Cysteamine Self-assembled Monolayer Research Articles

A QCM-D-based biosensor for E. coli O157:H7 highlighting the relevance of the dissipation slope as a transduction signal

A biosensor for detection of viable Escherichia coli (E. coli) O157:H7 is developed using a quartz crystal microbalance with dissipation monitoring (QCM-D). The detection platform is based on the immobilization of affinity purified polyclonal antibodies onto gold-coated QCM-D quartz crystals via a cysteamine self-assembled monolayer. QCM-D measurements conducted over a broad range of bacterial cell concentrations show that the optimal biosensor response is the initial slope of the dissipation shift as a function of elapsed time (Dslope). A highly log–log linear response in the initial Dslope is obtained for detection of E. coli O157:H7 over a wide range of cell concentrations from 3×105 to 1×109cells/mL. The prepared biosensor also exhibits a log–log linear working range from 107 to 109cells/mL for E. coli K12 D21, a non-pathogenic model organism and further shows satisfactory selectivity using Bacillus subtilis. To our knowledge, this is the first study demonstrating the use of the initial Dslope as a sensor response when using QCM-D technology.

Multifunctional Polypeptide EQCN Sensors: Probing the Cysteamine-Glutathione Film Permeability with Hg(II) Ions.

Multifunctional films are the basis of biosensors and play an important role in the emerging field of nanobioelectronics. In this work, films of a tripeptide glutathione (GSH) immobilized on a self-assembled monolayer of cysteamine (CA-SAM) on a quartz crystal Au piezosensor have been synthesized and characterized using electrochemical quartz crystal nanogravimetry (EQCN) with a Hg(II) ion probe. It has been found that in contrast to previously studied Au/GSH films, the Au/CA-GSH films strongly hinder the formation of Hg0 with bulk properties while still allowing for relatively easy permeation by Hg(II) ions. This results in complete disappearance of the sharp Hg0 electrodissolution peak which is observed on bare Au and Au/GSH piezosensors. The multiple-peak anodic behavior of Au/CA and bare Au is replaced by a single high-field anodic peak of mercury reoxidation in the case of Au/CA-GSH sensors. The mass-to-charge plots indicate predominant ingress/egress of Hg(II) to/from the film. The strong hindrance of CA-SAM to bulk-Hg0 formation is attributed to film-stabilizing formation of surface (CA)2Hg2+ complexes with conformation evaluated by ab initio quantum mechanical calculations of electronic structure using Hartree-Fock methods. The associates CA-GSH provide an additional functionality of the side sulfhydryl group which is free for interactions, e.g. with heavy metals. It is proposed that in the film, the CA-GSH molecules can assume open (extended) conformation or bent hydrogen-bonded conformation with up to four possible internal hydrogen bonds.

Culture of skeletal muscle cells in unprecedented proximity to a gold surface

Culturing of skeletal muscle cells on conductive surfaces is required to develop electronic device-muscle junctions for tissue engineering and medical applications. We characterized from a molecular and morphological point of view myogenic cells cultured on gold and on cysteamine-coated gold, as compared to the standard plastic for cell culture. Our results show that cell proliferation and survival are comparable between cells grown on either of the gold surface or plastic. The majority of the cells cultured on gold surfaces retain the ability to respond to differentiation cues, as shown by nuclear translocation of myogenin. Following terminal differentiation, the myotubes cultured on cysteamine-coated gold resemble myotube cultures obtained on plastic for the size and orientation of the myotube bundles retaining most of myosin expression; on the contrary, the myotube cultures on gold show a clumped morphology, likely due to repulsive cell-substratum interaction resulting in aberrant differentiation. On the basis of the aforementioned evidences, the culture of muscle cells on cysteamine-coated gold represents an advance with respect to previously reported substrata. The cysteamine self-assembled monolayer coating is a simple approach to accomplish cultures of myotubes in unprecedented tight proximity to conductive surfaces.

CdTe nanocrystal-based electrochemical biosensor for the recognition of neutravidin by anodic stripping voltammetry at electrodeposited bismuth film

CdTe quantum dots (QDs)-based electrochemical sensor for recognition of neutravidin, as a model protein, using anodic stripping voltammetry at electrodeposited bismuth film is presented. This biosensor involves the immobilization of the captured QDs conjugates which was dissolved with 1 M HCl solution to release cadmium ions and metal components were quantified by anodic stripping voltammetry after a 3-min accumulation at −1.2 V on bismuth-film electrode (BiFE) of the biotin, served as recognition element, onto the gold surface in connection with a cysteamine self-assembled monolayer. The modification procedure was characterized by electrochemical impedance spectroscopy and atomic force microscopy. We exploit QDs as labels for amplifying signal output and monitoring the extent of competition process between CdTe-labeled neutravidin and the target neutravidin for the limited binding sites on biotin. As expected for the competitive mechanism, the recognition event thus yields distinct cadmium stripping voltammetric current peak, whose response decreases upon increasing the level of target neutravidin concentrations. Under optimal conditions, the voltammetric response is highly linear over the range of 0.5–100 ng L −1 neutravidin and the limit of detection is estimated to be 0.3 ng L −1 (5 nM). Unlike earlier two-step sandwich bioassays, the present protocol relies on a one-step competitive assay, which is more accurate and sensitive, showing great promise for rapid, simple and cost-effective analysis of protein.

Development of acetylcholinesterase biosensor based on CdTe quantum dots modified cysteamine self-assembled monolayers

In this paper, a simple method for immobilization of acetylcholinesterase (AChE) on cysteamine self assembled monolayers modified gold electrode coupled with CdTe quantum dots (QDs) was proposed and thus a sensitive, fast and stable amperometric biosensor for quantitative determination of carbaryl was developed. The fabrication procedure was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and fluorescence spectrum. The nanoparticles of CdTe QDs led to an increased effective surface area for immobilization of enzyme and their electrocatalytic activity promoted electron transfer reactions and catalyzed the electro-oxidation of thiocholine, thus amplifying the detection sensitivity. Due to the notable decrease in voltammetric signal of the immobilized AChE, a simple method for determination of carbaryl was established. The inhibition of carbaryl was proportional to its concentration in two ranges, from 1 to 50 ng mL −1 and from 50 to 500 ng mL −1, with a detection limit of 0.6 ng mL −1. The constructed biosensor which processes prominent characteristics and performance, such as good precision and reproducibility, acceptable stability and accuracy, fast response and low detection limit, has potential application in the characterization of enzyme inhibitors and detection of toxic compounds against to enzyme.

Hydroxamation of gold surface via in-situ layer-by-layer functionalization of cysteamine self-assembled monolayer: Preparation and electrochemical characterization

A new, simple, and easy method for introducing hydroxamic acid group onto the surface of polycrystalline gold electrode by means of in-situ layer-by-layer functionalization is described. The fabrication was performed in a four-step method: (i) modification of gold by cysteamine self-assembled monolayer, Au–CA SAM, (ii) activation of nitrilotriacetic acid (NTA) by 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), (iii) immobilization of activated NTA onto Au–CA to form Au–CA–NTA, and (iv) conversion of the remaining activated carboxylic acid groups of Au–CA–NTA (terminals) to hydroxamic acid groups by using hydroxylamine hydrochloride (HAH) to form Au–CA–NDHA modified electrode. The resulting modified electrode was successfully tested for accumulation of zirconium ion (Zr(IV)) from an aqueous acidic solution. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were used to trace the events in each step, characterize the surface, determine the surface pKas, and find the affinity of the prepared electrode towards the Zr(IV). Surface pKas equal to 6.5 and 5.5 were estimated for Au–CA–NTA, and Au–CA–NDHA SAM electrodes, respectively. These values allowed estimating the charge-state of the surface at any pHs. The modified surface showed a large affinity for selective accumulation of Zr(IV) from acidic solution.

Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.

Application of DNA Hybridization Biosensor as a Screening Method for the Detection of Genetically Modified Food Components.

An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe) specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5'-phosphate end to amino group of cysteamine self-assembled monolayer (SAM) on gold electrode surface with the use of activating reagents - water soluble 1-ethyl-3(3'- dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-sulfosuccinimide (NHS). The hybridization reaction on the electrode surface was detected via methylene blue (MB) presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean) and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs) give a broad perspectives for analytical application of the biosensor.

Self-assembled monolayers as a base for immunofunctionalisation: unequal performance for protein and bacteria detection

Biosensor development strongly depends on the optimisation of surface functionalisation strategies. When gold surfaces are considered, immunofunctionalisation by modification of self-assembled monolayers (SAMs) is one of the preferred approaches. In this respect, SAM-based antibody (Ab) incorporation has shown better performance than Ab physisorption for the detection of proteins and small targets. Reports on bacteria detection are less frequent. In this work, we assess the performance of various SAM-based gold immunofunctionalisation strategies, currently applied to protein detection, in the field of bacteria determination. We present the results for Ab chemical conjugation on mercaptopropanoic acid and mercaptoundecanoic acid SAMs, as well as on a dextranized cysteamine SAM. All the modified surfaces studied were shown to be appropriate for the direct detection of an enzyme-labelled protein, but none succeeded in detecting a bacterial target in a sandwich assay format. Conversely, gold functionalised by Ab physisorption allowed E. coli detection when a sandwich enzyme-linked assay was carried out. The implications of bacteria size and wall complexity are discussed. These results indicate that immunofunctionalisation strategies appropriate for protein detection are not necessarily transferable to work with more complex targets such as bacteria. In this respect, Ab physisorption appears to be a suitable alternative to SAM-based gold functionalisation for bacteria detection.

In situ monitoring of the orientated assembly of strep-tagged membrane proteins on the gold surface by surface enhanced infrared absorption spectroscopy

Surface enhanced infrared absorption spectroscopy (SEIRAS) has been employed to monitor the orientated assembly of a strep-tagged membrane protein on the gold surface via a streptavidin/biotin interlayer. The high surface sensitivity of SEIRAS allows for tracking the individual assembling steps on the molecular level. The sequence of surface modification steps comprises: (i) cross-linking of biotin to the self-assembled monolayer of cysteamine along the gold surface; (ii) adsorption of streptavidin to and desorption from the biotin layer; and (iii) adsorption of the strep-tagged membrane protein ecgltP (glutamate transporter of E. coli) on the streptavidin/biotin layer. The analysis of the SEIRA spectra reveals that the biotin layer undergoes a phase transition from an isotropic orientation to a densely packed layer during coupling to the cysteamine monolayer. Formation of the densely packed layer weakens the interaction between streptavidin and the biotin layer but yields a binding specificity of 80%. The specificity of strep-tagged ecgltP to the streptavidin layer is with 60% only modest. Nevertheless, the streptavidin/biotin interlayer reveals a higher regeneration propensity than the His-tag/Ni-NTA interlayer.

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 (Aucoll–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–Aucoll–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–Aucoll–Cyst–AuE biosensor provided a calibration plot for inulin in the (5–100)×10−6 M linear range, with a detection limit of 6.6×10−7 mol L−1. One single bienzyme biosensor responded within the control limits, set at ±3× 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.

Detection of uranium(VI) in aqueous solution by a calix[6]arene modified electrode

This work describes the attempts to develop a new type of modified electrode with high selectivity towards uranyl ions, UO 2 2 + . This goal has been addressed by binding derivatives of calix[6]arene, which is a macrocyclic molecule composed of six phenol units, known as “super uranophile”, to the electrode surface. Selective extraction of UO 2 2 + onto the electrode surface, followed by a voltammetric measurement provides a quantitative uranyl analysis. Our initial approach to binding calix[6]arene molecules onto electrodes has involved an ionic derivative, 4-sulfonic-calix[6]arene, which was electrostatically attached to a positively-charged self-assembled monolayer of cysteamine (2-aminoethanethiol) on a gold electrode. The attachment of the 4-sulfonic-calix[6]arene to the cysteamine monolayer was verified by XPS, IR spectroscopy and capacitance measurements. Voltammetric experiments showed that uranyl ions can be preconcentrated and measured by these modified electrodes.

Electrochemical fabrication and electrocatalytic characteristics studies of gold nanopillar array electrode (AuNPE) for development of a novel electrochemical sensor

Gold nanopillar array electrodes were prepared by electrochemical deposition of gold into the nanopores of anodic aluminum oxide membrane placed onto the gold thin film electrode surface, which was in advance modified with cysteamine self-assembled monolayer as an anchoring layer. The Au nanopillar electrode is electrochemically stable and consists of highly dense, upstanding pillars assembled on the cysteamine monolayer. The structural morphology and chemical composition of the nanoarray electrode was characterized by field emission scanning electron microscopy, X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction. Electrochemical measurements indicate that the Au nanopillar electrode possesses high electrocatalytic activities in the reduction of hydrogen peroxide and molecular oxygen, especially in glucose oxidation due to its higher electroactive surface area. The electro-oxidation studies of several electroactive neurotransmitters demonstrate that the nanopillar electrode can be utilized as a promising material for the construction of novel electrochemical sensor.

Functionalization of gold cysteamine self-assembled monolayer with ethylenediaminetetraacetic acid as a novel nanosensor

Electrochemical characterization of gold cysteamine self-assembled monolayer, in situ functionalized with ethylenediaminetetraacetic acid (Au–CA–EDTA SAM), is described by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and Osteryoung square wave voltammetry (OSWV). The results obtained by EIS and CV, in the presence of [Fe(CN) 6] 3−/4− redox probe, show that EDTA is successfully grafted to the surface of Au–CA electrode. Reproducible and reversible variation of the R ct and ΔEp as a function of solution pH show that Au–CA–EDTA SAM is stable in a wide range of pH and potentials. Accumulation of the Pb 2+ and Cu 2+ ions on the Au–CA–EDTA SAM electrode is investigated using faradaic currents or impedimetric effects measured by OSWV and EIS, respectively. These results reveal the presence of active complexing functional groups of EDTA on the surface, and thus, the formation of Au–CA–EDTA SAM electrode. The new sensor responds to the Pb 2+ and Cu 2+ separately and simultaneously in a wide linear range of concentrations.

Amperometric glucose biosensor based on electrodeposition of platinum nanoparticles onto covalently immobilized carbon nanotube electrode

A new amperometric biosensor for glucose was developed based on adsorption of glucose oxidase (GOx) at the gold and platinum nanoparticles-modified carbon nanotube (CNT) electrode. CNTs were covalently immobilized on gold electrode via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM). The fabricated GOx/Au nano/Pt nano/CNT electrode was covered with a thin layer of Nafion to avoid the loss of GOx in determination and to improve the anti-interferent ability. The immobilization of CNTs on the gold electrode was characterized by quartz crystal microbalance technique. The morphologies of the CNT/gold and Pt nano/CNT/gold electrodes have been investigated by scanning electron microscopy (SEM), and the electrochemical performance of the gold, CNT/gold, Pt nano/gold and Pt nano/CNT/gold electrodes has also been studied by amperometric method. In addition, effects of electrodeposition time of Pt nanoparticles, pH value, applied potential and electroactive interferents on the amperometric response of the sensor were discussed. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for glucose in the absence of a mediator. The linear range was from 0.5 to 17.5 mM with correction coefficient of 0.996. The biosensor had good reproducibility and stability for the determination of glucose.

Copper(II) Nanosensor Based on a Gold Cysteamine Self-Assembled Monolayer Functionalized with Salicylaldehyde

Fabrication and electrochemical characterization of a novel nanosensor for determination of Cu2+ in subnanomolar concentrations is described. The sensor is based on gold cysteamine self-assembled monolayer functionalized with salicylaldehyde by means of Schiff's base formation. Cyclic voltammetry, Electrochemical impedance spectroscopy (EIS), and electrochemical quartz crystal microbalance were used to probe the fabrication and characterization of the modified electrode. The sensor was used for quantitative determination of Cu2+ by the EIS in the presence of parabenzoquinone in comparison with stripping Osteryoung square wave voltammetry (OSWV). The attractive ability of the sensor to efficiently preconcentrate trace amounts of Cu2+ allowed a simple and reproducible method for copper determination. A wide range linear calibration curve was observed, 5.0 x 10(-11)-5.0 x 10(-6) and 5.0 x 10(-10)-5.0 x 10(-6) M Cu2+, by using the EIS and OSWV, respectively. Moreover, the sensor presented excellent stability with lower than 10% change in the response, as tested for more than three months daily experiments, and a high repeatability with relative standard deviations of 6.1 and 4.6% obtained for a series of eight successive measurements in 5.0 x 10(-7) M Cu2+ solution, by the EIS and OSWV, respectively.

Impedimetric sensing of uranyl ion based on phosphate functionalized cysteamine self-assembled monolayers

A phosphate functionalized cysteamine self-assembled monolayer based on gold electrode is designed for uranyl ion (UO 2 2+) detection. The response of the modified electrode is studied by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry. The EIS data are approximated using constant phase element (CPE) model from which kinetic and analytical parameters are evaluated. Uranyl ion is recognized based on blocking effect against charge transfer between p-benzoquinone as a probe and the modified electrode. This effect is detected from linear variation of charge transfer resistance ( R ct) as a function of UO 2 2+ concentration. From the analysis of the EIS data and approximated parameters, a method is developed for UO 2 2+ determination based on impedimetric measurements.

