Cysteine Self-assembled Monolayers Research Articles

Exploring the pH Sensitivity of Ion-Pair Interactions on a Self-Assembled Monolayer by Scanning Electrochemical Microscopy

Insights into the chemical essence of weak interactions on the surface of biomacromolecules may help to regulate biological processes. In this work, the pH sensitivity of ion-pair interactions occurring on a cysteine self-assembled monolayer (Cys SAM) that simulates the local surface of a protein was probed by scanning electrochemical microscopy (SECM). Cys SAM and the ion-pair interactions subsequently formed with the introduced aspartic acid (Asp) were both pH-sensitive, as confirmed by the tip current changes in the feedback mode. After continuous pH measurements, the most significant negative feedback was observed at pH 5.50, indicating the most robust ion-pair interactions, which were simultaneously identified by voltammetry. In this case, the extra addition of the inorganic cation (i.e., Ca2+) did not disrupt the existing ion-pair interactions, and the binding constant (K) and Gibbs free energy (ΔGo) of the ion pair were finally determined to be 6.44 × 105 M-1 and -33.14 kJ mol-1, respectively. Overall, the pH sensitivity of ion-pair interactions was found to be mainly attributable to pH-induced changes in the deprotonated/protonated states of the α-amino acid moieties, which may provide insights into the artificial manipulation of complex binding events at the molecular level on the biological surface.

Disposable superoxide dismutase biosensors based on gold covered polycaprolactone fibers for the detection of superoxide in cell culture media.

A novel and disposable biosensor based on superoxide dismutase (SOD) immobilized on gold metallized polycaprolactone electrospun polymeric fibers (PCl/Au) has been developed for the determination of superoxide (O2•-) in cell culture media. SOD biosensors were constructed employing three immobilization methods: cross-linking with EDC/NHS at a cysteine self-assembled monolayer (PCl/Au/SODCYS), biopolymer encapsulation with chitosan (PCl/Au/SODCHI) and cross-linking with glutaraldehyde (PCl/Au/SODGA). Scanning electron microscopy was performed at the three different biosensors to evaluate their surface morphologies. Biosensors were employed for the electrochemical detection of superoxide by fixed potential amperometry at different applied potentials, with two distinct enzymatic mechanisms being proposed: i) the reduction of the enzymatically generated peroxide, at -0.3V, for which the PCl/Au/SODCHI biosensor presented the highest value of sensitivity of 40.1μAmM-1cm-2, and ii) the regeneration of the enzyme catalytic copper centre, at +0.3V, for which the PCl/Au/SODCYS biosensor had the highest sensitivity value of 16.1μAmM-1cm-2. The proposed recognition mechanisms were further confirmed by cyclic voltammetric measurements, which enabled also to determine the amount of immobilized electroactive SOD, with highest value corresponding to the PCl/Au/SODCYS biosensor. The biosensors with best analytical performance, PCl/Au/SODCYS and PCl/Au/SODCHI, were further investigated for stability and selectivity, with best results for the PCl/Au/SODCYS, chosen for superoxide monitoring in cell culture media. The study is promising for future application of PCl/Au/SODCYS for the on-line superoxide monitoring of superoxide in cell cultures, grown directly on the biosensor itself.

Investigations into Spin- and Unpolarized Secondary Electron-Induced Reactions in Self-Assembled Monolayers of Cysteine.

Cysteine is the simplest thiolated, chiral amino acid and is often used as the anchor for studies of self-assembled monolayers (SAMs) of complex biomolecules such as peptides. Understanding the interaction of SAMs of cysteine with low-energy secondary electrons (SEs) produced by X-rays can further our understanding of radiation damage in biomolecules. In particular, if the electrons are polarized, chiral-selective chemistry could have bearing on the origin of homochirality in nature. In the present paper, we use synchrotron radiation-based X-ray photoelectron spectroscopy to determine the changes that occur in the bonding of self-assembled layers of cysteine on gold as a result of soft X-ray irradiation. To investigate the possibility of chiral selectivity resulting from the interaction of low-energy, spin-polarized SEs (SPSEs), measurements were conducted on cysteine adsorbed on a 3 nm-thick gold layer deposited on a CoPt thin-film multilayer with perpendicular magnetic anisotropy. Time-dependent measurements of the C 1s, N 1s, O 1s, S 2p, and Au 4f core levels are used to follow the changes in surface chemistry and determine reaction cross-sections as a function of SE exposure. Analysis of the data results in cross-sections in the range of 5-7 Mb and suggests possible reaction pathways. Changing the magnetization direction of the CoPt multilayer produces SPSEs with opposite polarity. Some evidence of spin-dependent reactions is indicated but is inconclusive. Possible reasons for the discrepancy are posited.

