Glutaraldehyde Activation Research Articles

Concanavalin A immobilized poly(ethylene glycol dimethacrylate) based affinity cryogel matrix and usability of invertase immobilization

Concanavalin A (Con A) immobilized supermacroporous poly(ethylene glycol dimethacrylate) [poly(EGDMA)] monolithic cryogel column was prepared by radical cryocopolymerization of EGDMA as a monomer and N,N′-methylene-bisacrylamide as a crosslinker. Bioligand Con A was then immobilized by covalent binding onto poly(EGDMA) cryogel via glutaraldehyde activation [Con A–poly(EGDMA)]. Con A–poly(EGDMA) cryogel was characterized by swelling studies and scanning electron microscopy. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50μm size. The equilibrium swelling degree of the cryogel was 15.01g H2O/g dry cryogel. Con A–poly(EGDMA) cryogel was used in the adsorption/desorption of invertase from aqueous solutions. The maximum amount of invertase adsorption from aqueous solution in acetate buffer was 55.45mg/g polymer at pH 5.0. Con A–poly(EGDMA) cryogels were used for repetitive adsorption/desorption of invertase without noticeable loss in invertase adsorption capacity after 10 cycles.

β-Galactosidase of Aspergillus oryzae immobilized in an ion exchange resin combining the ionic-binding and crosslinking methods: Kinetics and stability during the hydrolysis of lactose

The strong dependence of the stability of the immobilized enzyme on the temperature. ► The use of ionic exchange resins for immobilization is used food industry. ► Glutaraldehyde activation of the carriers is one technique to immobilize enzymes. ► β-galactosidase e immobilized on the ionic exchange resin followed by crosslinking. ► The hydrolysis kinetics of lactose by β-galactosidase immobilized. ► The influence of temperature in the enzymatic activity of immobilized enzyme. In this work, the hydrolysis kinetics of lactose by Aspergillus oryzae β-galactosidase was studied using the ionic exchange resin Duolite A568 as a carrier. The enzyme was immobilized using a β-galactosidase concentration of 16 g/L in pH 4.5 acetate buffer and an immobilization time of 12 h at 25 ± 0.5 °C. Next, the immobilized β-galactosidase was crosslinked using glutaraldehyde concentration of 3.5 g/L for 1.5 h. The influence of lactose concentration was studied for a range of 5–140 g/L, and the Michaelis–Menten model was fitted well to the experimental results with V m and K m values of 0.71 U and 35.30 mM, respectively. The influence of the product galactose as an inhibitor on the hydrolysis reaction was studied. The model that was best fitted to the experimental results was the competitive inhibition by galactose with V m , K m and K i values of 0.77 U, 35.30 mM and 27.44 mM, respectively. The influence of temperature on the enzymatic activity of the immobilized enzyme was studied in the range of 10–80 °C, in which the temperature of the maximum activity was 60 °C, with an activation energy of 5.32 kcal/mol of lactose, using an initial concentration of lactose of 50 g/L in a pH 4.5 sodium acetate buffer solution. The thermal stability of the immobilized biocatalyst was determined to be in the range 55–65 °C. The first-order model described well the kinetics of thermal deactivation for all the temperatures studied. The activation energy of thermal deactivation from immobilized biocatalyst was 66.48 kcal/mol with a half-life of 8.9 h at 55 °C.

Evaluation of glycine as an inactivator of glutaraldehyde.

Glycine was evaluated as an inactivator of the sporicidal activity of glutaraldehyde. Spores from glucose depleted cultures of Bacillus stearothermophilus grown in a chemically defined medium were used. When glycine is used as an inactivator of glutaraldehyde, it lowers the pH value of the solution. Glycine 1% failed to inactivate 0.5% or higher concentrations of alkaline glutaraldehyde in sporicidal studies. If viable counts cannot be performed within the first hour after inactivation, the concentration of glycine should be at least 2% to inactivate effectively 2% alkaline glutaraldehyde.

Immobilization of horseradish peroxidase on silane-modified ceramics and their properties: potential for oily wastewater treatment

Horseradish peroxidase (HRP) is proved being effective in eliminating oil from aqueous solutions, but the elimination is expensive because free HRP can not be reused. In present work, HRP was successfully immobilized on cordierite porous ceramics support with a novel method of N-beta-aminoethyl-gamma-aminopropyl-trimethoxysilane modification and glutaraldehyde activation. Under the optimized immobilized conditions, the actual immobilized HRP was 1.16 mg/g support, the activity of the immobilized HRP could reach as high as 1379.4 U/g support. Experiment results showed that the properties of storage stability, acid-base stability and the tolerance to the pH fluctuation of the immobilized HRP were better than those of the free HRP. The operation stability of the immobilized HRP was also good. The immobilized HRP is suitable for the oily wastewater treatment because of its reusability proved in this work.

Glutaraldehyde Mediated Conjugation of Amino-Coated Magnetic Nanoparticles with Albumin Protein for Nanothermotherapy

A novel bioconjugation of amino saline capped Fe3O4 magnetic nanoparticles (MNPs) with bovine serum albumin (BSA) was developed by applying glutaraldehyde as activator. Briefly, Fe3O4 MNs were synthesized by the chemical co-precipitation method. Surface modification of the prepared MNPs was performed by employing amino saline as the coating agent. Glutaraldehyde was further applied as an activation agent through which BSA was conjugated to the amino-coated MNPs. The structure of the BSA-MNs was confirmed by FTIR analysis. Physico-chemical characterizations of the BSA-MNPs, such as surface morphology, surface charge and magnetic properties were investigated by Transmission Electron Microscopy (TEM), zeta-Potential and Vibrating Sample Magnetometer (VSM), etc. Magnetic inductive heating characteristics of the BSA-MNPs were analyzed by exposing the MNPs suspension (magnetic fluid) under alternative magnetic field (AMF). The results demonstrate that BSA was successfully conjugated with amino-coated MNs mediated through glutaraldehyde activation. The nanoparticles were spherical shaped with approximately 10 nm diameter. Possessing ideal magnetic inductive heating characteristics, which can generate very rapid and efficient heating while upon AMF exposure, BSA-MNPs can be applied as a novel candidature for magnetic nanothermotherapy for cancer treatment. In vitro cytotoxicity study on the human hepatocellular liver carcinoma cells (HepG-2) indicates that BSA-MNP is an efficient agent for cancer nanothermotherapy with satisfied biocompatibility, as rare cytotoxicity was observed in the absence of AMF. Moreover, our investigation provides a methodology for fabrication protein conjugated MNPs, for instance monoclonal antibody conjugated MNPs for targeting cancer nanothermotherapy.

Surface functionalization of silica-coated magnetic nanoparticles for covalent attachment of cholesterol oxidase

A systematic approach towards the fabrication of highly functionalized silica shell magnetic nanoparticles, presently used for enzyme immobilization, is herein fully presented. The synthesis of bare maghemite (γ-Fe 2O 3) nanoparticles was accomplished by thermal co-precipitation of iron ions in ammonia alkaline solution at harsh reaction conditions, respectively. Primary surface engineering of maghemite nanoparticles was successfully performed by the proper deposition of silica onto nanoparticles surface under strictly regulated reaction conditions. Next, the secondary surface functionalization of the particles was achieved by coating the particles with organosilane followed by glutaraldehyde activation in order to enhance protein immobilization. Covalent immobilization of cholesterol oxidase was attempted afterwards. The structural and magnetic properties of magnetic silica nanocomposites were characterized by TEM and vibrating sample magnetometer (VSM) instruments. X-ray diffraction measurements confirmed the spinel structure and average size of uncoated maghemite nanoparticles to be around 20 nm in diameter. SEM–EDS spectra indicated a strong signal for Si, implying the coating procedure of silica onto the particles surface to be successfully accomplished. Fourier transform infrared (FT-IR) spectra analysis confirmed the binding of amino silane molecules onto the surface of the maghemite nanoparticles mediated Si–O–Si chemical bonds. Compared to the free enzyme, the covalently bound cholesterol oxidase retained 50% of its activity. Binding of enzyme onto chemically modified magnetic nanoparticles via glutaraldehyde activation is a promising method for developing biosensing components in biomedicine.

A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents

Three different approaches to the immobilization of proteins at surfaces have been compared. All rely on the creation of surface groups that bind primary amines on lysine residues. Carboxylic acid terminated self-assembled monolayers (SAMs) have been activated using a water soluble carbodiimide to yield an active ester functionalized surface and with trifluoroacetic anhydride to yield a surface anhydride, and amine terminated SAMs have been activated using glutaraldehyde. Although the degree of surface derivatization by n-alkylamines was greater using the carbodiimide and anhydride methods under anhydrous conditions, the glutaraldehyde activation of amine terminated SAMs yielded significantly greater attachment of streptavidin than is achieved using either of the other methods. This is attributed to the susceptibility to hydrolysis of the active species formed by activation of the carboxylic acid terminated monolayers. Patterned protein structures may be formed by using both glutaraldehyde activation of amine terminated thiols and carbodiimide activation of carboxylic acid terminated thiols, in conjunction with selective photo-oxidation of oligo(ethylene glycol) terminated SAMs.

Ellipsometric characterization of flagellin films for biosensor applications

AbstractThe quality of flagellin films in terms of thickness and homogeneity was investigated by spectroscopic ellipsometry. Flagellin films were prepared in three steps: silanization, glutaraldehyde activation and finally the coating with proteins. The process of film preparation was optimized by varying the duration of the silanization and by testing sticking on different substrates including Si wafer covered with different thickness of silicon‐oxide and also covered with a thin film of tantallum pentoxide. Spectroscopic ellipsometry was applied to gain in‐depth information on the film properties for the optimization of the immobilization. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde

Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality-controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time-duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde-based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.

Preparation of poly(glycidylmethacrylate–methylmethacrylate) magnetic beads: Application in lipase immobilization

Magnetic bead was prepared from the monomers glycidylmethacrylate (GMA) and methylmethacrylate (MMA) via suspension copolymerization in the presence of ferric ions. The magnetic beads were characterized with scanning electron microscope (SEM), FT-IR and ESR spectrophotometers. The beads were sieved and 100–150 μm size of fraction was used in enzyme immobilization. The specific surface area of the magnetic beads was measured by the BET method and was found to be 16.2 m 2/g beads. The reactive character of the epoxy groups allowed the attachment of the amino groups during thermal precipitation reaction. The resulting magnetic beads were used for the covalent immobilization of Candida rugosa lipase via glutaraldehyde activation and glutaraldehyde was also acted a 5-carbon spacer arm. The maximum lipase immobilization on magnetic poly(GMA–MMA) was 23.4 mg g −1. The activity yield of the lipase immobilized on the spacer-arm attached magnetic beads was up to 81%. Kinetic analysis shows that the dependence of lipolytic activity of both free and immobilized lipase on trybutyrin substrate concentration can be described by Michaelis-Menten model with good agreement. The estimated Michaelis constants ( K m) for the free and immobilized lipase are 2.6 and 12.3 mM, respectively. The V max values of free and immobilized enzymes were calculated as 984 and 773 U/mg enzymes, respectively. Employment of immobilization seemed to result in an increase in K m and a decrease in V max. Optimal operational temperature was 5 °C higher for immobilized enzyme than that of the free counterpart. Thermal and storage stabilities were found to be increase with immobilization.

Enzyme immobilization on anodic aluminum oxide/polyethyleneimine or polyaniline composites

Anodic aluminum oxide (AAO) membranes were coated with either polyethyleneimine (PEI) or polyaniline (PANI) and used as supports for enzyme immobilization. The AAO–PEI composite was prepared by cross-flow filtration of the polymer solution through the membrane, whereas AAO–PANI was synthesized using surface chemical polymerization, where MnO 2 was used as oxidizing agent for aniline polymerization. After glutaraldehyde activation, horseradish peroxidase and trypsin were immobilized on the composites. The immobilized enzyme activities were determined either in a batch mode or flow-through system. The AAO–PEI–trypsin derivative yielded filtering membrane catalytically active with initial velocities of 2.95 nmol min −1 and 3.8 nmol min −1 measured in batch mode and flow-through system (0.46 mL min −1), respectively. Furthermore, AAO–PEI–trypsin prepared with membranes of different porosities showed that the enzymatic activity increased as the inside diameter of the pore enlarged. Additionally, it was demonstrated that covalent coupling presents higher activity as compared to attachment by adsorption on a membrane without PEI functionalities, which had 2.9 times less activity. Horseradish peroxidase was successfully immobilized onto the AAO–PANI and retained 74% of its initial activity after five batch cycles and 98% following 24 min under continuous flow. It was also observed by electron microscopy that membrane pores remained unclog after polyaniline coating. Therefore, these results showed the viability of these composites as attractive nano-structured matrices for biotechnological applications.

