Cysteamine-modified Gold Electrode Research Articles

Highly efficient direct electron transfer bioanode containing glucose dehydrogenase operating in human blood

Energy generation from bodily fluids (i. e. blood) is a significant challenge, which could be solved by designing high-performance direct electron transfer (DET) enzymatic electrodes. In this paper we report a highly efficient enzymatic DET bioanode based on glucose dehydrogenase, which is uniquely wired to polyaniline, gold nanoparticles and cysteamine modified gold electrode. The designed electrode demonstrates an exceptional performance by generating high chronoamperometric current density reaching 1 mA cm−2 at 0.1 V vs. SCE in the presence of 5 mM glucose, while in human blood samples the electrode generates average current density of 0.65 mA cm−2 at 0.1 V vs. SCE, which is the highest ever reported. Moreover, the electrodes demonstrate a good stability retaining 79 % and 23 % of the initial activity after the constant operation for 24 h in buffer solutions and blood samples, respectively. The designed electrode opens new possibilities for the development of improved implantable low-power electronics.

Electrochemical sensing and fluorescence imaging of E. coli O157:H7 based on aptamer-conjugated semiconducting nanoparticles

Aptamers are selective molecules against to various sizes of targets from small molecules to mammalian cells. Here, we reported QDs containing electrochemical aptasensor for the detection of E. coli O157:H7. The electrode surfaces were modified by cysteamine (CysN), which has amine and thiol groups, via self-assembled monolayer formation. The carboxyl-functionalized quantum dots (QD) and aptamers (Apt) were conjugated to cysteamine modified gold electrodes. Linear range for E. coli O157:H7 was from 1 to 10 2 CFU/mL after incubation on CysN/QD/Apt modified Au surfaces. QDs provide fluorescence surface, so that adhesion of cells was followed using fluorescence microscope. Adhered cells were also imaged by scanning electron microscopy. Finally, cell analysis was carried out in real samples.

Electrochemical Label-Free Aptasensor for Specific Analysis of Dopamine in Serum in the Presence of Structurally Related Neurotransmitters

Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, and as such, in vivo analysis of dopamine in the presence of structurally related neurotransmitters (NT) represents a holy grail of neuroscience. Interference from those NTs generally does not allow selective electroanalysis of dopamine, which redox transformation overlaps with those of other catecholamines. In our previous work, we reported an electrochemical RNA-aptamer-based biosensor for specific analysis of dopamine (Analytical Chemistry, 2013; Vol. 85, p 121). However, the overall design of the biosensor restricted its stability and impeded its operation in serum. Here, we show that specific biorecognition and electroanalysis of dopamine in serum can be performed by the RNA aptamer tethered to cysteamine-modified gold electrodes via the alkanethiol linker. The stabilized dopamine aptasensor allowed continuous 20 h amperometric analysis of dopamine in 10% serum within the physiologically important 0.1-1 μM range and in the presence of catechol and such dopamine precursors and metabolites as norepinephrine and l-DOPA. In a flow-injection mode, the aptasensor response to dopamine was ∼1 s, the sensitivity of analysis, optimized by adjusting the aptamer surface coverage, was 67 ± 1 nA μM(-1) cm(-2), and the dopamine LOD was 62 nM. The proposed design of the aptasensor, exploiting both the aptamer alkanethiol tethering to the electrode and screening of the catecholamine-aptamer electrostatic interactions, allows direct monitoring of dopamine levels in biological fluids in the presence of competitive NT and thus may be further applicable in biomedical research.

Tailored carbon nanotube immunosensors for the detection of microbial contamination.

The use of carbon nanotubes (CNTs) as building blocks in the design of electrochemical biosensors has been attracting attention over the last few years, mainly due to their high electrical conductivity and large surface area. Here, we present two approaches based on tailored single-walled CNTs (SWCNTs) architectures to develop immunosensors for the bacteriophage MS2, a virus often detected in sewage-impacted water supplies. In the first approach, SWCNTs were used in the bottom-up design of sensors as antibody immobilization support. Carboxy-functionalised SWCNTs were covalently tethered onto gold electrodes via carbodiimide coupling to cysteamine-modified gold electrodes. These SWCNTs were hydrazide functionalized by electrochemical grafting of diazonium salts. Site-oriented immobilization of antibodies was then carried out through hydrazone bond formation. Results showed microarray electrode behavior, greatly improving the signal-to-noise ratio. Excellent sensitivity and limit of detection (9.3 pfu/mL and 9.8 pfu/mL in buffer and in river water, respectively) were achieved, due to the combination of the SWCNTs' ability to promote electron transfer reactions with electroactive species at low overpotentials and their high surface-to-volume ratio providing a favorable environment to immobilize biomolecules. In the second approach, SWCNTs were decorated with iron oxide nanoparticles. Diazonium salts were electrochemically grafted on iron-oxide-nanoparticle-decorated SWCNTs to functionalize them with hydrazide groups that facilitate site-directed immobilization of antibodies via hydrazone coupling. These magnetic immunocarriers facilitated MS2 separation and concentration on an electrode surface. This approach minimized non-specific adsorptions and matrix effects and allowed low limits of detection (12 pfu/mL and 39 pfu/mL in buffer and in river water, respectively) that could be further decreased by incubating the magnetic immunocarriers with larger volumes of sample. Significantly, both approaches permitted the detection of MS2 to levels regularly encountered in sewage-impacted environments.

Nanohybrid Materials Consisting of Poly[(3‐aminobenzoic acid)‐co‐(3‐aminobenzenesulfonic acid)‐co‐aniline] and Multiwalled Carbon Nanotubes for Immobilization of Redox Active Cytochrome c

AbstractThe development of a new surface architecture for the efficient direct electron transfer of positively charged redox proteins is presented. For this reason different kinds of polyaniline terpolymers consisting of aminobenzoic acid (AB), aminobenzenesulfonic acid (ABS) and aniline (A) with different monomer ratios were synthesized. The P(AB‐ABS‐A) were grafted to the surface of multiwalled carbon nanotubes (MWCNTs). FTIR measurements prove the covalent binding to the carboxylic groups of the MWCNTs while conductivity tests show an increase in the conductivity of the nanohybrid in comparison to the polymers. The [MWCNT‐P(AB‐ABS‐A)] nanohybrids were used for the immobilization of redox active cytochrome c (cyt.c). The positively charged protein can electrostatically interact with the negatively charged nanohybrid. Cyclic voltammetry (CV) shows an increase in the protein loading on [MWCNT‐P(AB‐ABS‐A)] coupled to cysteamine modified gold electrodes in comparison to non‐grafted MWCNTs. A further increase in the sulfonation degree of P(AB‐ABS‐A) leads to an enhanced current output of the modified electrodes. The redox activity of the polymer decreases after the immobilization of the cyt.c on the nanohybrid. For the first time polymers covalently grafted to the surface of MWCNTs are used in a biosensor.

Bacteria Screening, Viability, And Confirmation Assays Using Bacteriophage-Impedimetric/Loop-Mediated Isothermal Amplification Dual-Response Biosensors

Here, we integrate two complementary detection strategies for the identification and quantification of Escherichia coli based on bacteriophage T4 as a natural bioreceptor for living bacteria cells. The first approach involves screening and viability assays, employing bacteriophage as the recognition element in label-free electrochemical impedance spectroscopy. The complementary approach is a confirmation by loop-mediated isothermal amplification (LAMP) to amplify specifically the E. coli Tuf gene after lysis of the bound E. coli cells, followed by detection using linear sweep voltammetry. Bacteriphage T4 was cross-linked, in the presence of 1,4-phenylene diisothiocyanate, on a cysteamine-modified gold electrode. The impedimetric biosensor exhibits specific and reproducible detection with sensitivity over the concentration range of 10(3)-10(9) cfu/mL, while the linear response of the LAMP approach was determined to be 10(2)-10(7) cfu/mL. The limit of detection (LOD) of 8 × 10(2) cfu/mL in less than 15 min and 10(2) cfu/mL within a response time of 40 min were achieved for the impedimetric and LAMP method, respectively. This work provides evidence that integration of the T4-bacteriophage-modified biosensor and LAMP can achieve screening, viability, and confirmation in less than 1 h.