Low-Fouling Characteristics of Ultrathin Zwitterionic Cysteine SAMs.

Surface fouling remains an exigent issue for many biological implants. Unwanted solutes adsorb to reduce device efficiency and hasten degradation while increasing the risks of microbial colonization and adverse inflammatory response. To address unwanted fouling in modern implants in vivo, surface modification with antifouling polymers has become indispensable. Recently, zwitterionic self-assembled monolayers, which contain two or more charged functional groups but are electrostatically neutral and form highly hydrated surfaces, have been the focus of many antifouling coatings. Reports using various compositions of zwitterionic polymer brushes have demonstrated ultralow fouling in the ng/cm2 range. These coatings, however, are thick and can hinder the target application of biological devices. Here, we report an ultrathin (8.52 Å) antifouling self-assembled monolayer composed of cysteine that is amenable to facile fabrication. The antifouling characteristics of the zwitterionic surfaces were evaluated against bovine serum albumin, fibrinogen, and human blood in real time using quartz crystal microbalance and surface plasmon resonance imaging. Compared to untreated gold surfaces, the ultrathin cysteine coating reduced the adsorption of bovine serum albumin by 95% (43 ng/cm2 adsorbed) after 3 h and 90% reduction after 24 h. Similarly, the cysteine self-assembled monolayer reduced the adsorption of fibrinogen as well as human blood by >90%. The surfaces were further characterized using scanning electron microscopy: protein-enhanced adsorption and cellular adsorption in human blood was found on untreated surfaces but not on the cysteine SAM-protected surfaces. These findings suggest that surfaces can be functionalized with an ultrathin layer of cysteine to resist the adsorption of key proteins, with performance comparable to zwitterionic polymer brushes. As such, cysteine surface coatings are a promising methodology to improve the long-term utility of biological devices.

Oriented immobilization of antibody through carbodiimide reaction and controlling electric field

A novel and simple approach for oriented immobilization of antibody (Ab) was proposed through carbodiimide reaction and controlling electric field (CEF). The electrode was modified by self-assembled monolayer of cysteine (CYS SAM) with amino group stretching outside, and Fc region of Ab was activated via carbodiimide reaction. By adjusting pH of solution and applying a proper electric field, Abs were manipulated owing to their charged nature and immobilized on the electrode via the activated Fc region. The stepwise modified electrodes were characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results proved that Abs could be successfully immobilized on the surface of electrode. Under optimal experimental parameters, the immunosensor could detect antigen (Ag) in a range from 0.04 to 400 ng/mL. Furthermore, the immunosensors fabricated with and without CEF were comparatively studied, and the results showed that the former had better detection properties than the later. Finally, the effect of CEF on Ab was studied by polarization modulation infrared reflection–absorption spectroscopy (PM-IRRAS) and scanning electron microscope (SEM); the results indicated that Abs were arranged in better order under electric field, this may be the reason for the better properties of the immunosenor. In a word, Ab immobilization through carbodiimide reaction and CEF was very simple, fast, and easy to perform, and it would find wide application in the immune-based assay systems.

Electrochemical immunosensor for dengue virus serotypes based on 4-mercaptobenzoic acid modified gold nanoparticles on self-assembled cysteine monolayers

Currently, Dengue is a worldwide health problem and no effective vaccine or specific medication. Thus, the development of new diagnostic methods has been the focus of efforts to combat the disease. Immunosensor based on gold nanoparticles (AuNPs) and self-assembled monolayers of cysteine was developed for dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4) detection. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor the signal response of the each dengue serotype in different dilutions. Scanning electron microscopy was used to characterize the surface of the immunosensor before and after exposure to DENV. In this work, four different immunosensors with specificity for DENV1-4 were obtained. EIS and CV measurements demonstrate the ability of the immunosensor to recognize all four studied DENV serotypes showing sensitivity and selectivity. The real part of the impedance was higher for Cys–MBA–AuNPs–anti-DENV antibody sensor system after interaction to different concentrations of DENV (1-4) compared to the sensor system without immunoreaction on their surface, revealing an increased blockage of the electron transfer. In addition, the surface coverage values indicate a good recognition of DENV by anti-DENV antibody. The new sensor platform consists of a single method, more affordable than conventional immunoassays and creates expectation on future production of new real-time devices for diagnosis of pathologies caused by virus.

Introducing a new method for evaluation of the interaction between an antigen and an antibody: Single frequency impedance analysis for biosensing systems

This paper illustrates the application of an antibody, anti-parathyroid hormone (anti-PTH), as a bioreceptor in a biosensor system for the first time, and demonstrates how this biosensor can be used in parathyroid hormone (PTH) determination. The interaction between the biosensor and parathyroid hormone was firstly investigated by a novel electrochemical method, single frequency impedance analysis. The biosensor was based on the gold electrode modified by cysteine self-assembled monolayers. Anti-PTH was covalently immobilized onto cysteine layer by using an EDC/NHS couple. The immobilization of anti-PTH was monitored by cyclic voltammetry and electrochemical impedance spectroscopy techniques. The performance of the biosensor was evaluated in terms of linearity, sensitivity, repeatability and reproducibility, after a few important optimization studies were carried out. In particular, parathyroid hormone was detected within a linear range of 10–60fg/mL. Kramers–Kronig transform was also performed on the impedance data. The specificity of the biosensor was also evaluated. The biosensor was validated by using a complementary reference technique. Lastly the developed biosensor was used to monitor PTH levels in artificial serum samples.

Nanostructured Screen-Printed Electrodes Modified with Self-Assembled Monolayers for Determination of Metronidazole in Different Matrices

This paper describes the development of a sensor for the determination of metronidazole using a simple method involving the creation of nanoporous structures on gold electrodes followed by modification with self-assembled monolayers (SAMs) of thiols. The initial formation of oxidized gold species at pH 7.0 was followed by potentiodynamic reduction in order to obtain a nanostructured surface that was then modified with a SAM of cysteine. The sensor was characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical techniques. Quantification of metronidazole was performed using optimized square wave voltammetry. Under the best analytical conditions, the sensor operated at -0.36 V showed a linear range of 50-300 µmol L-1 and a limit of detection (LOD) of 2.6 µmol L-1. Evaluation of selectivity demonstrated that the electrode provided higher sensitivity towards nitroimidazoles. The sensor responses showed that it could be used for detection of metronidazole in different types of matrix.

Penicillinase-based amperometric biosensor for penicillin G

A biosensor for penicillin G was created by immobilizing penicillinase to a gold electrode by means of a cysteine self-assembled monolayer. The biosensor amperometrically monitored the catalytic hydrolysis of penicillin in a very sensible manner. Furthermore, it was successfully used to measure the Michaelis–Menten enzymatic constant and a low limit of detection of 4.5nM was obtained.

An electrocatalytic oxygen reduction by copper nanoparticles-modified Au(100)-rich polycrystalline gold electrode in 0.5 M KOH

The electrocatalytic oxygen reduction reaction (ORR) at copper nanoparticles (nano-Cu) modified Au(100)-rich polycrystalline gold electrode (nano-Cu/Aur) in 0.5 M KOH is studied using cyclic and rotating disk voltammetry. The nano-Cu/Aur with a relatively enriched free Au(100) facet compared with the bare poly-Au electrode is prepared by a controlled electrodeposition of nano-Cu only on the Au(111) facet of the poly-Au electrode the other low-index facets (i.e., Au(100) and Au(110)) of which are previously covered with the self-assembled monolayer of cysteine. The electrocatalytic behavior of the nano-Cu/Aur is compared with those of the bare Au and the copper nanoparticles-modified gold electrodes (nano-Cu/Au) in which the nano-Cu is directly electrodeposited onto the poly-Au electrode. The nano-Cu/Aur shows a remarkable electrocatalysis, comparable to that of the platinum electrode, toward the ORR i.e., the ORR proceeds exclusively via a 1-step 4-electron reduction pathway at ca. 20–65 mV more positive potentials than at the bare Pt electrode, while the ORR at the nano-Cu/Au proceeds via a two-electron pathway. X-ray diffraction spectra confirms the relative enrichment of nano-Cu/Aur electrode in Au(100) facet. The relative enrichment in the free Au(100) facet of the nano-Cu/Aur electrode is thought to be behind the extraordinary electrocatalytic activity.

Fabrication of an amperometric tyramine biosensor based on immobilization of tyramine oxidase on AgNPs/l-Cys-modified Au electrode

An amperometric tyramine biosensor was constructed based on covalent immobilization of black gram tyramine oxidase onto citric acid-capped silver nanoparticles bound to surface of Au electrode through cysteine self-assembled monolayer. The enzyme electrode was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and cyclic voltametry. The biosensor showed optimum response within 8 s, when polarized at 0.25 V, pH 8.5, and 35 °C, with linearity from 0.017 to 0.25 mM and a detection limit of 0.01 mM. The biosensor was employed for measurement of tyramine in beer and sauce. The mean analytical recovery of added tyramine in beer at 0.36 and 0.72 mM were 97.2 ± 2.7 and 95.8 ± 4.1 %, respectively, and within and between batches coefficients of variation were 0.33–0.38 and 0.34–0.62 %, respectively. The enzyme electrode lost 35 % of its initial activity after its 100 uses, over a period of 2 months, when stored at 4 °C.

Study on the Reversible Changes of the Surface Properties of an l-Cysteine Self-Assembled Monolayer on Gold As a Function of pH

A stimuli-response biological surface of L-cysteine was prepared on a polycrystalline gold surface from aqueous solution. The effect of the pH value of the rinsing solution on the surface composition was studied with X-ray photoelectron spectroscopy (XPS). Qualitative and quantitative analysis of the amino, carboxyl, and thiol functional groups of these self-assembled monolayers indicate that L-cysteine molecules exist in the neutral and zwitterionic forms and that they are sensitive to the pH of the rinsing solution. In addition, the wetting properties of the functionalized surface were studied by contact angle (CA) analysis: they were also dependent on the pH of the rinsing solution. Furthermore, it was shown that this functionalization process was reversible.

Probing cysteine self-assembled monolayers over gold nanoparticles – Towards selective electrochemical sensors

Cysteine forms self-assembled monolayers over gold nanoparticles. Based on this knowledge, a novel electrochemical sensor (Au-Aunano-Cys-SDS) has been constructed by the formation of self-assembly monolayer (SAM) of cysteine on gold-nanoparticles modified gold electrode (Au-Aunano-Cys) to be utilized for determination of dopamine in the presence of sodium dodecyl sulfate (SDS). Electrochemical investigation and characterization of the modified electrode sensor was achieved using cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron, and atomic force microscopies. Au-Aunano-Cys electrode in the presence of SDS gave comparable high current response to that of the gold nanoparticles modified gold electrode (Au-Aunano). The Au-Aunano-Cys-SDS electrode current signal was remarkably stable via repeated cycles and long term stability due to the strong AuS bond. Very small peak separation, almost zero or 15mV peak separation was also obtained by repeated cycles indicating unusual high reversibility. The oxidation peak current was determined to be linearly dependent on the dopamine concentration. A resulting calibration curve using square wave voltammetry (SWV) was obtained over concentration range of 30–100μmolL−1 and 120–320μmolL−1 with correlation coefficients of 0.996 and 0.994 and a limit of detection of 16 and 57nmolL−1, respectively. Using differential pulse voltammetry (DPV), a highly selective and simultaneous determination of tertiary mixture of ascorbic acid AA, dopamine, and acetaminophen APAP was explored at this modified electrode. It has been demonstrated that Au-Aunano-Cys-SDS electrode can be used as a sensor with excellent reproducibility, sensitivity, and long term stability.

