Redox Properties of Ferricyanide Ion on Layer‐by‐Layer Thin Film‐Coated Gold Electrodes; Effects of Type of Polycationic Components in the Film

The surface of a gold (Au) electrode was coated with layer‐by‐layer (LbL) thin films composed of poly(vinyl sulfate) (PVS) and different type of poly(amine)s including poly(allylamine) (PAH), poly(ethyleneimine) (PEI) and poly(diallyldimethylammonium chloride) (PDDA) and redox properties of ferricyanide ion ([Fe(CN)₆]^3?) on the LbL film‐coated Au electrodes were studied. The LbL film‐coated electrodes exhibited redox response to [Fe(CN)₆]^3? ion when the outermost surface of the LbL film was covered with the cationic poly(amine)s while virtually no response was observed on the LbL film‐coated electrodes whose outermost surface was covered with PVS due to an electrostatic repulsion between [Fe(CN)₆]^3? ion and the negatively‐charged PVS layer. The redox properties of [Fe(CN)₆]^3? ion on the LbL film‐coated electrodes significantly depended on the type of polycationic materials in the LbL film. The LbL film‐coated electrodes which had been immersed in the [Fe(CN)₆]^3? solution for 15 min exhibited redox response even in a [Fe(CN)₆]^3? ion‐free buffer solution, suggesting that [Fe(CN)₆]^3? ion is confined in the films. In the buffer solution, redox peaks were observed between +0.1 and 0.4 V depending on the type of polycations in the film. Thus, [Fe(CN)₆]^3? ion can be confined in the film and the redox potential is polycation‐dependent.     

pH‐Switchable Bioelectrocatalysis Based on Weak Polyelectrolyte Multilayers

Weak polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were assembled into {PAH/PAA}_n layer‐by‐layer films on electrodes. The cyclic voltammetry (CV) response of ferrocenecarboxylic acid (Fc(COOH)) at {PAH/PAA}₅ film electrodes assembled under the specific condition showed pH‐sensitive “on‐off” switching property. This property was further used to control the electrocatalytic oxidation of glucose by glucose oxidase (GOD) with Fc(COOH) as the electron transfer mediator, so that the pH‐switchable bioelectrocatalysis could be realized. The mechanism of pH‐sensitive behavior of the system was explored and believed to originate from the pH‐dependent structure change of the films.     

A Facile Preparation of H2O2 Sensors Using Layer‐by‐Layer Deposited Thin Films Composed of Poly(ethyleneimine) and Carboxymethyl Cellulose as Matrices for Immobilizing Hemin

A layer‐by‐layer (LbL) thin film composed of poly(ethyleneimine) (PEI) and carboxymethyl cellulose (CMC) was prepared on the surface of a gold (Au) disk electrode and the LbL layer was impregnated with hemin to fabricate amperometric hydrogen peroxide (H₂O₂) sensors. Hemin can be easily immobilized in the LbL layer by immersing the LbL film‐coated electrode in the hemin solution. The hemin‐modified electrode thus prepared exhibited an amperometric response to H₂O₂ on the basis of the electrochemical reduction catalyzed by hemin. The output current of the hemin‐modified electrode depended on the concentration of H₂O₂ over the range of 0.005–1.0 mM. Thus, the LbL film composed of PEI and CMC was found to be an excellent material for the facile preparation of hemin‐based H₂O₂ sensors.     

Layer—by—layer Multilaryer Films Self—assembled from a Rare—earth—containing Poly—oxometalate,Na9[Eu（W5O18）2] and Poly （allylamine hydrochloride） and Their Photoluminescent Properties

Ultrathin multilayer films of a rare-earth-containing polyoxometalate Na9[Eu(W5O18)2](EW) and poly (allymamine hydrochloride)(PAH) have been prepared by layer-by-layer self-assembly from dilute aqueous solution.The fabrication process of the EW/PAH multilaryer films was followed by UV-vis spectroscopy and ellipsometry,which show that the deposition process is linear and highly reproducible from layer to layer.An average EW/PAH bilayer thickness of ca.2.1nm was determined by ellipsometry.In addition,the scanning electron microscopy(SEM) image of the EW/PAH film indicates that the film surface is relatively uniform and smooth.The photoluminescent properties of these films were also investigated by fluorescence spectroscopy.     

Polyelectrolyte Multilayer Films Containing Ferrocene: Effects of Polyelectrolyte Type and Ferrocene Contents in the Film on the Redox Properties

Electrochemical properties of Fc‐PEM films have been studied by changing the chemical structure of the polymer chains and the content of Fc moiety in the film systematically. We have prepared a series of PEM films by a layer‐by‐layer deposition of polycations, Fc‐modified poly(allylamine) (Fc‐PAA) and poly(ethyleneimine) (Fc‐PEI), and polyanionic poly(vinyl sulfate) (PVS) on the surface of a gold electrode. The redox properties of the Fc‐PAA/PVS and Fc‐PEI/PVS films depended significantly on the content of Fc moiety in the polymer chains and on the polymer type. Fc‐ PAA and Fc‐PEI polymer chains can penetrate 3 or 4 PAA/PVS bilayers inserted between the redox polymers and electrode. The Fc‐PAA film‐modified electrode can be used for electrocatalytic oxidation of ascorbic acid.     

Long Distance Electron Transfer Across >100 nm Thick Au Nanoparticle/Polyion Films to a Surface Redox Protein

Glutathione‐decorated 5 nm gold nanoparticles (AuNPs) and oppositely charged poly(allylamine hydrochloride) (PAH) were assembled into {PAH/AuNP}_n films fabricated layer‐by‐layer (LbL) on pyrolytic graphite (PG) electrodes. These AuNP/polyion films utilized the AuNPs as electron hopping relays to achieve direct electron transfer between underlying electrodes and redox proteins on the outer film surface across unprecedented distances >100 nm for the first time. As film thickness increased, voltammetric peak currents for surface myoglobin (Mb) on these films decreased but the electron transfer rate was relatively constant, consistent with a AuNP‐mediated electron hopping mechanism.     

Electrochemical Preparation of Brilliant‐Blue‐Modified Poly(diallyldimethylammonium Chloride) and Nafion‐Coated Glassy Carbon Electrodes and Their Electrocatalytic Behavior Towards Oxygen and L‐Cysteine

Brilliant blue FCF‐modified glassy carbon electrodes have been prepared by cycling the Nafion (or poly(diallyldimethylammonium chloride) (PDDAC)) coated electrodes repeatedly 15 cycles in brilliant blue FCF (BB FCF) dye solution. The BB FCF molecules are incorporated into Nafion coating by cycling the film‐covered electrode between +0.3 to 1.2 V (vs. Ag/AgCl) in pH 1.5 BB FCF solution while PDDAC‐coated electrode cycled between 0 to ?1.0 V (vs. Ag/AgCl) in pH 6.5 BB FCF solution to immobilize the dye. Electrostatic interaction between dye molecule and PDDAC was predominant in PDDAC coating whereas immobilization of dye in Nafion film attributed to the combined effect of electrostatic and hydrophobic interactions. The voltammetric features of BB FCF‐modified electrodes resemble that of surface‐confined redox couples. The peak potentials of BB FCF‐incorporated PDDAC‐coated electrode were shifted to more positive potential region with decreasing pH of contacting solution. BB FCF‐modified electrodes showed electrocatalytic activity towards reduction of oxygen and oxidation of L ‐cysteine with significant decease of overvoltage compared to unmodified electrode. The BB FCF‐modified Nafion‐coated electrode was tested for its analytical applications toward determination of L ‐cysteine. The linear range of calibration plot at BB FCF‐modified Nafion‐coated electrode is 10 to 100 μM, which coincides with L ‐cysteine levels in biological fluids. Sensitivity and detection limit of the electrode are 111 nA μM^?1 and 0.5 μM, respectively.     

Cross‐Linked Multilayers of Poly(vinyl amine) as a Single Component and Their Interaction with Proteins

Novel multilayer thin films that consist solely of cross‐linked single component layers are generated by a selective cross‐linking of the poly(vinyl amine) (PVAm) layers in [PVAm/poly(acrylic acid) (PAA)]_n thin films constructed either on silica particles or silicon wafers, followed by the removal of PAA. The surface topography of the (PVAm)_n multilayer thin films, before and after the adsorption of human serum albumin (HSA), has been studied by atomic force microscopy on the freeze‐dried films. The decrease of the average roughness of the film after the adsorption of HSA showed the protein was adsorbed into the (PVAm)_n film making these films potential reservoirs for proteins.

     

Degradation of poly(acrylates) under SF5+ primary ion bombardment studied using time‐of‐flight secondary ion mass spectrometry. 2. Poly(n‐alkyl methacrylates)

Polyatomic primary ions offer low penetration depth and high damage removal rates in some polymers, facilitating their use in the molecular depth profiling of these polymers by secondary ion mass spectrometry (SIMS). This study is the second in a series of systematic characterizations of the effect of polymer chemistry on degradation under polyatomic primary ion bombardment. In this study, time‐of‐flight SIMS (ToF‐SIMS) was used to measure the damage of ～90 nm thick spin‐cast poly(methyl methacrylate), poly(n‐butyl methacrylate), poly(n‐octyl methacrylate) and poly(n‐dodecyl methacrylate) films under extended (～2 × 10¹⁴ ions cm^?2) 5 keV SF₅⁺ bombardment. The degradation of the poly(n‐alkyl methacrylates) were compared to determine the effect of the length of the alkyl pendant group on their degradation under SF₅⁺ bombardment. The sputter rate and stability of the characteristic secondary ion intensities of these polymers decreased linearly with alkyl pendant group length, suggesting that lengthening the n‐alkyl pendant group resulted in increased loss of the alkyl pendant groups and intra‐ or intermolecular cross‐linking under SF₅⁺ bombardment. These results are partially at variance with the literature on the thermal degradation of these polymers, which suggested that these polymers degrade primarily via depolymerization with minimal intra‐ or intermolecular cross‐linking. Copyright © 2004 John Wiley & Sons, Ltd.     

Ferrocene-containing polyelectrolyte multilayer film-covered electrodes: electrocatalytic determination of ascorbic acid and use of inner blocking layers to improve the upper detection limit of the electrodes

A multilayer film composed of ferrocene(Fc)-appended poly(allylamine hydrochloride) (Fc-PAH) and poly(potassium vinylsulfate) (PVS) has been prepared on the surface of a gold(Au) electrode by using a layer-by-layer self-assembly technique. Fc-containing polyelectrolyte multilayer (PEM) film-modified electrodes can electrochemically catalyze the oxidation of ascorbic acid successfully. For a 2 (Fc-PAH/PVS) bilayer-covered electrode the catalytic current increased linearly with increasing concentration of ascorbic acid over the concentration range 6 mol L^–1–3 mmol L^–1. To extend the dynamic range for ascorbic acid, the surface of the Au electrode was first covered with a (PAH/PVS)₂ film on which an additional (Fc-PAH/PVS)₅ film was coated. This strategy successfully extended the dynamic range of the electrode up to 25 mmol L^–1 ascorbic acid, because the (PAH/PVS)₂ layer blocked access of ascorbic acid to the electrode surface. The upper detection limit of the (PAH/PVS)₂ (Fc-PAH/PVS)₅ film-modified electrode is much higher than those of Fc-based ascorbic acid sensors reported so far. Electron transfer is diffusion-controlled within the (PAH/PVS)₂(Fc-PAH/PVS)₅ film.     

Voltammetric Studies of Cadmium‐ and Zinc‐Containing Metallothioneins at Nafion‐Coated Mercury Thin Film Electrodes

Voltammetric studies of rabbit liver metallothioneins (MTs, containing both Zn and Cd ions) and Zn₇‐MT were carried out at Nafion‐coated mercury film electrodes (NCMFEs). The accumulation of MT molecules into the NCMFEs enhances the voltammetric signals and the electrostatic interaction between the Nafion membrane and MT facilitates facile electron transfer reactions. Two well‐defined redox waves, with reduction potential (E_pc) values at ?0.740 and ?1.173 V, respectively, were observed. The peak at E_pc =?0.740 V is attributable to the reduction of the Cd‐MT complex, whereas that at E_pc=?1.173 V was assigned to the reduction of the Zn‐MT complex. Zn₇‐MT exhibits only one redox wave with E_pc=?1.198 V. The NCMFE was found to be more advantageous than thin mercury film electrode (MFE), because the pristine metal ions in MTs (e.g., Cd²⁺ and/or Zn²⁺) are not significantly replaced by Hg²⁺. The NCMFE is also complementary to Nafion‐coated bismuth film electrode in that it has a greater hydrogen overpotential, which allows the reduction of the Zn‐MT complex to be clearly observed. Moreover, intermetallic compound formation between Cd and Zn appears to be less serious at NCMFEs. Consequently, the amounts of Cd and Zn deposited into the electrode upon the reduction reactions can be quantified more accurately.     

