Encoded and enzyme-activated nanolithography of gold and magnetic nanoparticles on silicon

A C18 monolayer-functionalized Si surface is electrochemically patterned to yield a carboxylic acid-terminated pattern. Tyramine is covalently linked to the pattern to yield an encoded nanostructure for the enzyme tyrosinase. The biocatalytic oxidation of the tyramine residues yields catechol moieties that control the assembly of boronic acid-functionalized Au nanoparticles (NPs) or magnetic NPs. The different NPs are linked to the patterns by the formation of complexes between the boronic acid residues or Fe3+ ions and the catechol ligands.     

A glassy carbon electrode modified with graphene and tyrosinase immobilized on platinum nanoparticles for sensing organophosphorus pesticides

An amperometric biosensor is described for the detection of organophosphorus pesticides. It is based on the enzyme tyrosinase immobilized on platinum nanoparticles and the use of a glassy carbon electrode modified with graphene. Tyrosinase was immobilized on the electrode surface via electrostatic interaction between a monolayer of cysteamine and the enzyme. In the presence of catechol as a substrate, the pesticides chlorpyrifos, profenofos and malathion can be determined as a result of their inhibition of the enzyme which catalyzes the oxidation of catechol to o-quinone. Platinum nanoparticles and graphene effectively enhance the efficiency of the electrochemical reduction of o-quinone, thus improving sensitivity. Under optimum experimental conditions, the inhibition effect of the pesticides investigated is proportional to their concentrations in the lower ppb-range. The detection limits are 0.2, 0.8 and 3?ppb for chlorpyrifos, profenofos and malathion, respectively. The biosensor displays good repeatability and acceptable stability.

Boronic Acid functionalized core-shell polymer nanoparticles prepared by distillation precipitation polymerization for glycopeptide enrichment

The boronic acid-functionalized core-shell polymer nanoparticles, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@4-vinylphenylboronic acid (poly(MBA-co-MAA)@VPBA), were successfully synthesized for enriching glycosylated peptides. Such nanoparticles were composed of a hydrophilic polymer core prepared by distillation precipitation polymerization (DPP) and a boronic acid-functionalized shell designed for capturing glycopeptides. Owing to the relatively large amount of residual vinyl groups introduced by DPP on the core surface, the VPBA monomer was coated with high efficiency, working as the shell. Moreover, the overall polymerization route, especially the use of DPP, made the synthesis of nanoparticles facile and time-saving. With the poly(MBA-co-MAA)@VPBA nanoparticles, 18?glycopeptides from horseradish peroxidase (HRP) digest were captured and identified by MALDI-TOF mass spectrometric analysis, relative to eight glycopeptides enriched by using commercially available meta-aminophenylboronic acid agarose under the same conditions. When the concentration of the HRP digest was decreased to as low as 5?nmol, glycopeptides could still be selectively isolated by the prepared nanoparticles. Our results demonstrated that the synthetic poly(MBA-co-MAA)@VPBA nanoparticles might be a promising selective enrichment material for glycoproteome analysis.     

Electrochemical Determination of Malathion on an Acetylcholinesterase-Modified Glassy Carbon Electrode

An acetylcholinesterase biosensor based on glassy carbon electrode modified with carbon black and pillar[5]arene was used for the determination of malathion after its preliminary oxidation. The contributions of enzyme immobilization and oxidation conditions to the improvement of analytical characteristics of the biosensor were considered and quantified. In optimal conditions, the acetylcholinesterase biosensor allows the determination of 40 pM of malathion with 10?min of incubation and 15 pM with 30?min of incubation. The sensitivity of immobilized enzyme was found to be higher than that the free enzyme due to sorbtional accumulation in the modifier layer. Incomplete oxidation of malathion decreased the sensitivity of the assay. The developed acetylcholinesterase biosensor was validated for the determination of malathion residues in grapes, wine, and peanuts. The recoveries calculated against a high-performance liquid chromatography assay were between 80 and 120% due to possible matrix effects and the simplified extraction protocols.     

Au掺杂Fe3O4纳米粒子酶传感器的制备及其应用于有机磷农药检测的研究

合成了金掺杂的四氧化三铁纳米粒子(Au-Fe₃O₄), 以壳聚糖为交联剂, 制备了电流型乙酰胆碱酯酶(AChE)生物传感器, 并将其应用于有机磷农药(OPs)的检测. 实验表明, Au-Fe₃O₄纳米粒子具有良好的生物兼容性, 能够有效地促进电子传递, 修饰了Au-Fe₃O₄纳米粒子的酶传感器, 响应速度快, 检测灵敏度高, 稳定性好; 固定在传感器上的乙酰胆碱酯酶有良好的酶动力学响应, 其表观米氏常数( )为10.3 mmol/L. 利用有机磷农药对乙酰胆碱酯酶的抑制作用, 以硫代乙酰胆碱(ATCh)为底物, 对有机磷农药敌敌畏进行了检测, 检测限达到4.0×10^－13 mol/L.     