Development of an amperometric immunosensor for detection of staphylococcal enterotoxin type A in cheese

This paper reports a novel electrochemical immunosensor for the sensitive detection of staphylococcal enterotoxin A (SEA) based on self-assembly monolayer (SAM) and protein A immobilization on gold electrode. Three different methods of protein A immobilization were tested: physical adsorption, cross-linking using glutaraldehyde and covalent binding after activation with N-hydroxysuccinimide (NHS)/N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) on cysteamine-modified gold electrode. The EDC/NHS method for protein A immobilization was selected to lead development of the biosensor. The coating steps of the surface modification were characterized by cyclic voltammetry and the biosensor response by chronoamperometry. The advantages of the immunosensor were exposed in its high sensitivity and specificity. The proposed amperometric immunosensor was successfully used for determination of SEA in contaminated and non-contaminated cheese samples with excellent responses.

Improvement of antibody immobilization using hyperbranched polymer and protein A

For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 μg mL −1. The detection limit was estimated to be 0.53 μg mL −1. The immunosensor holds good selectivity, sensitivity, and repeatability.

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine-modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at -0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4 degrees C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation.

A Novel Sensor Based on Layer‐by‐Layer Hybridized Phosphomolybdate and Poly(ferrocenylsilane) on a Cysteamine Modified Gold Electrode

AbstractA novel sensor have been constructed by layer‐by‐layer hybridizing phosphomolybdate (POM) and poly(ferrocenylsilane) (PFS) on a cysteamine modified gold electrode. The properties and performance of the sensor have been measured by electrochemistry and atomic force microscopy in detail. The results showed that the constructed multilayers modified gold electrode combined the properties of POM and PFS, and exhibited good electrocatalytic ability to a series of inorganic ions, including BrO$\rm{ {_{3}^{-}}}$, IO$\rm{ {_{3}^{-}}}$, NO$\rm{ {_{2}^{-}}}$, Fe3+, ascorbic acid and SO$\rm{ {_{3}^{2-}}}$. The well catalytic activity of the sensor was ascribed to the porous structure of hybrid POM‐PFS multilayer. The resulted sensor exhibited extremely fast amperometric response, low detection limit, high selectivity and wide linear range to these analyses.

Electrochemical behavior and its electrocatalytic properties of chemically modified electrode with Keggin-type [SiNi(H 2O)W 11O 39] 6−

A monolayer of Keggin-type heteropolyanion [SiNi(H 2O)W 11O 39] 6− was fabricated by electrodepositing [SiNi(H 2O)W 11O 39] 6− on cysteamine modified gold electrode. The monolayer of [SiNi(H 2O)W 11O 39] 6− modified gold electrode was characterized by atomic force microscopy (AFM) and electrochemical method. AFM results showed the [SiNi(H 2O)W 11O 39] 6− uniformly deposited on the electrode surface and formed a porous monolayer. Cyclic voltammetry exhibited one oxidation peak and two reduction peaks in 1.0 M H 2SO 4 in the potential range of −0.2 to 0.7 V. The constructed electrode could exist in a large pH (0–7.6) range and showed good catalytic activity towards the reduction of bromate anion (BrO 3 −) and nitrite (NO 2 −), and oxidation of ascorbic acid (AA) in acidic solution. The well catalytic active of the electrode was ascribed to the porous structure of the [SiNi(H 2O)W 11O 39]6 − monolayer.

Immobilization of cytochrome c on cysteamine-modified gold electrodes with EDC as coupling agent

Cyclic voltammetry has been applied for the characterization of cross-linked horse heart cytochrome c (HHC) on cysteamine-modified gold electrodes. The cross-linking, i.e. amide bond formation, between the proteins was achieved by using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as coupling reagent. The optimal conditions for the formation of the HHC film were determined by varying the HHC concentration. In addition the reproducibility, stability and the influence of the scan rate upon these films were investigated with cyclic voltammetry. The protein film stability in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution was tested by UV/vis absorption spectroscopy.

