Bienzyme amperometric biosensor using gold nanoparticle-modified electrodes for the determination of inulin in foods

A biosensor design involving coimmobilization of fructose dehydrogenase (FDH) and inulinase (INU) on a gold nanoparticle-cysteamine (Cyst) self-assembled monolayer (SAM)-modified gold electrode (Au(coll)-Cyst-AuE), for the determination of the carbohydrate inulin in foodstuffs, is reported. Tetrathiafulvalene (TTF), used as the mediator, was also coimmobilized by crosslinking with glutaraldehyde. INU catalyzes the hydrolysis of inulin, forming fructose that is detected through the fructose dehydrogenase system by the electrochemical oxidation of TTF at the bioelectrode. The variables involved in the preparation and performance of both the single enzyme FDH biosensor and the bienzyme inulin biosensor were optimized. The FDH-Au(coll)-Cyst-AuE biosensor exhibited rapid and sensitive response to fructose, allowing the obtention of improved analytical characteristics for the determination of fructose with respect to other FDH electrochemical biosensors. Moreover, the lifetime of this biosensor was 35 days. The bienzyme INU/FDH-Au(coll)-Cyst-AuE biosensor provided a calibration plot for inulin in the (5-100)x10(-6) M linear range, with a detection limit of 6.6 x 10(-7) mol L(-1). One single bienzyme biosensor responded within the control limits, set at +/-3x the standard deviation of the currents measured on the first day of use, for more than 5 months. Furthermore, the biosensor exhibited high selectivity with respect to other carbohydrates. The usefulness of the biosensor was evaluated by the rapid determination of inulin in food products involving minimization of the fructose interference.     

Molecular imprinted nanoelectrodes for ultra sensitive detection of ovarian cancer marker

The relentless discovery of cancer biomarkers demands improved methods for their detection. In this work, we developed protein imprinted polymer on three-dimensional gold nanoelectrode ensemble (GNEE) to detect epithelial ovarian cancer antigen-125 (CA 125), a protein biomarker associated with ovarian cancer. CA 125 is the standard tumor marker used to follow women during or after treatment for epithelial ovarian cancer. The template protein CA 125 was initially incorporated into the thin-film coating and, upon extraction of protein from the accessible surfaces on the thin film, imprints for CA 125 were formed. The fabrication and analysis of the CA 125 imprinted GNEE was done by using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The surfaces of the very thin, protein imprinted sites on GNEE are utilized for immunospecific capture of CA 125 molecules, and the mass of bound on the electrode surface can be detected as a reduction in the faradic current from the redox marker. Under optimal conditions, the developed sensor showed good increments at the studied concentration range of 0.5-400 U mL(-1). The lowest detection limit was found to be 0.5 U mL(-1). Spiked human blood serum and unknown real serum samples were analyzed. The presence of non-specific proteins in the serum did not significantly affect the sensitivity of our assay. Molecular imprinting using synthetic polymers and nanomaterials provides an alternative approach to the trace detection of biomarker proteins.     

Highly sensitive amperometric immunosensors for microcystin detection in algae

The presence of cyanobacterial toxins in water and algae pose a health hazard for animals and humans, due to their tumour-promoting activity and carcinogen effects. The use of simple, rapid and reliable tools for routine analysis is becoming a necessity. With this purpose, our group has developed two electrochemical immunosensors for the detection of microcystin-LR (MC-LR) based on the affinity between this cyanotoxin and the corresponding monoclonal and polyclonal antibodies. A competitive direct enzyme-linked immunosorbent assays (ELISAs) was designed and, after validation of the approach on microtiter wells, screen-printed graphite electrodes were used as supports. Colorimetry was used to optimise the experimental parameters and to compare the performance of monoclonal and polyclonal antibodies. Afterwards, electrochemical measurements were performed at -200 mV (versus Ag/AgCl) using 5-methyl-phenazinium methyl sulfate (MPMS) as mediator for horseradish peroxidase (HRP), the enzymatic label of the competitor. The IC(50) values were 0.10 and 1.73 microgL(-1) for MAb and PAb, respectively. Whereas Mab provided higher sensitivities, the reproducibility was better when using PAb. The developed amperometric immunosensors were applied to the analysis of cyanobacterial samples from the Tarn River (Midi-Pyrénées, France) and the presence of MC was confirmed by the colorimetric protein phosphatase inhibition (PPI) assay and high performance liquid chromatography (HPLC). The limits of detection attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of the immunosensors as screening tools for routine use in the assessment of water quality and the control of toxins in algae.     