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi–CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi–CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of ?0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0–400 μmol L^?1, with a very low limit of detection of 1.0 μmol L^?1 (s/n?=?3). The operational stability of the uricase/Chi–CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis–Menten constant of 0.21 mmol L^?1 indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P?>?0.05).

Fenugreek hydrogel–agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection

A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel–agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10–20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel–agarose–acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples.     

Organophosphorus pesticides detection using acetylcholinesterase biosensor based on gold nanoparticles constructed by electroless plating on vertical nitrogen-doped single-walled carbon nanotubes

An acetylcholinesterase (AChE) biosensor was constructed based on gold nanoparticles (AuNPs) using electroless plating on vertical nitrogen-doped single-walled carbon nanotubes (VNSWCNTs) for detecting organophosphorus pesticides (OPs). AChE was immobilised on AuNPs via Au–S bonding, and VNSWCNTs were produced by spontaneous chemical adsorption of NSWCNTs on gold electrode, also via Au–S bonding. This modified electrode exhibited excellent electron transfer capacity due to the synergy between AuNPs and VNSWCNTs. The developed biosensor showed good linear relations at concentrations of 10^?5 – 1 ppb, and the detection limits were 3.04 × 10^?6 ppb for methyl parathion, 1.96 × 10^?6 ppb for malathion and 2.06 × 10^?6 ppb for chlorpyrifos, respectively. The AChE biosensor revealed satisfactory stability, excellent sensitivity and good repeatability. These results suggest that this biosensor has good application prospects and can function as a sensitive device in OPs analysis.     

Amperometric Biosensor Based on Immobilization Acetylcholinesterase on Manganese Porphyrin Nanoparticles for Detection of Trichlorfon with Flow‐Injection Analysis System

A highly sensitive amperometric biosensor for the detection of organophosphate pesticides (OPs) is developed. The biosensor was fabricated by immobilized acetylcholinesterase (AChE) on manganese (III) meso‐tetraphenylporphyrin (MnTPP) nanoparticles (NPs)‐modified glassy carbon (GC) electrode. The MnTPP NPs used in this article were synthesized by mixing solvent techniques. AChE enzyme was immobilized on the MnTPP NPs surface by conjugated with chitosan (CHIT). The electrocatalytic activity of MnTPP NPs led to a greatly improved performance for thiocholine (TCh) product detection. The developed AChE‐CHIT/MnTPPNP/GC biosensor integrated with a flow‐injection analysis (FIA) system was used to monitor trichlorfon (typical OP). A wide linear inhibition response for trichlorfon is observed in the range of 1.0 nM–1.0 mM, corresponding to 10–83% inhibition for AChE with a detection limit of 0.5 nM.     

Acetylcholine Biosensor Based on Dendrimer Layers for Pesticides Detection

We tested a new design of an enzyme biosensor based on acetylcholinesterase (AChE) and choline oxidase (ChO) immobilized on the supported monomolecular layer composed of poly(amidoamine) (PAMAM) dendrimers of the fourth generation (G4) mixed with 1‐hexadecanethiol (HDT). The resulting enzymatic activity, measured amperometrically, was substantially depressed in the presence of the organophosphate pesticide dimethyl‐2,2‐dichlorovinylphosphate (DDVP, Dichlorvos), carbamate pesticides carbofuran and carbamate drug eserine. The detection limits (1.3×10^?3 ppb for DDVP, 0.01 ppb for carbofuran and 0.03 for eserine) were considerably lower than so far reported for AChE based amperometric and potentiometric sensors. The relative simple protocol of biosensor preparation, high sensitivity and stability is very promising for determination of environmental pollutants in field conditions.     

Magnetic particles–based biosensor for biogenic amines using an optical oxygen sensor as a transducer

We have developed a fibre optic biosensor with incorporated magnetic microparticles for the determination of biogenic amines. The enzyme diamine oxidase from Pisum sativum was immobilized either on chitosan-coated magnetic microparticles or on commercial microbeads modified with a ferrofluid. Both the immobilized enzyme and the ruthenium complex were incorporated into a UV-cured inorganic–organic polymer composite and deposited on a lens that was connected, by optical fibres, to an electro-optical detector. The enzyme catalyzes the oxidation of amines under consumption of oxygen. The latter was determined by measuring the quenched fluorescence lifetime of the ruthenium complex. The limits of detection for the biogenic amines putrescine and cadaverine are 25–30 μmol?L^?1, and responses are linear up to a concentration of 1 mmol L^?1.

Fe2O3@Au core/shell nanoparticle-based electrochemical DNA biosensor for Escherichia coli detection

A Fe₂O₃@Au core/shell nanoparticle-based electrochemical DNA biosensor was developed for the amperometric detection of Escherichia coli (E. coli). Magnetic Fe₂O₃@Au nanoparticles were prepared by reducing HAuCl₄ on the surfaces of Fe₂O₃ nanoparticles. This DNA biosensor is based on a sandwich detection strategy, which involves capture probe immobilized on magnetic nanoparticles (MNPs), target and reporter probe labeled with horseradish peroxidase (HRP). Once magnetic field was added, these sandwich complexes were magnetically separated and HRP confined at the surfaces of MNPs could catalyze the enzyme substrate and generate electrochemical signals. The biosensor could detect the concentrations upper than 0.01 pM DNA target and upper than 500 cfu/mL of E. coli without any nucleic acid amplification steps. The detection limit could be lowered to 5 cfu/mL of E. coli after 4.0 h of incubation.     

Reagentless Glucose Biosensor Based on the Direct Electrochemistry of Glucose Oxidase on Carbon Nanotube‐Modified Electrodes

The direct electrochemistry of glucose oxidase (GOD) was revealed at a carbon nanotube (CNT)‐modified glassy carbon electrode, where the enzyme was immobilized with a chitosan film containing gold nanoparticles. The immobilized GOD displays a pair of redox peaks in pH 7.4 phosphate buffer solutions (PBS) with the formal potential of about ?455 mV (vs. Ag/AgCl) and shows a surface‐controlled electrode process. Bioactivity remains good, along with effective catalysis of the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, which could be used for reagentless detection of glucose with a linear range from 0.04 to 1.0 mM. The proposed glucose biosensor exhibited high sensitivity, good stability and reproducibility, and was also insensitive to common interferences such as ascorbic and uric acid. The excellent performance of the reagentless biosensor is attributed to the effective enhancement of electron transfer between enzyme and electrode surface by CNTs, and the biocompatible environment that the chitosan film containing gold nanoparticles provides for immobilized GOD.     

Electrochemical DNA biosensor for the detection of Trichoderma harzianum based on a gold electrode modified with a composite membrane made from an ionic liquid, ZnO nanoparticles and chitosan, and by using acridine orange as a redox indicator

An electrochemical DNA biosensor was developed that is based on a gold electrode modified with a nanocomposite membrane made from an ionic liquid, ZnO nanoparticles and chitosan. A single-stranded DNA probe was immobilized on this electrode. Acridine orange was used as the hybridization probe for monitoring the hybridization of the target DNA. The biosensor was capable of detecting target DNA in the concentration range from 1.0?×?10?C14 to 1.8?×?10?C4?mol?L-1, with a detection limit of 1.0?×?10?C15?mol?L-1. The approach towards constructing a DNA biosensor allows studies on the hybridization even with crude DNA fragments and also to analyze sample obtained from real samples. The results show that the DNA biosensor has the potential for sensitive detection of a specific sequence of the Trichoderma harzianum gene and provides a quick, sensitive and convenient method for the study of microorganisms.

Amperometric biosensing of carbamate and organophosphate pesticides utilizing screen-printed tyrosinase-modified electrodes

A tyrosinase (Tyr) screen-printed biosensor based on the electroreduction of enzymatically generated quinoid products was electrochemically characterized and optimized for determination of carbamates and organophosphorus pesticides. A composite electrode prepared by screen-printing a cobalt (II) phthalocyanine (CoPc) modified cellulose-graphite composite on a polycarbonate support was employed as electrochemical transducer. The Tyr biosensor was prepared by immobilization of enzyme on the composite electrode surface by cross-linking with glutaraldehyde and bovine serum albumin. Parameters affecting the biosensor response such as response time, enzyme loading, concentration and pH of the buffer solution were optimized utilizing catechol as substrate. The maximum response for o-quinone enzymatically generated was obtained after 2 min of reaction. A good reproducibility and high operational stability were found for Tyr biosensor (60 units) at 50 mM phosphate buffer, pH 6.50. Under these conditions, the useful lifetime of biosensor was 10 days. After 15 days, the biosensor could be used with 20% of the initial value. Inhibition studies on the o-quinone steady-state current (at −0.20 V versus Ag/AgCl) were performed to investigate the inhibition kinetics of the pesticides in the enzymatic activity of mushroom tyrosinase. The results shown that the methyl parathion and carbofuran can lead to competitive inhibition process of the enzyme, while diazinon and carbaryl act as mixed inhibitors. Linear relationships were found for methyl parathion (6-100 ppb), diazinon (19-50 ppb), carbofuran (5-90 ppb) and carbaryl (10-50 ppb). Analysis of natural river water samples spiked with 30 ppb of each pesticide showed recoveries between 92.50% and 98.50% and relative standard deviations of 2%.     