Electrolyte Effects on Charge Transport Behavior of [Os(bpy)2(PVP)10Cl]Cl and [Ru(bpy)2(PVP)10Cl]Cl Redox Polymers in Ultra‐Thin Films of Polyions

Metallopolymer films have important applications in electrochemical catalysis. The alternate electrostatic layer‐by‐layer method was used to assemble films of [Ru(bpy)₂(PVP)₁₀Cl]Cl (denoted as ClRu‐PVP) and [Os(bpy)₂(PVP)₁₀Cl]Cl (ClOs‐PVP) metallopolymers onto pyrolytic graphite electrodes. Film thickness estimated by quartz crystal microbalance was 6–8 nm. The effects of pH, electrolyte species and concentration on the electrochemical properties of these electroactive polymers were studied using cyclic voltammetry (CV). Behavior in various electrolytes was compared. Also the mass changes within the ultra‐thin film during redox of Os^2+/3+ were characterized by in situ electrochemical quartz crystal microbalance (EQCM). The results indicate rapid reversible electron transfer, and show that both ClRu‐PVP and ClOs‐PVP have compact surface structures while ClOs‐PVP is a little denser than ClRu‐PVP. Although hydrogen ions do not participate in the chemical reaction of either film, the movement of Na⁺ cation and water accompanies the redox process of ClOs‐PVP films.     

Electrochemistry of and Redox‐Induced Metal Release from Metallothioneins at a Nafion‐Coated Bismuth Film Electrode

Nafion‐coated bismuth film electrodes (NCBiFEs) were used to investigate the redox behavior of and metal release from rabbit liver metallothioneins (MTs) in an acetate buffer. Owing to the permselective exchange between positively charged MT molecules and cations in Nafion and the absence of detectable MT adsorption at bismuth surface, a diffusion‐controlled Nernstian redox wave of MTs (E_pc=?0.869 V) was observed for the first time. The Nernstian behavior of the diffusing MTs is in contrast to the voltammetric responses of MTs at thin mercury films or Nafion‐coated mercury film electrodes, which either result in the replacement of the metals originally present in MTs by mercury or lead to a noticeable MT adsorption. By avoiding these undesirable features, the NCBiFE provides an excellent milieu for voltammetric studies of different types of MTs and related isoforms, paving the way for studying the redox‐modulated metal transfer of MTs and understanding the biological role of MTs in heavy metal detoxification and essential metal regulations.     

Electropreparation of Poly(benzophenone‐4) Film Modified Electrode and Its Electrocatalytic Behavior Towards Dopamine,Ascorbic Acid and Nitrite

The oxidation of benzophenone‐4 (2‐hydroxy‐4‐methoxybenzophenone‐5‐sulfonic acid) at glassy carbon electrode gives rise to stable redox active electropolymerized film during repetitive potential cycling between 0 to 1.3 V (Ag/AgCl). Cyclic voltammogram of poly(benzophenone‐4) film shows a redox couple with well‐defined peaks. The redox response of the modified electrode was found to be depending on the pH of the contacting solution. The peak potentials were shifted to a less positive region with increasing pH and the dependence of the peak potential was found to be 51 mV/pH. The electrocatalytic behavior of poly(benzophenone‐4) film modified electrode towards oxidation of dopamine, ascorbic acid and reduction of nitrite was investigated. The oxidation of dopamine and ascorbic acid occurred at less positive potential on poly(benzophenone‐4) film compared to bare glassy carbon electrode. For dopamine, the overpotential was reduced about 180 mV. Feasibility of utilizing poly(benzophenone‐4) film coated electrode in analytical estimation of dopamine, ascorbic acid and nitrite was also demonstrated.     

