Parkinson biomarker determination with an Au microflower-enhanced electrochemical immunosensor using non-Faradaic capacitance measurements.

This work comprehends the development and characterization of a carbon black-based electrode modified with Au microflowers to increase its effect as a capacitance biosensor for the determination of PARK7/DJ-1. Due to its high surface-to-volume ratio and biocompatibility, Au particles are suitable for antibody binding, and by monitoring surface capacitance, it is possible to identify the immune-pair interaction. Au microflowers allowed the adequate immobilization of Parkinsonian-related proteins: PARK7/DJ-1 and its antibody. The protein is associated with several antioxidant mechanisms, but its abnormal concentrations or mutations can be the cause of the loss of dopaminergic neurons, leading to Parkinson's disease. The device was characterized by scanning electron microscopy and cyclic voltammetry, revealing the flower-like structures and the electrochemically-interest enhancements they provide, such as increased heterogeneous electron transfer rate coefficient and electroactive area. The self-assembled monolayers of different molecules were optimized with the aid of 22 central composite experiments and a linear calibration curve was obtained between 0.700 and 120 ng mL-1 of PARK7/DJ-1, with a limit of detection of 0.207 ng mL-1. The data confirms that the addition of Au microflowers enhanced the electrochemical signal of the device, as well as allowed for the determination of an early stage Parkinson's disease biomarker with appreciable analytical performance.

Electrochemical Sensor Based on Glassy Carbon Electrode Modified with Carbon Nanohorns (SWCNH) for Determination of Cr(VI) via Adsorptive Cathodic Stripping Voltammetry (AdCSV) in Tap Water.

In this study, a new and simple glassy carbon electrode modified with carbon nanohorns (SWCNH/GCE) was used for the determination of Cr(VI) in aqueous matrices via adsorptive cathodic stripping voltammetry (AdCSV). The modified electrode was characterized via field emission scanning electron microscopy and cyclic voltammetry, which revealed a homogeneous distribution of spherical agglomerates of SWCNH on the electrode surface. The modification increased the electrochemically active area from 0.10 cm2 ± 0.01 (GCE) to 0.16 cm2 ± 0.01 (SWCNH/GCE). The optimized analytical conditions were as follows: a supporting electrolyte (0.15 mol L-1 HCl), an accumulation potential of 0.8 V versus Ag/AgCl, and an accumulation time of 240 s. Validation of the analytical methodology was performed, obtaining a linear range between 20 and 100 µg L-1, a limit of detection of 3.5 µg L-1, and a limit of quantification of 11.6 µg L-1 with good accuracy and precision. The method was applied to the analysis of spiked tap water samples, and the results were compared using a flame atomic absorption spectrophotometer (FAAS) with no significant statistical differences.

Anodic stripping voltammetry of arsenic determination with edible mushroom-nafion-modified glassy carbon electrode.

An edible Mushroom-Nafion modified glassy carbon electrode (M2N5-GCE) was prepared using a homogeneous mixture varying the concentrations of these, in addition to the origin of the mushroom (Shiitake, Lentinula edodes, M1 and Abrantes, Agariscus bisporus, M2) and applied to the As(III) determination by anodic stripping voltammetry. After choosing the optimal conditions in the preparation of the electrode, the second stage was to study the effects of various parameters such as supporting electrolyte, pH, accumulation potential, and time (Eacc, tacc). The optimum experimental conditions chosen were Britton Robinson buffer 0.01 mol L-1 pH:4.6; Eacc: -1.0 and tacc: 60 s obtaining a signal of oxidation of As(0) to As(III) about 0.08 V. Peak current was proportional to arsenic concentration over the 19.6-117.6 µg L-1 range, with a 3σ detection limit of 13.4 µg L-1. The method was validated using As(III) spiked tap water from the laboratory with satisfactory results (RE:3.0 %). Finally, the method was applied to the determination of As(III) in water samples from the Loa River (Northern Chile) in the presence of As(V) in a concentration >20 times higher (RE: 2.3 %).

Development of recycled and miniaturized electroanalytical sensor: Probing isoniazid determination in environmental water matrices.

Surface water pollution has become relevant because growing population and intense industrial activities. Thus, to protect the environment from contamination, recently the electroanalytical sensors that require small sample volume and easy preparation have shown a prominent performance for pharmaceuticals monitoring. For this purpose, a miniaturized electrochemical platform was developed based on recycling obsolete computer integrated circuits (microchips), fitting with the ideals of green chemistry and circular economy. The gold microelectrodes array (Au-µEA) was easily exposed by polishing the device surface and then characterized by optical microscopy, scanning electron microscopy and cyclic voltammetry. To enhance the analytical performance for isoniazid detection, the Au-µEA was modified with electrochemically reduced graphene oxide (ERGO). The developed sensor presented a linear range between 5 and 100 µmol L-1 and a limit of detection of 1.38 µmol L-1 demonstrating a reliable performance. Looking to its environmental application, the ERGO/Au-µEA sensor was used for isoniazid quantification in lagoon, river, tap water and synthetic effluent spiked samples with recovery values between 92.5 and 108.4%. Thus, this research field opens up new possibilities in global water-related issues contributing with innovative sustainable solutions.

Recent progress and perspectives of metal organic frameworks (MOFs) for the detection of food contaminants.

Over the past decades, increasing research in metal-organic frameworks (MOFs) being a large family of highly tunable porous materials with intrinsic physical properties, show propitious results for a wide range of applications in adsorption, separation, electrocatalysis, and electrochemical sensors. MOFs have received substantial attention in electrochemical sensors owing to their large surface area, active metal sites, high chemical and thermal stability, and tunable structure with adjustable pore diameters. Benefiting from the superior properties, MOFs and MOF-derived carbon materials act as promising electrode material for the detection of food contaminants. Although several reviews have been reported based on MOF and its nanocomposites for the detection of food contaminants using various analytical methods such as spectrometric, chromatographic, and capillary electrophoresis. But there no significant review has been devoted to MOF/and its derived carbon-based electrodes using electrochemical detection of food contaminants. Here we review and classify MOF-based electrodes over the period between 2017 and 2022, concerning synthetic procedures, electrode fabrication process, and the possible mechanism for detection of the food contaminants which include: heavy metals, antibiotics, mycotoxins, and pesticide residues. The merits and demerits of MOF as electrode material and the need for the fabrication of MOF and its composites/derivatives for the determination of food contaminants are discussed in detail. At last, the current opportunities, key challenges, and prospects in MOF for the development of smart sensing devices for future research in this field are envisioned.

Synergistic effect of electrotrophic perchlorate reducing microorganisms and chemically modified electrodes for enhancing bioelectrochemical perchlorate removal.

Perchlorate has been described as an emerging pollutant that compromises water sources and human health. In this study, a new electrotrophic perchlorate reducing microorganism (EPRM) isolated from the Atacama Desert, Dechloromonas sp. CS-1, was evaluated for perchlorate removal in water in a bioelectrochemical reactor (BER) with a chemically modified electrode. BERs were operated for 17 days under batch mode conditions with an applied potential of -500 mV vs. Ag/AgCl. Surface analysis (i.e., SEM, XPS, FT-IR, RAMAN spectroscopy) on the modified electrode demonstrated heterogeneous transformation of the carbon fibers with the incorporation of nitrogen functional groups and the oxidation of the carbonaceous material. The BERs with the modified electrode and the presence of the EAM reached high cathodic efficiency (90.79 ± 9.157%) and removal rate (0.34 ± 0.007 mol m-3-day) compared with both control conditions. The observed catalytic enhancement of CS-1 was confirmed by a reduction in the charge transfer resistance obtained by electrochemical impedance spectroscopy (EIS). Finally, an electrochemical kinetic study revealed an eight-electron perchlorate bioreduction reaction at -638.33 ± 24.132 mV vs. Ag/AgCl. Therefore, our results show the synergistic effect of EPRM and chemically modified electrodes on perchlorate removal in a BER.

PEDOT: PSS/AuNPs-Based Composite as Voltammetric Sensor for the Detection of Pirimicarb.

An electrochemical sensor for the pesticide Pirimicarb (PMC) has been developed. A screen-printed electrode (SPCE) was used and modified with the conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) and gold nanoparticles (AuNPs) to enhance electrochemical proprieties. Electrode characterizations were performed using scattering electron microscopy (SEM) and cyclic voltammetry (CV). With the SPCE/PEDOT:PSS/AuNPs modified electrode, a new peak at 1.0 V appeared in the presence of PMC related to the PMC oxidation. To elucidate the mechanism of PMC oxidation, Gas Chromatography-Mass Spectrometry (GC-MS), where two major peaks were identified, evidencing that the device can both detect and degrade PMC by an electro-oxidation process. Exploring this peak signal, it was possible the sensor development, performing detection from 93.81-750 µmol L-1, limits of quantification (LOQ) and detection (LOD) of 93.91 µmol L-1 and 28.34 µmol L-1, respectively. Thus, it was possible to study and optimization of PMC degradation, moreover, to perform detection at low concentrations and with good selectivity against different interferents using a low-cost printed electrode based on graphite modified with conductive polymer and AuNPs.

A mechanothermal approach for the synthesis of Fe3O4 nanoparticles as dopant on mesoporous TiO2 for electrochemical determination of catechol.

The subject of water contamination and how it gets defiled to the society and humans is confabulating from the past decades. Phenolic compounds widely exist in the water sources and it is emergent to determine the toxicity in natural and drinking water, because it is hazardous to the humans. Among these compounds, catechol has sought a strong concern because of its rapid occurrence in nature and its potential toxicity to humans. The present work aims to develop an effective electrochemical sensing of catechol using mesoporous structure of Fe3O4-TiO2 decorated on glassy carbon (GC) electrode. The creation of pure TiO2 using the sol-gel technique was the first step in the synthesis protocol for binary nanocomposite, which was then followed by the loading of Fe3O4 nanoparticles on the surface of TiO2 using the thermal decomposition method. The resultant Fe3O4-TiO2 based nanocomposite exhibited mesoporous structure and the cavities were occupied with highly active magnetite nanoparticles (Fe3O4) with high specific surface area (90.63 m2/g). When compared to pure TiO2, catechol showed a more prominent electrochemical response for Fe3O4-TiO2, with a significant increase in anodic peak current at a lower oxidation potential (0.387 V) with a detection limit of 45 µM. Therefore, the prepared magnetite binary nanocomposite can serve as an efficient electroactive material for sensing of catechol, which could also act as a promising electrocatalyst for various electrocatalytic applications.

Electrochemical Sensor Based on Multi-Walled Carbon Nanotubes and N-Doped TiO2 Nanoparticles for Voltametric Simultaneous Determination of Benserazide and Levodopa.

An electrochemical sensor for simultaneous determination of Benserazide (BEZ) and levodopa (L-dopa) was successfully developed using a glassy carbon electrode (GCE) modified with multi-walled carbon nanotube and nitrogen-doped titanium dioxide nanoparticles (GCE/MWCNT/N-TiO2). Cyclic voltammetry and square wave voltammetry were employed to investigate the electrochemical behavior of different working electrodes and analytes. In comparison with unmodified GCE, the modified electrode exhibited better electrocatalytic activity towards BEZ and L-dopa and was efficient in providing a satisfactory separation for oxidation peaks, with a potential difference of 140 mV clearly allows the simultaneous determination of these compounds. Under the optimized conditions, linear ranges of 2.0-20.0 and 2.0-70.0 µmol L-1 were obtained for BEZ and L-dopa, respectively, with a limit of detection of 1.6 µmol L-1 for BEZ and 2.0 µmol L-1 for L-dopa. The method was applied in simultaneous determination of the analytes in pharmaceutical samples, and the accuracy was attested by comparison with HPLC-DAD as the reference method, with a relative error lower than 4.0%.

Development of Folate-Group Impedimetric Biosensor Based on Polypyrrole Nanotubes Decorated with Gold Nanoparticles.