Glutaraldehyde activation of polymer Nylon-6 for lipase immobilization: Enzyme characteristics and stability

An extracellular alkaline lipase of a thermo tolerant Bacillus coagulans BTS-3 was immobilized onto glutaraldehyde activated Nylon-6 by covalent binding. Under optimum conditions, the immobilization yielded a protein loading of 228 μg/g of Nylon-6. Immobilized enzyme showed maximum activity at a temperature of 55 °C and pH 7.5. The enzyme was stable between pH 7.5–9.5. It retained 88% of its original activity at 55 °C for 2 h and also retained 85% of its original activity after eight cycles of hydrolysis of p-NPP. Kinetic parameters K m and V max were found to be 4 mM and 10 μmol/min/ml, respectively. The influence of organic solvents on the catalytic activity of immobilized enzyme was also evaluated. The bound lipase showed enhanced activity when exposed to n-heptane. The substrate specificity of immobilized enzyme revealed more efficient hydrolysis of higher carbon length (C-16) ester than other ones.

A low, particle‐sized, nonporous support for enzyme immobilization: Uniform poly(glycidyl methacrylate) latex particles

AbstractThe uniform and nonporous poly(glycidyl methacrylate) (poly(GMA)) latex particles, 1.7 μm in size, were first tried as a support in enzyme immobilization. For this purpose, α‐chymotrypsin (CT) was selected as the model enzyme. The low particle size and nonporous character of the selected support allowed to carry out the enzyme–subtrate interaction on a sufficiently large surface area (3.36 m2/g) and in the absence of intraparticular diffusion limitations. This property is particularly important when the immobilized CT is used for the substrates with high molecular weights (i.e., proteins). The latex particles were synthesized by dispersion polymerization of GMA. The reactive character of poly(GMA) allowed the direct attachment of primary amine groups onto the particles. Confocal laser scanning microscopy (CLSM) showed that primary amine groups were preferentially located on the particle's surface. Hence, the selected enzyme, CT was immobilized on the surface of nonporous particles via glutaraldehyde activation. For CT‐immobilized poly(GMA) particles, the maximum activity (rm) and Michealis constant (Km) were found to be 17.2 μmol/mg CT min and 121.6 μm, respectively. No significant loss was observed in the activity of immobilized CT, during the course of experiments. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 818–824, 2006

Improved stability of urease upon coupling to alkylamine and arylamine glass and its analytical use

Biocatalyst stability is a major concern in almost all bioprocesses, because it may affect the overall cost of the process. Immobilization is a cost-effective approach and in the present work, pigeonpea urease was covalently coupled to alkylamine glass via glutaraldehyde activation and arylamine glass via diazotation. This coupling resulted in 92.5% and 90% immobilization, respectively. The immobilized urease showed optimum activity at 77 °C and retained 50% of its activity when incubated at this temperature for 90 min. This immobilized enzyme was quite stable at higher temperatures and over a broad pH range; unusually stable upon storing at 4 °C and also retained 50% activity even after 25 reuses. Hence, the obtained results are much better compared to other matrixes used so far for urease immobilization. This preparation was also successfully used in potentiometric biosensing for the estimation of blood urea from clinical patients. Hence, coupling of urease to glass via this mode of coupling could therefore be a versatile tool for immobilization of proteins and would have great promise in clinical as well as industrial use.

Rapid protein identification using monolithic enzymatic microreactor and LC‐ESI‐MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Immunoassay using the probe-labeled Au/Ag core-shell nanoparticles based on surface-enhanced Raman scattering

Au/Ag core-shell nanoparticles labeled by monoclonal antibody and surface-enhanced Raman scattering (SERS) probes were used for immunoassay analysis. Mouse polyclonal antibodies against hepatitis B surface antigen (mouse Anti-HBsAg PAb) were covalently immobilized on the silicon chips modified by a self-assembled monolayer of (3-amino-propyl) trimethoxysilane via glutaraldehyde (GA) activation. The Au/Ag core-shell nanoparticles were labeled by the mouse monoclonal antibody against hepatitis B surface antigen (mouse Anti-HBsAg MAb) and the probe molecule, p-mercaptobenzoic acid (MBA). Immunoreactions between the labeled Au/Ag core-shell nanoparticles and the corresponding antigens captured by the Anti-HBsAg PAb on the silicon chips were investigated by SERS.

Microfluidic biosensing systems. Part I. Development and optimisation of enzymatic chemiluminescent micro-biosensors based on silicon microchips.

