biophysical research and nanotoxicity studies.

Improving surface plasmon resonance imaging of DNA by creating new gold and silver based surface nanostructures

The use of nanoparticles (NPs) can substantially improve the analytical performance of surface plasmon resonance imaging (SPRi) in general, and in DNA sensing in particular. In this work, we report on the modification of the gold surface of commercial biochips with gold nanospheres, silica-coated gold nanoshells, and silver nanoprisms, respectively. The NPs were tethered onto the surface of the chip and functionalized with a DNA probe. The effects of tethering conditions and varying nanostructures on the SPRi signals were evaluated via hybridization assays. The results showed that coupling between planar surface plasmons and electric fields, generated by localized surface plasmons of the NPs, is mandatory for signal enhancement. Silver nanoprisms gave the best results in improving the signal change at a target DNA concentration of <50 nM by +50 % (compared to a conventional SPRi chip). The limit of detection for the target DNA was 0.5 nM which is 5 times less than in conventional SPRi.

Preparation of a nanocomposite film from poly(diallydimethyl ammonium chloride) and gold nanoparticles by in-situ electrochemical reduction, and its application to SERS spectroscopy and sensing of ascorbic acid

A nanocomposite film is described that is composed of alternating layers of poly(diallydimethyl ammonium chloride) and gold nanoparticles that interact through electrostatic forces. The films of varying thickness were prepared by the layer-by-layer technique, and Au-NPs were generated by electrochemical reduction of hexachloroauric acid. The composite films were characterized by UV?Cvis spectroscopy, X-ray photoelectron spectroscopy, and cyclic voltammetry. Most nanocomposite films exhibit linear, uniform, and regular layer-by-layer growth during the process of formation. The films exhibit unique performance in terms of surface enhanced Raman scattering and electrocatalytic activitiy towards the oxidation of ascorbic acid.

Electrochemical synthesis of a graphene sheet and gold nanoparticle-based nanocomposite, and its application to amperometric sensing of dopamine

We describe a simple, green and controllable approach for electrochemical synthesis of a nanocomposite made up from electrochemically reduced graphene oxide (ERGO) and gold nanoparticles. This material possesses the specific features of both gold nanoparticles and graphene. Its morphology was characterized by scanning electron microscopy which reveals a homogeneous distribution of gold nanoparticles on the graphene sheets. Cyclic voltammetry was used to evaluate the electrochemical properties of this nanocomposite towards dopamine by modification of it on surface of glassy carbon electrode (GCE). Compared to the bare GCE, the electrode modified with gold nanoparticles, and the electrode modified with ERGO, the one modified with the nanocomposite displays better electrocatalytic activity. Its oxidation peak current is linearly proportional to the concentration of dopamine (DA) in the range from 0.1 to 10?μM, with a detection limit of 0.04?μM (at S/N?=?3). The modified electrode also displays good storage stability, reproducibility, and selectivity.

Amperometric determination of sulfite using screen-printed electrodes modified with metallic nanoparticles

We report on the amperometric determination of sulfite using screen-printed carbon electrodes (SPCEs) modified with gold and silver nanoparticles that were deposited on the electrode to improve the capabilities of detection. The electrode is fairly selective and responds to sulfite with an oxidation current (at 300 mV and pH 6) in the 9.80 to 83.33 μM concentration range. The precision in terms of repeatability and reproducibility is 14.4 % and 10.7 % in the case of SPCEs modified by gold nanoparticles. The method was applied to the determination of sulfite in drinking water, pickle juice and vinegar. Recoveries ranged from 96 % to 104 %.

Glucose biosensor based on a platinum electrode modified with rhodium nanoparticles and with glucose oxidase immobilized on gold nanoparticles

We have developed an enzymatic glucose biosensor that is based on a flat platinum electrode which was covered with electrophoretically deposited rhodium (Rh) nanoparticles and then sintered to form a large surface area. The biosensor was obtained by depositing glucose oxidase (GOx), Nafion, and gold nanoparticles (AuNPs) on the Rh electrode. The electrical potential and the fractions of Nafion and GOx were optimized. The resulting biosensor has a very high sensitivity (68.1 μA mM^?1 cm^?2) and good linearity in the range from 0.05 to 15 mM (r?=?0.989). The limit of detection is as low as 0.03 mM (at an SNR of 3). The glucose biosensor also is quite selective and is not interfered by electroactive substances including ascorbic acid, uric acid and acetaminophen. The lifespan is up to 90 days. It was applied to the determination of glucose in blood serum, and the results compare very well with those obtained with a clinical analyzer.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Gold electrodes modified with gold nanoparticles and thio compounds for electrochemical sensing of dopamine alone and in presence of potential interferents. A comparative study

Gold electrodes were modified with self assembled layers (SAMs) composed of mercaptopropionic acid, thiodipropionic acid, dithiodipropionic acid, cysteamine and gold nanoparticles and used to study the electrooxidation of dopamine (DA) in solution at pH 7. SAMs endowed with gold nanoparticles gave the highest catalytic effect. The results showed that such electrodes are capable of resolving the oxidation peaks of DA, ascorbic acid, and uric acid which is most favourable with respect to the detection of DA in physiological matrices.

Visual detection of silver(I) ions by a chromogenic reaction catalyzed by gold nanoparticles

We report on a novel method for visual detection silver(I) ion. It is based on the finding that Ag(I) ions are rapidly reduced by hydroquinone to form a shell of silver on the surface of gold nanoparticles (AuNPs) which act as catalysts for this reaction. This leads to a color change from red to yellow which can be seen with bare eyes. This scheme is sensitive and highly specific for Ag(I) ions. The detection limits are 5 μM for visual inspection and 1 μM for photometric readout, respectively. The method was successfully applied to the determination of Ag(I) ions in spiked lake water and soil.

Disposable electrochemical immunosensor for myeloperoxidase based on the indium tin oxide electrode modified with an ionic liquid composite film containing gold nanoparticles, poly(o-phenylenediamine) and carbon nanotubes

A disposable electrochemical myeloperoxidase (MPO) immunosensor was fabricated based on the indium tin oxide electrode modified with a film composed of gold nanoparticles (AuNPs), poly(o-phenylenediamine), multi-walled carbon nanotubes and an ionic liquid. The composite film on the surface of the electrode was prepared by in situ electropolymerization using the ionic liquid as a supporting electrolyte. Negatively charged AuNPs were then adsorbed on the modified electrode via amine-gold affinity and to immobilize MPO antibody. Finally, bovine serum albumin was employed to block possible remaining active sites on the AuNPs. The modification of the electrode was studied by cyclic voltammetry and scanning electron microscopy. The factors affecting the performance of the immunosensor were investigated in detail using the hexacyanoferrate redox system. The sensor exhibited good response to MPO over two linear ranges (from 0.2 to 23.4 and from 23.4 to 300 ng.mL^?1), with a detection limit of 0.05 ng.mL^?1 (at an S/N of 3).

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H₂O₂ and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L^-1 of target DNA.

Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces.