Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 μg L⁻¹. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (1) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future.     

On-line ionic imprinted polymer selective solid-phase extraction of nickel and lead from seawater and their determination by inductively coupled plasma-optical emission spectrometry

Nickel(II) and lead(II) ionic imprinted 8-hydroxyquinoline polymers were synthesized by a precipitation polymerization technique and were used as selective solid phase extraction supports for the determination of nickel and lead in seawater by flow injection solid phase extraction on-line inductively coupled plasma-optical emission spectrometry. An optimum loading flow rate of 2.25 mL min⁻¹ for 2 min and an elution flow rate of 2.25 mL min⁻¹ for 1 min gave an enrichment factor of 15 for nickel. However, a low dynamic capacity and/or rate for adsorption and desorption was found for lead ionic imprinted polymer and a flow rate of 3.00 mL min⁻¹ for 4-min loading and a flow rate of 2.25 mL min⁻¹ for 1-min elution gave a enrichment factor of 5. The limit of detection was 0.33 μg L⁻¹ for nickel and 1.88 μg L⁻¹ for lead, with a precision (n = 11) of 8% (2.37 μg Ni L⁻¹) for nickel and 11% (8.38 μg Pb L⁻¹) for lead. Accuracy was also assessed by analyzing SLEW-3 (estuarine water) and TM-24 (lake water) certified reference materials, and the values determined were in good agreement with the certified concentrations.     

Effect of Substrate Concentration on Dark Fermentation Hydrogen Production Using an Anaerobic Fluidized Bed Reactor

The effect of substrate (glucose) concentration on the stability and yield of a continuous fermentative process that produces hydrogen was studied. Four anaerobic fluidized bed reactors (AFBRs) were operated with a hydraulic retention time (HRT) from 1 to 8 h and an influent glucose concentration from 2 to 25 g L⁻¹. The reactors were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C with an influent pH around 5.5 and an effluent pH of about 3.5. The AFBRs with a HRT of 2 h and a feed strength of 2, 4, and 10 g L⁻¹ showed satisfactory H₂ production performance, but the reactor fed with 25 g L⁻¹ of glucose did not. The highest hydrogen yield value was obtained in the reactor with a glucose concentration of 2 g L⁻¹ when it was operated at a HRT of 2 h. The maximum hydrogen production rate value was achieved in the reactor with a HRT of 1 h and a feed strength of 10 g L⁻¹. The AFBRs operated with glucose concentrations of 2 and 4 g L⁻¹ produced greater amounts of acetic and butyric acids, while AFBRs with higher glucose concentrations produced a greater amount of solvents.     

Production and analytical characterization of neopterin immunoreagents for biosensor developments

Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins. The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 μg L⁻¹, respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 μg L⁻¹. The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations ranging from 3.2 to 103 μg L⁻¹ could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5–16 μg L⁻¹ (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into multianalyte biochip detection systems.     

Competitive capacitive biosensing technique (CCBT): A novel technique for monitoring low molecular mass analytes using glucose assay as a model study

A novel technique for monitoring of low molecular mass analytes using a flow-injection capacitive biosensor is presented. The method is based on the ability of a small molecular mass analyte to displace a large analyte–carrier conjugate from the binding sites of an immobilized biorecognition element with weak affinity to both compounds. A model study was performed on glucose as the small molecular mass analyte. In the absence of glucose, binding of a glucose polymer or a glycoconjugate to concanavalin A results in a capacitance decrease. Upon introduction of glucose, it displaces a part of the bound glucose polymer or glycoconjugate leading to a partial restoration of capacitance. Experimental results show that the change in capacitance depends linearly on glucose concentration within the range from 1.0 × 10⁻⁵ to 1.0 × 10⁻¹ M, corresponding to 1.8 μg ml⁻¹ to 18 mg ml⁻¹ in a logarithmic plot, with a detection limit of 1.0 × 10⁻⁶ (0.18 μg ml⁻¹) under optimized conditions. In addition, by modifying the molecular mass of the glucose polymer, amount of biorecognition element, and buffer composition, we were able to tune the analyte-sensing range. The developed technique has the benefits of expanded dynamic range, high sensitivity, and excellent reusability.     

Stannum film electrode for square wave voltammetric determination of trace copper(II)

An anodic stripping voltammetric procedure for the determination of Cu(II) at an in situ-plated stannum film electrode (SnFE) was described. The results indicated that the SnFE had an attractive electroanalytical performance, with two distinct voltammetric stripping signals for copper and stannum, and showed the superior advantage for the determination of copper compared with the bismuth film electrode. Several experimental parameters were optimized. The SnFE exhibited highly linear behavior in the concentration range from 1.0 to 100.0 μg L⁻¹ of Cu(II) (r = 0.994) with the detection limit of 0.61 μg L⁻¹ (S/N = 3), and the relative standard deviation for a solution containing 40.0 μg L⁻¹ Cu(II) was 2.2% (n = 8). The procedure has been successfully applied for the determination of Cu(II) in lake water sample.     

Ultra-fast HPLC-ICP-MS analysis of oxaliplatin in patient urine

A novel method for rapid HPLC-ICP-MS analysis of oxaliplatin in human urine was developed implementing a stationary HPLC phase with a particle size of 1.8 μm. The method allowed a cycle time of <1 min at a HPLC flow rate of 0.9 mL min⁻¹. Procedural limits of detection of 0.05 μg L⁻¹ oxaliplatin (150 fg on column) were obtained. Analysis of oxaliplatin in patient urine showed that accurate quantification of the intact drug demanded for storage at −80 °C and rapid measurement after thawing.     

Effect of organic load on the performance and methane production of an AnSBBR treating effluent from biodiesel production

Currently, there is an increasing demand for the production of biodiesel and, consequently, there will be an increasing need to treat wastewaters resulting from the production process of this biofuel. The main objective of this work was, therefore, to investigate the effect of applied volumetric organic load (AVOL) on the efficiency, stability, and methane production of an anaerobic sequencing batch biofilm reactor applied to the treatment of effluent from biodiesel production. As inert support, polyurethane foam cubes were used in the reactor and mixing was accomplished by recirculating the liquid phase. Increase in AVOL resulted in a drop in organic matter removal efficiency and increase in total volatile acids in the effluent. AVOLs of 1.5, 3.0, 4.5 and 6.0 g COD L⁻¹ day⁻¹ resulted in removal efficiencies of 92%, 81%, 67%, and 50%, for effluent filtered samples, and 91%, 80%, 63%, and 47%, for non-filtered samples, respectively, whereas total volatile acids concentrations in the effluent amounted to 42, 145, 386 and 729 mg HAc L⁻¹, respectively. Moreover, on increasing AVOL from 1.5 to 4.5 g COD L⁻¹ day⁻¹ methane production increased from 29.5 to 55.5 N mL CH₄ g COD⁻¹. However, this production dropped to 36.0 N mL CH₄ g COD⁻¹ when AVOL was increased to 6.0 g COD L⁻¹ day⁻¹, likely due to the higher concentration of volatile acids in the reactor. Despite the higher concentration of volatile acids at the highest AVOL, alkalinity supplementation to the influent, in the form of sodium bicarbonate, at a ratio of 0.5–1.3 g NaHCO₃ g COD_fed⁻¹, was sufficient to maintain the pH near neutral and guarantee process stability during reactor operation.     

Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis–Menten constant (K _M) of the enzyme and V _max of the reaction. The obtained K _M of the reaction was 14 ± 3 g L⁻¹ (41 μM). Furthermore, inhibition by adenosine 5′-monophosphate (AMP) was evaluated, and the K _M^App value was determined to be 12 ± 2 g L⁻¹ (35 μM) for 7.5 and 15 μM AMP, respectively, with V _max decreasing from 0.1 ± 0.03 to 0.05 ± 0.01 g L⁻¹ min⁻¹. Therefore, AMP exerted a non-competitive inhibition.     

Flow injection on-line hydrophobic sorbent extraction for flame atomic absorption spectrometric determination of cadmium in water samples

A flow injection system was developed for on-line sorbent extraction preconcentration and flame atomic absorption spectrometric determination of cadmium in natural water samples. The non-charged cadmium complex with diethyl-dithiophosphate (DDPA) was formed on-line in 0.1 mol L⁻¹ HNO₃ and retained on the hydrophobic poly-chlorotrifluoroethylene (PCTFE) sorbent material. The adsorbed complex was eluted with isobutyl methylketone (IBMK) and injected directly into the nebulizer via a flow compensation unit. All major chemical and flow parameters affecting the complex formation adsorption and elution as well as interference were studied and optimized. By processing 2.4 mL of sample, the enhancement factor was 39 and the sampling frequency was 50 h⁻¹. For 30 s preconcentration time the detection limit was 0.3 μg L⁻¹ and the relative standard deviation at 5.0 μg L⁻¹ Cd concentration level was 2.9%. The calibration curve was linear in the range 0.8–40.0 μg L⁻¹. The accuracy of the method was estimated by analyzing a certified reference material NIST-CRM 1643d (Trace elements in water). Good recoveries were obtained for spiked natural-water and waste-water samples. Correspondence: Aristidis N. Anthemidis, Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University, GR-Thessaloniki 54124, Greece     

Scale up and Application of Biosurfactant from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in Enhanced Oil Recovery

There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y _p/x), biosurfactant on sucrose (Y _p/s), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g⁻¹, 0.18 g g⁻¹, and 0.03 g l⁻¹ h⁻¹, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y _x/s, Y _p/x, Y _p/s, and Y of 0.42 g g⁻¹, 0.595 g g⁻¹, 0.25 g g⁻¹, and 0.057 g l⁻¹ h⁻¹, respectively. The biosurfactant maximum production, 2.5 g l⁻¹, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K _L a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s⁻¹, respectively. Comparison of K _L a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K _L a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.     

A hydrogen peroxide biosensor based on direct electrochemistry of hemoglobin in palladium nanoparticles/graphene-chitosan nanocomposite film

Thermally two-dimensional lattice graphene (GR) and biocompatibility chitosan (CS) act as a suitable support for the deposition of palladium nanoparticles (PdNPs). A novel hydrogen peroxide (H₂O₂) biosensor based on immobilization of hemoglobin (Hb) in thin film of CS containing GR and PdNPs was developed. The surface morphologies of a set of representative membranes were characterized by means of scanning electron microscopy and showed that the PdNPs are of a sphere shape and an average diameter of 50 nm. Under the optimal conditions, the immobilized Hb showed fast and excellent electrocatalytic activity to H₂O₂ with a small Michaelis–Menten constant of 16 μmol L⁻¹, a linear range from 2.0 × 10⁻⁶ to 1.1 × 10⁻³ mol L⁻¹, and a detection limit of 6.6 × 10⁻⁷ mol L⁻¹. The biosensor also exhibited other advantages, good reproducibility, and long-term stability, and PdNPs/GR–CS nanocomposites film would be a promising material in the preparation of third generation biosensor.     

Acetylcholinesterase-based biosensors for quantification of carbofuran, carbaryl, methylparaoxon, and dichlorvos in 5% acetonitrile

Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes—AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 × 10⁻¹¹ and 2.7 × 10⁻⁹ mol L⁻¹, respectively), the B1 enzyme for carbofuran (4.5 × 10⁻⁹ mol L⁻¹), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 × 10⁻⁷ mol L⁻¹). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.     

The Fed-Batch Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase (DERA) in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Exponential Feeding Strategy Control

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (μ _set) was evaluated at 0.20, 0.15, and 0.10 h⁻¹, respectively. The DERA concentration and volumetric productivity were associated with μ _set. The cells synthesized the enzyme most efficiently at μ _set = 0.15 h⁻¹. The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L⁻¹ h⁻¹) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.     

Flow injection analysis of volatile phenols in environmental water samples using CdTe/ZnSe nanocrystals as a fluorescent probe

On the basis of flow injection analysis technology, a simple, accurate, and sensitive method has been developed for the determination of volatile phenols in environmental water samples by using CdTe/ZnSe nanocrystals as a fluorescent probe. The influences of coexisting metal ions and volatile phenol substitutes were also investigated. The method developed for analysis of volatile phenols displayed very good linearity in the range from 1.0 × 10⁻⁸ to 4.0 × 10⁻⁷ g L⁻¹, with a correlation coefficient greater than 0.995 and a detection limit down to 2.7 × 10⁻⁹ g L⁻¹ (signal-to-noise ratio 3). The proposed method was successfully applied to determine the content of volatile phenols in environmental water samples, and the quantitative recoveries were 93.4–106.1%. A possible reaction mechanism for the quenching of fluorescence is discussed using UV–vis absorption spectra, fluorescence spectra, and time-resolved luminescence spectra of volatile phenols obtained by titrating a CdTe/ZnSe nanocrystal aqueous solution and zeta potential data.     