Selective Detection of Ascorbic Acid Using Octacyanomolybdate‐Doped‐Glutaraldehyde‐Cross‐Linked Poly‐L‐Lysine Film Modified Glassy Carbon Electrode

The present work describes oxidation of ascorbic acid (AA) at octacyanomolybdate‐doped‐glutaraldehyde‐cross‐linked poly‐L ‐lysine (PLL‐GA‐Mo(CN) film modified glassy carbon electrode in 0.1 M H₂SO₄. The modified electrode has been successfully prepared by means of electrostatically trapping Mo(CN) mediator in the cationic film of glutaraldehyde‐cross‐linked poly‐L ‐lysine. The dependence of peak current of modified electrode in pure supporting indicates that the charge transfer in the film was a mixed process at low scan rates (5 to 200 mV s^?1), and kinetically restrained at higher scan rates (200 to 1000 mV s^?1). Cyclic voltammetry and rotating disk electrode (RDE) techniques are used to investigate the electrocatalytic oxidation of ascorbic acid and compared with its oxidation at bare and undoped PLL‐GA film coated electrodes. The rate constant of catalytic reaction k obtained from RDE analysis was found to be 9.5×10⁵ cm³ mol^?1 s^?1. The analytical determination of ascorbic acid has been carried out using RDE technique over the physiological interest of ascorbic acid concentrations with a sensitivity of 75 μA mM^?1. Amperometric estimation of AA in stirred solution shows a sensitivity of 15 μA mM^?1 over the linear concentration range between 50 and 1200 μM. Interestingly, PLL‐GA‐Mo(CN) modified electrode facilitated the oxidation of ascorbic acid but not responded to other electroactive biomolecules such as dopamine, uric acid, NADH, glucose. This unique feature of PLL‐GA‐Mo(CN) modified electrode allowed for the development of a highly selective method for the determination of ascorbic acid in the presence of interferents.     

Zinc Oxide/Zinc Hexacyanoferrate Hybrid Film‐Modified Electrodes for Guanine Detection

An electroactive polynuclear hybrid films of zinc oxide and zinc hexacyanoferrate (ZnO/ZnHCF) have been deposited on electrode surfaces from H₂SO₄ solution containing Zn(NO₃)₂ and K₃[Fe(CN)₆] by repetitive potential cycling method. Simultaneous cyclic voltammetry and electrochemical quartz crystal microbalance (EQCM) measurements demonstrate the steady growth of hybrid film. There are two redox couples present in the voltammograms of hybrid film and it is obvious in the case of pH 2. Surface morphology of hybrid film was investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Energy dispersive spectrometer (EDS) data confirm existence of zinc oxide in the hybrid film. The effect of type of monovalent cations on the redox behavior of resulting film was investigated. In pure supporting electrolyte, electrochemical responses of modified electrode resemble with that of a surface immobilized redox couple. The electrocatalytic activity of ZnO/ZnHCF hybrid film was investigated towards guanine using cyclic voltammetry and rotating disc electrode (RDE) techniques. Finally, feasibility of using ZnO/ZnHCF hybrid film‐coated electrodes for guanine estimation in flow injection analysis (FIA) was also investigated.     

Electrochemical decomposition of layer-by-layer thin films composed of TEMPO-modified poly(acrylic acid) and poly(ethyleneimine)

The decomposition of layer-by-layer (LbL) thin films composed of 2,2,6,6-tetramethylpiperidine-1-oxyl free radical-appended poly(acrylic acid) (TEMPO-PAA) and poly(ethylenimine) (PEI) was studied by using a quartz crystal microbalance (QCM) and cyclic voltammetry. The electrode potential of the (PEI/TEMPO-PAA)₄/PEI film-coated Au resonator was scanned from +0.2 to +0.8 V vs Ag/AgCl. The CV showed that the oxidation peak current decreased as the number of scans increased. The change in the resonance frequency of the QCM increased after electrolysis, indicating that the film was decomposed by electrolysis. The positive charges originating from the oxoammonium ions probably destabilized the (PEI/TEMPO-PAA)₄/PEI film. Furthermore, the release of 5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonic acid (TPPS) from TPPS-loaded (PEI/TEMPO-PAA)₄/PEI-coated ITO electrodes was investigated. TPPS was released at electrode potentials greater than +0.6 V by the decomposition of the film. The results suggest that TEMPO-PAA/PEI LbL films are suitable for electrochemically controlled drug delivery systems.     