Highly sensitive amperometric immunosensor for the detection of Escherichia coli

With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH₂-anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole (Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl β-d-galactopyranoside (PAPG), the bacterial enzyme, β-d-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC) electrode at 0.22 V. The operational procedure consists in first adding the test sample containing the bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte (being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates an improved detection limit of more than 10 times less bacteria detected than other immunosensing techniques without the need for multi step pretreatments of the test sample and/or incubation as found in some of the existing methods.     

Covalent immobilization of cholesterol oxidase on self-assembled gold nanoparticles for highly sensitive amperometric detection of cholesterol in real samples

A novel scheme for the fabrication of gold nanoparticle modified cholesterol oxidase based bioelectrode is presented and its application potential for cholesterol biosensor is investigated. The fabrication procedure is based on the deposition of gold nanoparticles on the 1,6-hexanedithiol modified gold electrode, functionalization of the surface of deposited gold nanoparticles with carboxyl groups using 11-mercaptoundecanoic acid and then covalent immobilization of cholesterol oxidase on the surface of gold nanoparticle film using the N-ethyl-N'-(3-dimethylaminopropyl carbodimide) and N-hydroxysuccinimide ligand chemistry. The assembly process of the bioelectrode is investigated using atomic force microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticle film on the electrode surface provided an environment for the enhanced electrocatalytic activities and thus resulted in enhanced analytical response. The resulting bioelectrode is further applied to the amperometric detection of cholesterol and exhibited a linear response to cholesterol in the range of 0.04-0.22 mM with a detection limit of 34.6 μM, apparent Michaelis-Menten constant (K(m)(app)) of 0.062 mM and a high sensitivity of 9.02 μA mM(-1). The fabricated bioelectrode is successfully used for the selective determination of cholesterol in human serum samples.     

Highly sensitive amperometric detection of genomic DNA in animal tissues

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase– avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 µl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT–PCR mixture.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

A label-free amperometric immunosensor for fast and sensitive assay of human serum chorionic gonadotropin (hCG) is presented. hCG was immobilized on nanoporous gold (NPG) foils and using hydroquinone (HQ) redox species as indicator. The variation of amperometric response to the concentration of hCG, the target antigen, was evaluated by cyclic voltammetry in phosphate-buffered solution. Taking advantage of dual-amplification effects of the NPG foils and graphene sheets (GSs), the immunosensor exhibited a specific response to hCG in the range of 0.5–40.00 ng ml⁻¹ with a detection limit of 0.034 ng ml⁻¹ under optimal conditions. It was demonstrated that our proposed method possesses good accuracy, acceptable precision, and reproducibility. The NPG showed a better sensitizing effect and stability as immobilization matrices.     

Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene

Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.     

Immobilization of acetylcholinesterase on gold nanoparticles embedded in sol-gel film for amperometric detection of organophosphorous insecticide

A simple method to immobilize acetylcholinesterase (AChE) on silica sol-gel (SiSG) film assembling gold nanoparticles (AuNPs) was proposed, thus a sensitive, fast and stable amperometric sensor for quantitative determination of organophosphorous insecticide was developed. The large quantities of hydroxyl groups in the sol-gel composite provided a biocompatible microenvironment around enzyme molecule and stabilized its biological activity to a large extent. The immobilized AChE could catalyze the hydrolysis of acetylthiocholine chloride (ATCl) with a Kmapp value of 450 microM to form thiocholine, which was then oxidized to produce detectable single with a linear range of 10-1000 microM. AuNPs catalyzed the electro-oxidation of thiocholine, thus increasing detection sensitivity. Based on the inhibition of organophosphorous insecticide on the enzymatic activity of AChE, using monocrotophos as a model compound, the conditions for detection of the insecticide were optimized. The inhibition of monocrotophos was proportional to its concentration ranging from 0.001 to 1 microg/ml and 2 to 15 microg/ml, with the correlation coefficients of 0.9930 and 0.9985, respectively. The detection limit was 0.6 ng/ml at a 10% inhibition. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors.     