Determination of dopamine in the presence of high concentration of ascorbic acid by using gold cysteamine self-assembled monolayers as a nanosensor

A monolayers of cysteamine (CA) was prepared on a polycrystalline gold electrode through self-assembly procedure to produce a gold cysteamine self-assembled monolayers (Au-CA SAMs) modified electrode. Characterization of the modified electrode was performed by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The EIS was used to evaluate p K a of the adsorbed CA on the gold surface, and a value of 7.6 was obtained for the Au-CA surface p K a. The charged terminal groups of monolayers served for determination of dopamine (DA) in the presence of high concentration of ascorbic acid (AA) using differential pulse voltammetry (DPV). Well-separated DA and AA voltammetric waves (∼330 mV) were observed at the Au-CA SAMs electrodes in an acidic solution. A calibration curve with two linear parts was obtained for DA, 6.00 × 10 −6 to 3.84 × 10 −4 M and 3.36 × 10 −4 to 9.50 × 10 −3 M, with correlation coefficients 0.997 and 0.992, respectively. The detection limit for DA was found to be 2.31 μM in the presence of 1.0 mM AA. The apparent charge transfer rate constants ( k app) of AA and DA were evaluated by using EIS measurements on the modified electrode as 44.0 cm s −1 × 10 −8 cm s −1 and 2.45 cm s −1 × 10 −8 cm s −1, respectively.

Preparation and electrochemical characterization of a new nanosensor based on self-assembled monolayer of cysteamine functionalized with phosphate groups

Fabrication and electrochemical characterization of a self-assembled three layers modified gold electrode is described. The modification involves a three-step method; (i) preparation of cysteamine self-assembled monolayer, Au-CA, (ii) activation of Au-CA by glutaraldehyde to prepare Au-CA-GA, (iii) modification of Au-CA-GA by 2-Aminoethyl dihydrogen phosphate to functionalize the surface by phosphate groups, Au-CA-GA-AEDP. The resulting thin film modified electrode was tested successfully to recognize uranyl cations (UO 2 2+) in aqueous solution. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to trace events in each step. The high affinity of the film towards the UO 2 2+ was confirmed by EIS and CV results. The EIS data allowed recognition of UO 2 2+ based on variation of the charge-transfer resistance ( R ct) of the film as a function of the cation concentration. A dynamic range with more than three orders of magnitude was obtained.

Electrochemical sensor for immunoassay of carcinoembryonic antigen based on thionine monolayer modified gold electrode

A sensor based on thionine monolayer modified gold electrode for determination of carcinoembryonic antigen (CEA) in human serum is proposed. The sensor is prepared by covalently binding thionine to a cysteamine self-assembled monolayer with p-phthaloyl chloride as a linkage, which gives a surface coverage of 8.97 ± 3.28 × l0 −12 mol/cm 2 for thionine. The electrochemistry of the immobilized thionine displays a surface-controlled electrode process with an average electron transfer rate constant of 1.47 ± 0.84 s −1. Based on an electrochemical enzyme-linked immunoassay by using the immobilized thionine as an electron transfer mediator between the electrode and the horseradish peroxidase (HRP) labeled anti-CEA antibody, a calibration curve with two linear ranges from 0.6 to 17 and 17 to 200 ng/mL and a detection limit of 0.2 ng/mL for CEA determination is obtained in pH 4.2 PBS containing 2.0 mmol/L H 2O 2 and 0.5 mol/L NaCl. The sensor shows a good accuracy. The precision and reproducibility are acceptable with the intra-assay CV of 4.9% and 5.9% at 10 and 100 ng/mL CEA concentrations, respectively, and the inter-assays CV of 7.8% at 100 ng/mL CEA. The response of thionine modified electrode shows only 1.6% decrease after 100 replicate measurements and the storage stability is acceptable in a pH 7.0 PBS at 4 °C for 1 week. The method avoids the addition of electron transfer mediator to the solution, thus is much simpler. The proposed method would be valuable for the diagnosis and monitoring of carcinoma and its metastasis.