Electrochemical Acetylcholinesterase Biosensor Based on Polylactide–Nanosilver Composite for the Determination of Anti-dementia Drugs

A new acetylcholinesterase biosensor has been developed for the determination of anticholinesterase drugs applied for neurodegenerative disease treatment. For this purpose, silver nanosendrites were deposited by potentiostatic electrolysis on a glassy carbon electrode covered with oligolactides cross-linked with p-tert-butylthiacliax[4]arene core in the cone, partial cone, and 1,3-alternate configurations. The roles of macrocycle configuration and electrolysis conditions on the silver depostion were characterized and optimal conditions selected for the subsequent immobilization of acetylcholinesterase. Silver nanoparticles provide higher response at low working potential (0.05?V) due to the electrostatic accumulation of silver ions and prevention of their leaching after reoxidation. The biosensor allows the determination of 10^?12 – 10^?7 M donepezil, berberine, and huperzine A within 20-30?s by the relative decay of the current related to the oxidation of thiocholine formed in enzymatic reaction. The reversible inhibition of immobilized acetylcholinesterase with huperzine A was quantified for the first time. The developed biosensor was employed for the analysis of spiked urine samples.     

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.

Improved Performance of Lipase Immobilized on Tannic Acid-Templated Mesoporous Silica Nanoparticles

Mesoporous silica nanoparticles were synthesized by using tannic acid as a pore-forming agent, which is an environmentally friendly, cheap, and non-surfactant template. SEM and TEM images indicated that the tannic acid-templated mesoporous silica nanoparticles (TA-MSNs) are monodisperse spherical-like particles with an average diameter of 195?±?16 nm. The Brunauer–Emmett–Teller (BET) results showed that the TA-MSNs had a relatively high surface area (447 m²/g) and large pore volume (0.91 cm³/g), and the mean pore size was ca. 10.1 nm. Burkholderia cepacia lipase was immobilized on the TA-MSNs by physical adsorption for the first time, and the properties of immobilized lipase (BCL@TA-MSNs) were investigated. The BCL@TA-MSNs exhibited satisfactory thermal stability; strong tolerance to organic solvents such as methanol, ethanol, isooctane, n-hexane, and tetrahydrofuran; and high operational reusability when BCL@TA-MSNs were applied in esterification and transesterification reactions. After recycling 15 times in the transesterification reaction for biodiesel production, over 85 % of biodiesel yield can be maintained. With these desired characteristics, the TA-MSNs may provide excellent candidates for enzyme immobilization.     

Development of a tyrosinase biosensor based on gold nanoparticles-modified glassy carbon electrodes: Application to the measurement of a bioelectrochemical polyphenols index in wines

The preparation of a tyrosinase biosensor based on the immobilization of the enzyme onto a glassy carbon electrode modified with electrodeposited gold nanoparticles (Tyr-nAu-GCE) is reported. The enzyme immobilized by cross-linking with glutaraldehyde retains a high bioactivity on this electrode material. Under the optimized working variables (a Au electrodeposition potential of −200 mV for 60 s, an enzyme loading of 457 U, a detection potential of −0.10 V and a 0.1 mol l⁻¹ phosphate buffer solution of pH 7.4 as working medium) the biosensor exhibited a rapid response to the changes in the substrate concentration for all the phenolic compounds tested: phenol, catechol, caffeic acid, chlorogenic acid, gallic acid and protocatechualdehyde. A R.S.D. of 3.6% (n = 6) was obtained from the slope values of successive calibration plots for catechol with the same Tyr-nAu-GCE with no need to apply a cleaning procedure to the biosensor. The useful lifetime of one single biosensor was of at least 18 days, and a R.S.D. of 4.8% was obtained for the slope values of catechol calibration plots obtained with five different biosensors. The kinetic constants and the analytical characteristics were calculated for all the phenolic compounds tested. The Tyr-nAu-GCE was applied for the estimation of the phenolic compounds content in red and white wines. A good correlation of the results (r = 0.990) was found when they were plotted versus those obtained by using the spectrophotometric method involving the Folin-Ciocalteau reagent.