Graphene-assisted dual amplification strategy for the fabrication of sensitive amperometric immunosensor

A sensitive electrochemical immunosensor with graphene-assisted signal amplification has been developed. In order to construct the base of the immunosensor, a novel hybrid architecture was initially fabricated by combining poly (diallyldimethylammonium chloride) functionalized graphene nanosheets (PDDA-G) and gold nanoparticles (AuNPs) via a simple sonication-induced assembly. The formed hybrid architecture provided an effective matrix for antibody immobilization with good stability and bioactivity. Subsequently, a smart, multilabel, and graphene-based nanoprobe that contains gold nanoparticles functionalized exfoliated graphene oxide and horseradish peroxidase-secondary antibodies was designed for constructing a novel sandwiched electrochemical immunosensor. Enhanced sensitivity was obtained by combining the advantages of high-binding capability and excellent electrical conductivity of hybrid architecture with the multilabel signal amplification. On the basis of the dual signal amplification strategy of graphene-based architecture and the multilabel, the immunosensor displayed excellent analytical performance for the detection of human IgG (HIgG) range from 0.1 to 200 ng/mL with a detection limit of 0.05 ng/mL at 3σ. Moreover, the proposed method showed good precision, acceptable stability and reproducibility, and could be used for the detection of HIgG in real samples. Therefore, the present strategy definitely paves a way for the wide application of graphene in clinical research.     

Immobilization of enzyme on long period grating fibers for sensitive glucose detection

Glucose oxidase (GOD) immobilized long period grating (LPG) fibers have been proposed for the specific and sensitive detection of glucose. The treatment of LPG fibers with aminopropyl triethoxysilane has induced biding sites for the subsequent GOD immobilization. Field emission scanning electron microscopy, confocal laser scanning microscopy, infrared spectroscopy and Raman spectroscopy have provided detailed evidences about the effectiveness of the adopted biofunctionalization methodology. The enzyme activity is conserved during the immobilization step. Fabricated LPG sensor was tested on different glucose solutions to record the transmission spectra on an optical spectrum analyzer. The wavelength shifts in the transmission spectra are linearly correlated with the glucose concentration in the range of 10-300 mg dL(-1). The fabricated sensor gives fast response and is demonstrated to be of practical utility by determining glucose contents in blood samples. Proposed technique can further be extended to develop LPG fiber based novel, sensitive and label free nanosensors for disease diagnosis and clinical analysis.     

Biomolecule-stabilized Au nanoclusters as a fluorescence probe for sensitive detection of glucose

In this work, biomolecule-stabilized Au nanoclusters were demonstrated as a novel fluorescence probe for sensitive and selective detection of glucose. The fluorescence of Au nanoclusters was found to be quenched effectively by the enzymatically generated hydrogen peroxide (H(2)O(2)). By virtue of the specific response, the present assay allowed for the selective determination of glucose in the range of 1.0×10(-5) M to 0.5×10(-3) M with a detection limit of 5.0×10(-6) M. The absorption spectroscopy, X-ray photoelectron spectroscopy (XPS) and fluorescence decay studies were then performed to discuss the quenching mechanism. In addition, we demonstrated the application of the present approach in real serum samples, which suggested its great potential for diagnostic purposes.     

Label-free colorimetric immunoassay for the simple and sensitive detection of neurogenin3 using gold nanoparticles

Neurogenin3 (ngn3), as a marker for pancreatic endocrine precursor cells and an essential ingredient in the development of islet cells, was quantitatively detected for the first time. Based on a non-cross-linking specific interaction mechanism, a label-free colorimetric immunoassay for the synthetic peptide fragment of ngn3 (SKQRRSRRKKAND) using glutathione (-Glu-Cys-Gly, GSH) functionalized gold nanoparticles (GNPs) is reported. The anti-ngn3 antibody conjugated GNPs (GNP-Ab) was formed through electrostatic interaction upon the addition of anti-ngn3 antibody to the GSH-modified GNPs solution. Monobinding of the positively charged ngn3 to the negatively charged GNP-Ab will minimize the electrostatic repulsion between nanoparticles by neutralizing the surface charge, and then agglomeration is induced by an increasing salt concentration. Under the optimal conditions, the assay showed a linear response range of 50-300 ng/mL for the peptide with a detection limit being 20 ng/mL. The preliminary study on ngn3 opens up an innovative insight to detect short synthetic peptide fragment of antigen, and may own an opportunity for practical applications in clinical diagnosis and therapeutics.     