Chemiluminescent (CL) enzyme-based flow-through microchip biosensors (micro-biosensors) for detection of glucose and ethanol were developed for the purpose of monitoring real-time production and release of glucose and ethanol from microchip immobilised yeast cells. Part I of this study focuses on the development and optimisation of the micro-biosensors in a microfluidic sequential injection analysis (microSIA) system. Glucose oxidase (GOX) or alcohol oxidase (AOX) was co-immobilised with horseradish peroxidase (HRP) on porous silicon flow through microchips. The hydrogen peroxide produced from oxidation of the corresponding analyte (glucose or ethanol) took part in the chemiluminescent (CL) oxidation of luminol catalysed by HRP enhanced by addition of p-iodophenol (PIP). All steps in the microSIA system, including control of syringe pump, multiposition valve (MPV) and data readout, were computer controlled. The influence of flow rate and luminol- and PIP concentration were investigated using a 2(3)-factor experiment using the GOX-HRP sensor. It was found that all estimated single factors and the highest order of interaction were significant. The optimum was found at 250 microM luminol and 150 microM PIP at a flow rate of 18 microl min(-1), the latter as a compromise between signal intensity and analysis time. Using the optimised system settings one sample was processed within 5 min. Two different immobilisation chemistries were investigated for both micro-biosensors based on 3-aminopropyltriethoxsilane (APTS)- or polyethylenimine (PEI) functionalisation followed by glutaraldehyde (GA) activation. GOX-HRP micro-biosensors responded linear in a log-log format within the range 10-1000 microM glucose. Both had an operational stability of at least 8 days, but the PEI-GOX-HRP sensor was more sensitive. The AOX-HRP micro-biosensors responded linear (log-log) in the range between 1 and 10 mM ethanol, but the PEI-AOX-HRP sensor was in general more sensitive. Both sensors had an operational stability of at least 8 h, but with a half-life of 2-3 days.

Immobilization of lipase on chitin and its use in nonconventional biocatalysis.

Chitin was functionalized with hexamethylenediamine followed by glutaraldehyde activation, and its capacity to bind Candida rugosa lipase was investigated. The loading of 250 units g(-1) support showed to be effective, resulting in a uniform enzyme fixation with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, and thermal stability. For the immobilized lipase, the influence of the reaction temperature and substrate polarity in nonconventional biocatalysis was also analyzed. Production of butyl esters was found to be dependent on the substrate partition coefficient, which accounts the greatest value for the system butanol and butyric acid. The highest enzyme activity was found for the system butanol and caprylic acid at a reaction temperature of 40 degrees C. Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 12 batches, revealing a biocatalyst half-life of 426.7 h.

Potential biological indicators for glutaraldehyde and formaldehyde sterilization processes.

The present study aimed to isolate, select, and evaluate bacterial isolates with potential for use as biological indicators for sterilization with glutaraldehyde and/or formaldehyde. A total of 340 local Bacillus isolates were screened for glutaraldehyde and/or formaldehyde resistance by determination of minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and extinction time and were compared with B. subtilis (var. niger) ATCC 9372, the biological indicator for ethylene oxide sterilization, as reference. Of these, 85 isolates had glutaraldehyde MICs of 0.5% or higher, while 29 had formaldehyde MICs of 0.04% or higher. Of the 29 resistant isolates, 15 had MBCs of 0.05% or more. Extinction times were used to evaluate the bactericidal/sporicidal activity of glutaraldehyde. Eight had inactivation times of more than 5 h in 2% glutaraldehyde (pH 8), whereas 12 had inactivation times of more than 3 h in l% formaldehyde, with one isolate in common. These 19 isolates were selected and evaluated as potential biological indicators for aldehydes by determination of the decimal reduction times ( D values), compared with the reference strain. Eight glutaraldehyde-resistant isolates exhibited D values 2.0- to 3.5-fold higher than the reference strain (30 min.). Only five of 12 formaldehyde resistant isolates had D values higher than that of the reference strain. Using six resistant isolates, temperature coefficient values between 2.11 and 3.02 were obtained for 2% formaldehyde. Finally, 14 isolates were tested for potential pathogenicity and were identified to species level. All of the eight glutaraldehyde-resistant isolates, including the isolate with dual resistance, and three formaldehyde-resistant isolates were B. licheniformis, while two other formaldehyde-resistant isolates were B. cereus. Six of the selected B. licheniformis isolates are potential biological indicators for sterilization processes using aldehydes. Three can be suggested for glutaraldehyde only and three for both aldehydes.

Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents

A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25×45×5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1–110 mM and the detection limit was 0.1 mM (3 σ).