Measurement of gold and sulfur mass fractions in l-cysteine-modified gold nanoparticles by ICP-DRC-MS after acid digestion: validation and uncertainty of results

Measurement of mass fractions of trace element sulfur and main element gold in L-cysteine-modified gold nanoparticles was performed by inductively coupled plasma mass spectrometry with dynamic reaction cell (DRC) technology after aqua regia digestion. Sulfur concentration was measured at m/z 48 as 32S16O+ in a reaction cell with O2 to reduce the effect of polyatomic interferences at m/z 32. Following a study of the main interferences affecting the signal from 32S16O+, optimization of instrument parameters by an experimental design along with an investigation of the effect of aqua regia in matrix on the signal-to-background ratio of 32S16O+ were carried out to improve the detection limit for sulfur. Under the optimum experimental conditions the detection limits were 1.43 μg L−1 for S and 0.033 μg L−1 for Au. 45Sc was selected as the internal standard for measurement of 32S16O+. The presence of Au at high concentrations showed a negligible effect on the intensity of 32S16O+ and 45Sc+ under DRC mode. The method was in-house validated and the combined uncertainty was estimated according to the Guide to the Expression of Uncertainty in Measurement. Preliminary studies show that the method may be transferred to other ICP-MS systems by re-optimizing cell gas flow (e.g. 1.8 mL min−1 used for NexION 300D). X-ray photoelectron spectroscopy confirmed that a self-assembled monolayer of cysteine was formed on the surface of colloidal gold nanoparticles via sulfur-to-gold bonding.

A novel sensor of cysteine self-assembled monolayers over gold nanoparticles for the selective determination of epinephrine in presence of sodium dodecyl sulfate

A novel sensor of cysteine self-assembled monolayers over gold nanoparticles modified gold electrode has been constructed for the determination of epinephrine in presence of sodium dodecyl sulfate (Au/Au(nano)-CysSDS). Electrochemical investigation and characterization of the modified electrode are achieved using cyclic voltammetry, linear sweep voltammetry, and scanning electron microscopy. The Au/Au(nano)-CysSDS electrode current signal is remarkably stable via repeated cycles and long term stability, due to the strong Au-S bond, compared to the Au/Au(nano) electrode. The catalytic oxidation peak currents obtained from linear sweep voltammetry (LSV) increased linearly with increasing epinephrine concentrations in the range of 2 to 30 μmol L(-1) and 35 to 200 μmol L(-1) with correlation coefficients of 0.9981 and 0.9999 and a limit of detection of 0.294 nmol L(-1) and 1.49 nmol L(-1), respectively. The results showed that Au/Au(nano)-CysSDS can selectively determine epinephrine in the coexistence of a large amount of uric acid and glucose. In addition, a highly selective and simultaneous determination of tertiary mixture of ascorbic acid, epinephrine, and acetaminophen is explored at this modified electrode. Excellent recovery results were obtained for determination of epinephrine in spiked urine samples at the modified electrode. Au/Au(nano)-CysSDS can be used as a sensor with excellent reproducibility, sensitivity, and long term stability.