In this study, polypyrrole nanotubes (PPy-NT) and gold nanoparticles (AuNPs) were electrochemically synthesized to form a hybrid material and used as an electroactive layer for the attachment of proteins for the construction of a high-performance biosensor. Besides the enhancement of intrinsic conductivity of the PPy-NT, the AuNPs act as an anchor group for the formation of self-assembly monolayers (SAMs) from the gold-sulfur covalent interaction between gold and Mercaptopropionic acid (MPA). This material was used to evaluate the viability and performance of the platform developed for biosensing, and three different biological approaches were tested: first, the Avidin-HRP/Biotin couple and characterizations were made by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), wherein we detected Biotin in a linear range of 100-900 fmol L-1. The studies continued with folate group biomolecules, using the folate receptor α (FR-α) as a bioreceptor. Tests with anti-FR antibody detection were performed, and the results obtained indicate a linear range of detection from 0.001 to 6.70 pmol L-1. The same FR-α receptor was used for Folic Acid detection, and the results showed a limit of detection of 0.030 nmol L-1 and a limit of quantification of 90 pmol L-1. The results indicate that the proposed biosensor is sensitive and capable of operating in a range of clinical interests.

Electrochemical Biosensor for SARS-CoV-2 cDNA Detection Using AuPs-Modified 3D-Printed Graphene Electrodes.

A low-cost and disposable graphene polylactic (G-PLA) 3D-printed electrode modified with gold particles (AuPs) was explored to detect the cDNA of SARS-CoV-2 and creatinine, a potential biomarker for COVID-19. For that, a simple, non-enzymatic electrochemical sensor, based on a Au-modified G-PLA platform was applied. The AuPs deposited on the electrode were involved in a complexation reaction with creatinine, resulting in a decrease in the analytical response, and thus providing a fast and simple electroanalytical device. Physicochemical characterizations were performed by SEM, EIS, FTIR, and cyclic voltammetry. Square wave voltammetry was employed for the creatinine detection, and the sensor presented a linear response with a detection limit of 0.016 mmol L-1. Finally, a biosensor for the detection of SARS-CoV-2 was developed based on the immobilization of a capture sequence of the viral cDNA upon the Au-modified 3D-printed electrode. The concentration, immobilization time, and hybridization time were evaluated in presence of the DNA target, resulting in a biosensor with rapid and low-cost analysis, capable of sensing the cDNA of the virus with a good limit of detection (0.30 µmol L-1), and high sensitivity (0.583 µA µmol-1 L). Reproducible results were obtained (RSD = 1.14%, n = 3), attesting to the potentiality of 3D-printed platforms for the production of biosensors.

Tailoring the heterojunction of TiO2 with multivalence CeO2 nanocrystals - for detection of toxic 2-aminophenol.

Enhancing the current signal response for semiconductors is the key factor for designing and fabrication of efficient electrode in electrochemical sensors. By the aid of doping with binary metal oxides, the conductivity of the resultant titanium oxide (TiO2) based nanocomposite will deliver fast electron transfer rate at the heterojunction interface. Herein, by taking advantage of mesoporous structure in TiO2, cubic shaped multivalent cerium oxide (CeO2) was incorporated into the porous cavity by simple ground assisted solvothermal process, which resulted in enormous enhancement in the current response towards detection of 2-aminophenol. The advantage of CeO2 on TiO2 not only involves the loading of binary metal oxide on its mesoporous sites, but also facilitates the formation of CeO2 nanocrystals which induce larger surface area and high electroactive sites with rapid diffusion of target species through pores. As a result, CeO2-TiO2 on modified GC electrode exhibits drastic enhancement in the current response for oxidation of 2-aminophenol with large decrease in the onset potential than TiO2/GC electrode. Furthermore, the CeO2-TiO2 modified electrode shows significant behavior for sensing of 2-aminophenol with wide linear range of 0.01-500 µM. The sensitivity and detection limit were calculated to be 0.603 µA µM cm-2 and 3.5 nM respectively. This work establishes the facile strategy for decoration of binary metal oxide-based nanocomposites as effective electrode and also possible to create new opportunities in the designing and fabrication of variety of efficient electrode in various electrochemical applications.

Development of polypyrrole (nano)structures decorated with gold nanoparticles toward immunosensing for COVID-19 serological diagnosis.

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion in humans is crucial for suitable infection control. In this sense, many studies have focused on increasing the sensibility, lowering the detection limits and minimizing false negative/positive results. Thus, biosensors based on nanoarchitectures of conducting polymers are promising alternatives to more traditional materials since they can hold improved surface area, higher electrical conductivity and electrochemical activity. In this work, we reported the analytical comparison of two different conducting polymers morphologies for the development of an impedimetric biosensor to monitor SARS-CoV-2 seroconversion in humans. Biosensors based on polypyrrole (PPy), synthesized in both globular and nanotubular (NT) morphology, and gold nanoparticles are reported, using a self-assembly monolayer of 3-mercaptopropionic acid and covalently linked SARS-CoV-2 Nucleocapsid protein. First, the novel hybrid materials were characterized by electron microscopy and electrochemical measurements, and the biosensor step-by-step construction was characterized by electrochemical and spectroscopic techniques. As a proof of concept, the biosensor was used for the impedimetric detection of anti-SARS-CoV-2 Nucleocapsid protein monoclonal antibodies. The results showed a linear response for different antibody concentrations, good sensibility and possibility to quantify 7.442 and 0.4 ng/mL of monoclonal antibody for PPy in the globular and NT morphology, respectively. The PPy-NTs biosensor was able to discriminate serum obtained from COVID-19 positive versus negative clinical samples and is a promising tool for COVID-19 immunodiagnostic, which can contribute to further studies concerning rapid, efficient, and reliable detections.

Antidepressants determination using an electroanalytical approach: A review of methods.

Antidepressants are the pharmaceutical compounds used in the treatment of depression, anxiety disorders and all related disturbances promoted by genetic factors, environmental problems or modern lifestyles. Nonetheless, the inadequate ingestion of antidepressants provokes adverse effects in the human body and can contaminate the environment. For this reason, it is necessary to identify and quantify these compounds in biological fluids, natural water, wastewater, and pharmaceutical formulations. Consequently, this review presents the main electroanalytical techniques used in the analysis of antidepressants, indicating the advantages, which include low cost, suitable analytical parameters, simplified sample preparation steps, easy operation and reduced time for completion of the analysis. Reports in specialized literature, published from 2000 to 2020, are presented and some are discussed, demonstrating that the electroanalytical techniques can be employed, with success, in the determination of antidepressants, indicating alternative methodologies to improve analytical parameters and minimize the use and generation of toxic residues.

Applicability of Cork as Novel Modifiers to Develop Electrochemical Sensor for Caffeine Determination.

This study aims to investigate the applicability of a hybrid electrochemical sensor composed of cork and graphite (Gr) for detecting caffeine in aqueous solutions. Raw cork (RAC) and regranulated cork (RGC, obtained by thermal treatment of RAC with steam at 380 °C) were tested as modifiers. The results clearly showed that the cork-graphite sensors, GrRAC and GrRGC, exhibited a linear response over a wide range of caffeine concentration (5-1000 µM), with R2 of 0.99 and 0.98, respectively. The limits of detection (LOD), estimated at 2.9 and 6.1 µM for GrRAC and GrRGC, suggest greater sensitivity and reproducibility than the unmodified conventional graphite sensor. The low-cost cork-graphite sensors were successfully applied in the determination of caffeine in soft drinks and pharmaceutical formulations, presenting well-defined current signals when analyzing real samples. When comparing electrochemical determinations and high performance liquid chromatography measurements, no significant differences were observed (mean accuracy 3.0%), highlighting the potential use of these sensors to determine caffeine in different samples.

Electrochemical detection of specific interactions between apolipoprotein E isoforms and DNA sequences related to Alzheimer's disease.

Apolipoprotein E4 (ApoE4) has a key role on the onset and progression of Alzheimer's disease (AD), since it favours the deposition of toxic amyloid-beta (Aß) aggregates in the brain. These effects might result from the interaction between ApoE4 and specific DNA promoters related to cellular autophagy pathways and to the expression of neuroprotective proteins, like sirtuin-1. Herein, we modified gold electrodes with mixed self-assembled monolayers of 6-mercapto-1-hexanol and thiolated DNA oligonucleotides related to CLEAR (associated with autophagic processes that enable the clearance of toxic species, such as Aß) and SirT1 (related to the expression of sirtuin-1) promoter sequences. The interactions of the immobilized DNA sequences with isoforms of ApoE (ApoE4/ApoE3/ApoE2) were investigated by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) measurements. By monitoring current and charge transfer resistance (Rct) variations, CLEAR showed to interact specifically with ApoE4, whereas SirT1 showed a higher affinity to ApoE4 compared to ApoE3 and ApoE2. To the best of our knowledge, this is the first report about the application of electrochemical techniques to investigate the sequence-specific interaction between ApoE isoforms and CLEAR and SirT1 oligonucleotides.