Enzyme Activity Control by Responsive Redoxpolymers

A new thermoresponsive poly-N-isopropylacrylamide (PNIPAM)-ferrocene polymer was synthesized and characterized. PNIPAMFoxy bears additional oxirane groups which were used for attachment by a self-assembly process on a cysteamine-modified gold electrode to create a thin hydrophilic film. The new redox polymer enabled electrical communication between the cofactor pyrrolinoquinoline quinone (PQQ) of soluble glucose dehydrogenase (sGDH) and the electrode for sensitive detection of this enzyme as a prospective protein label. The temperature influence on the redox polymer/enzyme complex was investigated. An inverse temperature response behavior of surface bound PNIPAMFoxy compared to the soluble polymer was found and is discussed in detail. The highest efficiency of mediated electron transfer for the immobilized PNIPAMFoxy with sGDH was observed at 24 degrees C, which was twice as high as that of its soluble counterpart. A steady-state electrooxidation current densitiy of 4.5 microA.cm-2 was observed in the presence of 10 nM sGDH and 5 mM glucose. A detection limit of 0.5 nM of soluble PQQ-sGDH was obtained.

Electrical wiring of Pseudomonas putida and Pseudomonas fluorescens with osmium redox polymers

Two different flexible osmium redox polymers; poly(1-vinylimidazole) 12-[Os-(4,4′-dimethyl-2,2′-di'pyridyl) 2Cl 2] 2+/+ (osmium redox polymer I) and poly(vinylpyridine)-[Os-( N, N′-methylated-2,2′-biimidazole) 3] 2+/3+ (osmium redox polymer II) were investigated for their ability to efficiently “wire” Pseudomonas putida ATCC 126633 and Pseudomonas fluorescens ( P. putida DSM 6521), which are well-known phenol degrading organisms, when entrapped onto cysteamine modified gold electrodes. The two Os-polymers differ in redox potential and the length of the side chains, where the Os 2+/3+-functionalities are located. The bacterial cells were adapted to grow in the presence of phenol as the sole source of organic carbon. The performance of the redox polymers as mediators was investigated for making microbial sensors. The analytical characteristics of the microbial sensors were evaluated for determination of catechol, phenol and glucose as substrates in both batch analysis and flow analysis mode.

Electrochemical detection of DNA immobilized on gold colloid particles modified self-assembled monolayer electrode with silver nanoparticle label

The target DNA was immobilized successfully on gold colloid particles associated with a cysteamine monolayer on gold electrode surface. Self-assembly of colloidal Au onto a cysteamine modified gold electrode can enlarge the electrode surface area and enhance greatly the amount of immobilized single stranded DNA (ssDNA). The electron-transfer processes of [Fe(CN) 6] 4−/[Fe(CN) 6] 3− on the gold surface were blocked due to the procedures of the target DNA immobilization, which was investigated by impedance spectroscopy. Then single stranded target DNA immobilized on the gold electrode hybridized with the silver nanoparticle–oligonucleotide DNA probe, followed by the release of the silver metal atoms anchored on the hybrids by oxidative metal dissolution, and the indirect determination of the released solubilized Ag I ions by anodic stripping voltammetry (ASV) at a carbon fiber microelectrode. The results show that this method has good correlation for DNA detection in the range of 10–800 pmol/l and allows the detection level as low as 5 pmol/l of the target oligonucleotides.

Immobilization of polyglutamate-glucose oxidase onto a cysteamine-modified gold electrode

Polyglutamate–glucose oxidase (GOD) complex was prepared, and the complex was dropped on a cysteamine-modified gold electrode to immobilize the complex; the electrostatic binding between the cysteamine on the electrode surface and the polyglutamate moiety of the complex resulted in the formation of a GOD-attached electrode. The enzyme electrode showed quick response (∼15 s), since the enzyme layer was quite thin (enzyme-complex monolayer structure). When we use un-modified GOD instead of polyglutamate–GOD, the sensitivity of the resulting enzyme electrode was quite low. This clearly shows that the presence of polyglutamate chain of the complex is essential for the immobilization of the modified electrode surface, i.e., the polyglutamate moiety acts as an anchor part for the complex. A linear relationship between the current response on the complex immobilized electrode and the glucose concentration was observed between 5 and 100 μM. The immobilization technique can widely be applied to prepare biosensors and biochips.

Direct electron transfer and characterization of hemoglobin immobilized on a Au colloid–cysteamine-modified gold electrode

Hemoglobin (Hb) was immobilized successfully on nanometer-sized gold colloid particles associated with a cysteamine monolayer on a gold electrode surface, and was characterized by atomic force microscopy (AFM), UV–vis spectroscopy (UV–vis), surface enhanced Raman spectroscopy (SERS) and electrochemical impedance spectroscopy (EIS). The immobilized Hb was shown to keep its biological activity well. Direct electron transfer (eT) between Hb and the modified electrode was achieved without the aid of any electron mediator. In pH 7.2 phosphate buffer solution, the formal potential ( E°′) of Hb was −0.051 V (vs. SCE) and the eT rate constant was 0.49 s −1. The average surface coverage of Hb immobilized on the gold colloid was about 6.71×10 −11 mol cm −2. The immobilized Hb displayed the features of a peroxidase and gave an excellent electrocatalytic response to the reduction of H 2O 2. The Michaelis–Menten constant ( K m) was 1.2×10 −4 M. The currents were proportional to the H 2O 2 concentration from 3.6×10 −7 to 8.6×10 −4 M and the detection limit was as low as 1.2×10 −7 M ( S/ N=3).

Colloid Au-enhanced DNA immobilization for the electrochemical detection of sequence-specific DNA

We use colloidal Au to enhance the DNA immobilization amount on a gold electrode and ultimately lower the detection limit of our electrochemical DNA biosensor. Self-assembly of approximately 16-nm diameter colloidal Au onto a cysteamine modified gold electrode resulted in an easier attachment of an oligonucleotide with a mercaptohexyl group at the 5′-phosphate end, and therefore an increased capacity for nucleic acid detection. Quantitative results showed that the surface densities of oligonucleotides on the Au colloid modified gold electrode were approximately (1–4)×10 14 molecules cm −2. Hybridization was induced by exposure of the ssDNA-containing gold electrode to ferrocenecarboxaldehyde labeled complementary ssDNA in solution. The detection limit is 5×10 −10 mol l −1 of complementary ssDNA, which is much lower than our previous electrochemical DNA biosensors. The Au nanoparticle films on the Au electrode provide a novel means for ssDNA immobilization and sequence-specific DNA detection.

Hydrogen peroxide sensor based on horseradish peroxidase-labeled Au colloids immobilized on gold electrode surface by cysteamine monolayer

A novel strategy to construct a sensitive amperometric sensor of H 2O 2 was described. Horseradish peroxidase (HRP) was successfully immobilized on nanometer-sized Au colloids, which were supported by thiol-tailed groups of cysteamine monolayer. The thiol-tailed groups were formed through the covalent binding of glutaraldehyde on a cysteamine-modified gold electrode. With the aid of the catechol mediator in the solution, HRP-labeled Au colloids displayed excellent electrocatalytical response to the reduction of H 2O 2, which increased with decreasing size of Au colloids. The sensors responded to H 2O 2 in the concentration range of 0.39 μM–0.33 mM, and reached 95% of the steady-state current in less than 5 s with the detection limit of 0.15 μM. The response showed a Michaelis–Menten behavior at larger H 2O 2 concentrations. The K app M values for the sensors based on different sized HRP-labeled Au colloids (colloid A–D) were found to be 0.11, 0.094, 0.054 and 0.064 mM, respectively. The low K app M values demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H 2O 2 with no loss of enzymatic activity.

Adsorption characteristics of Fe(CN)63−/4− on Au colloids as monolayer films on cysteamine-modified gold electrode

Abstract Self-assembled monolayers (SAMs) of chemisorbed cysteamine on gold electrode surfaces have been used as base interfaces for the deposition of the Au colloid. Different Au colloids, ranging in particles of diameter 2.6, 16, 42 and 51 nm, can be deposited on self-assembled cysteamine monolayers on a gold electrode. Fe(CN)64− adsorbed on Au colloid surfaces yields a pair of symmetric adsorption peaks with half-peak width (W1/2) of 90.2 mV and peak-to-peak separation (ΔEp) of 3 mV in 1.0 M KNO3 solution containing 1.00×10−3 M K3Fe(CN)6. The dependence of peak current on scan rate (ν) is linear. ΔEp of the adsorption wave increases while the Au colloid size decreases. The apparent specific capacitances of a bare gold electrode, the cysteamine-modified electrode and the Au colloid modified electrode measured by cyclic voltammetry at 500 mV are 57.8, 32.0 and 81.1 μF cm−2, respectively.