Rapid Diagnostic Device for Subclinical Mastitis Based on Electrochemical Detection of Superoxide Produced from Neutrophils in Fresh Milk

Electrochemical microdevices were fabricated to identify mastitic cows based on the increased number of neutrophils in raw milk. Because neutrophils produce superoxide (O2·-), the amount of O2·- can be used as an early indicator for subclinical mastitis. In the microdevices, O2·- was detected on a gold electrode using superoxide dismutase immobilized via a self-assembled monolayer of cysteine. In a preliminary test using xanthine oxidase to produce O2·-, one of the devices detected the production and rapid extinction of O2·-. When neutrophils obtained from a mastitic cow were concentrated by centrifugation and introduced into the device, a current increase distinctly different from the background was observed. Furthermore, a micropillar structure was fabricated on the gold electrode to trap and collect neutrophils, thereby facilitating the concentration of these cells around the electrode. The measured current clearly depended on the number of neutrophils in raw milk samples, demonstrating the applicability of the device for rapid diagnosis of subclinical mastitis.

Enantiomeric recognition of phenylalanine by self-assembled monolayers of cysteine: Surface enhanced Raman scattering evidence

The stereoselective interaction between enantiomers of two amino acids, cysteine and phenylalanine, on roughened silver surface was investigated by SERS spectroscopy. It was demonstrated that the surface coated with one cysteine enantiomer can recognize the phenylalanine enantiomer present in the solution. The change of conformation of some part of adsorbed cysteine molecules from P H to P N caused by the phenylalanine molecules of proper chirality was observed. This effect was attributed to difference in possibility of hydrogen bonding between the NH 3 + groups of the surface-attached cysteine molecule with the COO − groups of phenylalanine molecules. Our experiments demonstrate that the SERS spectra are sensitive to chirality of interacting cysteine and phenylalanine molecules at the silver electrode surface, thus indicating the possibility of using this method for enantiomeric recognition of amino acids.

Ascorbic acid biosensor based on laccase immobilized on an electrode modified with a self-assembled monolayer and coated with functionalized quantum dots

Laccase was immobilized on an electrode modified with a cysteine self-assembled monolayer and coated with functionalized quantum dots. The immobilized laccase is capable of directly transferring an electron. Immobilized laccase retained its activity to oxidize ascorbic acid (AA), and the apparent Michaelis–Menten constant was found to be 0.47 mM. The modified electrode was used to determine AA in the 10 to 140 μM concentration range, with linear response curve and a detection limit of 1.4 μM (s/n = 3).

Electrochemical study of self-assembled cysteine monolayers on polycrystalline gold electrodes and functionalization with microperoxidase MP-11

Self-assembled monolayers of cysteine (CYST) have been constructed on polycrystalline gold (polyAu) electrode at different immersion times (t immer) in order to characterize them by cyclic voltammetry and electrochemical impedance spectroscopy (EIS). Oxidative desorption experiments allowed the calculation of the surface coverage, whereas reductive desorption informed about the binding of CYST to different gold domains. Desorption products showed different diffusion ability. The maximum coverage was already achieved at short t immer, although progressive reordering of CYST molecules led to a relevant decrease of the defects at long time, as ascertained by EIS. Functionalization of polyAu-CYST electrodes with the enzyme microperoxidase MP-11 performed by following two different binding methods showed that the enzymatically catalyzed reduction of H2O2 is enhanced when the –COOH residues of the MP-11 undecapeptide chain are firstly activated and then linked to the –NH2 functional groups of the non-activated CYST layer formed on the polyAu surface.

Enantiodiscrimination between an N‐Acetyl‐L‐cysteine SAM and Proline: An In Situ Spectroscopic and Computational Study

A combination of attenuated total reflection infrared (ATR-IR) and modulation excitation spectroscopy (MES) is used to study the enantiodiscriminating interactions between proline and a chiral, self-assembled monolayer (SAM) of N-acetyl-L-cysteine on gold. The N-acetyl-L-cysteine SAM consists of a mixture of protonated and deprotonated molecules. Whereas both species are influenced by adsorbed proline, only the deprotonated molecules are involved in enantiodiscrimination. Density functional theory (DFT) calculations reveal that electrostatics dominates the interaction between the two molecules. By modulating the absolute configuration of proline over the chiral SAM, and a subsequent phase-sensitive detection of the periodically varying signals in the ATR-IR spectra, the small spectral differences between the diastereomeric complexes are spotted. The resulting difference spectrum is in qualitative agreement with the spectrum predicted by the DFT calculations.