Development of electrochemical DNA biosensor for Trichoderma harzianum based on ionic liquid/ZnO nanoparticles/chitosan/gold electrode

Electrochemical DNA biosensor was successfully developed by depositing the ionic liquid (e.g., 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM][Otf])), ZnO nanoparticles, and chitosan (CHIT) nanocomposite membrane on a modified gold electrode (AuE). The electrochemical properties of the [EMIM][Otf]/ZnO/CHIT/AuE for detection of DNA hybridization were studied. Under optimal conditions using cyclic voltammetry, the target DNA sequences could be detected in the concentration range of 1.0 × 10⁻¹⁸ to 1.82 × 10⁻⁴ mol L⁻¹, and with the detection limit of 1.0 × 10⁻¹⁹ mol L⁻¹. This DNA biosensor detection approaches provide a quick, sensitive, and convenient method to be used in the identification of Trichoderma harzianum.     

Development of a sequential injection–square wave voltammetry method for determination of paraquat in water samples employing the hanging mercury drop electrode

This work describes the development and optimization of a sequential injection method to automate the determination of paraquat by square-wave voltammetry employing a hanging mercury drop electrode. Automation by sequential injection enhanced the sampling throughput, improving the sensitivity and precision of the measurements as a consequence of the highly reproducible and efficient conditions of mass transport of the analyte toward the electrode surface. For instance, 212 analyses can be made per hour if the sample/standard solution is prepared off-line and the sequential injection system is used just to inject the solution towards the flow cell. In-line sample conditioning reduces the sampling frequency to 44 h⁻¹. Experiments were performed in 0.10 M NaCl, which was the carrier solution, using a frequency of 200 Hz, a pulse height of 25 mV, a potential step of 2 mV, and a flow rate of 100 μL s⁻¹. For a concentration range between 0.010 and 0.25 mg L⁻¹, the current (i _p, μA) read at the potential corresponding to the peak maximum fitted the following linear equation with the paraquat concentration (mg L⁻¹): i _p = (−20.5 ± 0.3)C _paraquat − (0.02 ± 0.03). The limits of detection and quantification were 2.0 and 7.0 μg L⁻¹, respectively. The accuracy of the method was evaluated by recovery studies using spiked water samples that were also analyzed by molecular absorption spectrophotometry after reduction of paraquat with sodium dithionite in an alkaline medium. No evidence of statistically significant differences between the two methods was observed at the 95% confidence level.     

Ethanol Production from Cashew Apple Bagasse: Improvement of Enzymatic Hydrolysis by Microwave-Assisted Alkali Pretreatment

In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L⁻¹ of NaOH (372 ± 12 and 355 ± 37 mg g_glucan⁻¹) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15–30 min) and microwave power (600–900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g_CAB-M⁻¹) increased glucose concentration to 15 g L⁻¹. The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L⁻¹ and 1.41 g L⁻¹ h⁻¹, respectively.     

Production of Lipids Containing High Levels of Docosahexaenoic Acid by a Newly Isolated Microalga, <Emphasis Type="Italic">Aurantiochytrium</Emphasis> sp. KRS101

In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.     

Retinol fluorescence in lecithin/<Emphasis Type="Italic">n</Emphasis>-butanol/water aggregates: a new improvement for its analysis in cosmetics without pretreatment

The possibilities of different media formed by lecithin/n-butanol (n-BuOH)/water ternary mixtures for the analysis of all-trans-retinol by fluorescence have been studied. Fluorescence intensity of retinol increases in the presence of different types of aggregates formed in these media. Analytical features are good, the detection limit and quantification limit have micrograms per liter levels, and the linear range and sensitivity are appropriate to determine retinol in cosmetic samples. The analysis of retinol in anti-wrinkle creams can be achieved directly without any pretreatment of the sample. The vesicles built up from a biocompatible surfactant (lecithin) in aqueous solution with a low amount of n-BuOH permit an appropriated media for a simple, rapid, and sensitive analytical method. This method has a linear range between 64.1 and 800 μg L⁻¹, a sensitivity of 202.3 L mg⁻¹, and a low detection and quantification limit at 19.2 and 64.1 μg L⁻¹, respectively.