A probe for NADH and H2O2 amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor applications

Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.     

Highly sensitive fluorescent detection of trypsin based on BSA-stabilized gold nanoclusters

In this study, fluorescent metal nanoclusters are presented as novel probes for sensitive detection of protease for the first time. The sensing mechanism is based on trypsin digestion of the protein template of BSA-stabilized Au nanoclusters. The decrease in fluorescence intensity of BSA-Au nanoclusters caused by trypsin allows the sensitive detection of trypsin in the range of 0.01-100 μg/mL. The detection limit for trypsin is 2 ng/mL (86 pM) at a signal-to-noise ratio of 3. The present nanosensor for trypsin detection possesses red emission, excellent biocompatibility, high selectivity, and good stability. In addition, we demonstrated the application of the present approach in real urine samples, which suggested its potential for diagnostic purposes.     

Electroless-plated gold films for sensitive surface plasmon resonance detection of white spot syndrome virus

The paper describes the rapid and label-free detection of the white spot syndrome virus (WSSV) using a surface plasmon resonance (SPR) device based on gold films prepared by electroless plating. The plating condition for obtaining films suitable for SPR measurements was optimized. Gold nanoparticles adsorbed on glass slides were characterized by transmission electron microscopy (TEM). Detection of the WSSV was performed through the binding between WSSV in solution and the anti-WSSV single chain variable fragment (scFv antibody) preimmobilized onto the sensor surface. Morphologies of the as-prepared gold films, gold films modified with self-assembled alkanethiol monolayers, and films covered with antibody were examined using an atomic force microscope (AFM). To demonstrate the viability of the method for real sample analysis, WSSV of different concentrations present in a shrimp hemolymph matrix was determined upon optimizing the surface density of the antibody molecules. The SPR device based on the electroless-plated gold films is capable of detecting concentration of WSSV as low as 2.5 ng/mL in 2% shrimp hemolymph, which is one to two orders of magnitude lower than the level measurable by enzyme-linked immunosorbant assays.     

A sensitive immunochromatographic assay using gold nanoparticles for the semiquantitative detection of prostate-specific antigen in serum

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.     

Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor

Glucose oxidase (GOD) was genetically modified by adding a poly-lysine chain at the C-terminal with a peptide linker inserted between the enzyme and poly-lysine chain. The poly-lysine chain was added in order to anchor more electron transfer mediator, ferrocenecarboxylic acid, to GOD for the purpose of improving sensitivity and stability of glucose biosensors. The modified GOD had similar K(m) and K(cat) to those of the wild type enzyme. After interacted with the electron transfer mediator, the modified enzyme retained 90.01% of its native activity, while the commercial GOD and the wild type GOD (Aspergillus niger) retained only 22.43 and 22.17%, respectively. Screen-printed electrodes coated with the modified GOD, wild type yeast-derived GOD or the commercial GOD were tested in glucose solution of different concentrations. Experimental results showed that the biosensor based on the modified GOD gave the largest signal among the three. In addition, the linear range of the biosensor prepared by the modified GOD could extend to 45 mM, while they were about 20 mM for the biosensors based on the wild type yeast-derived enzyme and the commercial enzyme.     

A novel NiO-Au hybrid nanobelts based sensor for sensitive and selective glucose detection

A novel amperometric nonenzymatic glucose sensor based on Au-doped NiO nanobelts has been successfully fabricated and applied to nonenzymatic glucose detection. Its electrochemical behavior towards the oxidation of glucose was compared with NiO nanofibers and Au microparticles prepared with a similar procedure. The NiO-Au hybrid nanobelts modified electrode displays greatly enhanced electrocatalytic activity towards glucose oxidation, revealing a synergistic effect between the matrix NiO and the doped Au. The as-prepared NiO-Au nanobelts based glucose sensor displays significantly lower onset potential, lower detection limit, higher sensitivity, and wider linear range than that of pristine NiO nanofibers modified electrode. Moreover, Au nanoparticles distributed in NiO nanofibers enabled amperometric glucose detection with insignificant interference from ascorbic acid and uric acid. These results indicate that the NiO-Au hybrid nanobelt is a promising candidate in the development of highly sensitive and selective nonenzymatic glucose sensors.     

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab₂) were performed by one-pot assembly of HRP and Ab₂ on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab₁) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml⁻¹, and the limit of detection was 30 pg ml⁻¹ (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.