Glassy carbon electrode modified with carbon black and cross-linked alginate film: a new voltammetric electrode for paraquat determination.

A new electrode based on glassy carbon modified with an alginate film cross-linked with glutaraldehyde containing immobilized carbon black particles was successfully developed and applied for the determination of paraquat (PQ), a herbicide widely used for broadleaf weed control. Different polysaccharides (alginate, cellulose, pectin, starch, and chitosan) were investigated for the immobilization process, and alginate presented the highest chemical modifier potential for PQ determination. Additionally, the influence of chemical cross-linking agents (glutaraldehyde and epichlorohydrin) on the morphology, electrochemical response, and film stability was investigated. All experimental conditions were optimized, including the supporting electrolyte conditions (composition, pH, and concentration) and square wave voltammetry technical parameters. Under the optimized experimental conditions, the PQ analytical curve was linear from 0.4 to 2.0 mg L-1 and the limits of detection and quantification were 0.06 and 0.19 mg L-1, respectively. The proposed electrode is easy to obtain, stable, selective, sensitive, and low cost and was successfully applied for PQ determination in environmental and beverage samples. Graphical abstract.

Effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin.

In this work, we have studied the effect of Crotalus basiliscus snake venom on the redox reaction of myoglobin (Mb), and by means of electrochemical techniques, we have shown that this reaction is undoubtedly affected following the interaction with the venom. Surface plasmon resonance, electrophoresis, UV-Vis, and circular dichroism showed that the interaction involves the attachment of some constituent of the venom to the protein, although not affecting its first and secondary structures. Mass spectra support this suggestion by showing the appearance of signals assigned to the Mb dimer and to a new species resulting from the interaction between Mb and the venom proteins. In addition, the mass spectra suggest the aromatic amino acids of myoglobin, mainly tryptophan and phenylalanine, are more exposed to the solvent medium upon the exposure to the venom solution. The results altogether indicate that the harmful effects of the venom of Crotalus basiliscus snake are likely connected to the blocking of the redox site of Mb.

Amperometric determination of ascorbic acid with a glassy carbon electrode modified with TiO2-gold nanoparticles integrated into carbon nanotubes.

A glassy carbon electrode was modified with a TiO2-gold nanoparticle hybrid integrated with multi-walled carbon nanotubes in a dihexadecylphosphate film (TiO2-Au NP-MWCNT-DHP/GCE) and applied to amperometric determination of ascorbic acid (AA). The modified sensor displays fast charge transfer and shows an irreversible anodic behavior for AA by cyclic voltammetry. Under optimal experimental conditions and using amperometry at 0.4 V, the analytical curve presented a statistical linear concentration range for AA from 5.0 to 51 µmol L-1, with a limit of detection of 1.2 µmol L-1. The electrode was successfully applied to the determination of AA in pharmaceutical and fruit juice without the need for major pretreatment of samples. Graphical abstract Schematic of a new sensing platform for ascorbic acid (AA). It is based on a glassy carbon electrode (GCE) modified with TiO2-Au nanoparticles integrated into carbon nanotubes in a dihexadecylphosphate film. The sensor was applied to amperometric determination of AA in juice and pharmaceutical samples.

Poly(methylene blue)-modified electrode for indirect electrochemical sensing of OH radicals and radical scavengers

ABSTRACT A new modified electrode for indirect sensing of OH· and radical scavengers was described. The electrochemical polymerization of methylene blue in aqueous solutions and the properties of the resulting films on a glassy carbon electrode were carried out using cyclic voltammetry. A surface coverage of 1.11 × 109mol cm2 was obtained, revealing a complete surface coverage of the polymeric film on the electrode surface. OH· was able to destroy the poly(methylene blue) film by exposure to a Fenton solution. The loss of the electrochemical signal of the residual polymeric film attached to the electrode surface was related to the extent of its dissolution. The applicability of the sensor was demonstrated by evaluating the OH radical scavenging effect on different concentrations of ascorbic acid. The obtained radical scavenging capacity were 31.4%, 55.7%, 98.9% and 65.7% for the ascorbic acid concentrations of 5, 10, 30 and 50 mM, respectively.