Spectroelectrochemical Sensing Based on Multimode Selectivity Simultaneously Achievable in a Single Device. 19. Preparation and Characterization of Films of Quaternized Poly(4‐vinylpyridine)‐silica

Quaternized poly(4‐vinylpyridine) (QPVP) has been incorporated as an anion exchanger into sol‐gel derived silica films for use in a spectroelectrochemical sensor. The preparation, characteristics and performance of these films are described. The films, which are spin‐coated onto the surface of a planar optically transparent electrode, are optically transparent and uniform. Scanning electron microscopy and spectroscopic ellipsometry have been used to examine film structure, thickness and optical properties. These films have been shown both spectroscopically and electrochemically to preconcentrate ferrocyanide, a model analyte for the sensor. The films can be regenerated for multiple measurements by exposure to 1 M KNO₃. The effects of polymer molecular weight and storage conditions on film performance are described. The overall response of this film is comparable to the poly(dimethyldiallylammonium chloride)‐silica films previously used for this sensor.     

Novel thermally stable poly(amine hydrazide)s and poly(amine‐1,3,4‐oxadiazole)s for luminescent and electrochromic materials

We describe the preparation, characterization, and luminescence of four novel electrochromic aromatic poly(amine hydrazide)s containing main‐chain triphenylamine units with or without a para‐substituted N,N‐diphenylamino group on the pendent phenyl ring. These polymers were prepared from either 4,4′‐dicarboxy‐4″‐N,N‐diphenylaminotriphenylamine or 4,4′‐dicarboxytriphenylamine and the respective aromatic dihydrazide monomers via a direct phosphorylation polycondensation reaction. All the poly(amine hydrazide)s were amorphous and readily soluble in many common organic solvents and could be solution‐cast into transparent and flexible films with good mechanical properties. These poly(amine hydrazide)s exhibited strong ultraviolet–visible absorption bands at 346–348 nm in N‐methyl‐2‐pyrrolidone (NMP) solutions. Their photoluminescence spectra in NMP solutions or as cast films showed maximum bands around 508–544 and 448–487 nm in the green and blue region for the two series of polymers. The hole‐transporting and electrochromic properties were examined by electrochemical and spectroelectrochemical methods. All obtained poly(amine hydrazide)s and poly(amine‐1,3,4‐oxadiazole)s exhibited two reversible oxidation redox couples at 0.8 and 1.24 V vs. Ag/AgCl in acetonitrile solution and revealed excellent stability of electrochromic characteristics, changing color from original pale yellow to green and then to blue at electrode potentials of 0.87 and 1.24 V, respectively. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3245–3256, 2005     

Non‐covalent Immobilization of Iron‐triazole (Fe(Htrz)3) Molecular Mediator in Mesoporous Silica Films for the Electrochemical Detection of Hydrogen Peroxide

Mesoporous silica thin films encapsulating a molecular iron‐triazole complex, Fe(Htrz)₃ (Htrz=1,2,4,‐1H‐triazole), have been generated by electrochemically assisted self‐assembly (EASA) on indium‐tin oxide (ITO) electrode. The obtained modified electrodes are characterized by well‐defined voltammetric signals corresponding to the Fe^II/III centers of the Fe(Htrz)₃ species immobilized into the films, indicating fast electron transfer processes and stable operational stability. This is due to the presence of a high density of redox probes in the material (1.6×10^?4 mol g^?1 Fe(Htrz)₃ in the mesoporous silica film) enabling efficient charge transport by electron hopping. The mesoporous films are uniformly deposited over the whole electrode surface and they are characterized by a thickness of 110 nm and a wormlike mesostructure directed by the template role played by Fe(Htrz)₃ species in the EASA process. These species are durably immobilized in the material (they are not removed by solvent extraction). The composite mesoporous material (denoted Fe(Htrz)₃@SiO₂) is then used for the electrocatalytic detection of hydrogen peroxide, which can be performed by amperometry at an applied potential of ?0.4 V versus Ag/AgCl and by flow injection analysis. The organic‐inorganic hybrid film electrode displays good sensitivity for H₂O₂ sensing over a dynamic range from 5 to 300 μM, with a detection limit estimated at 2 μM.