Entrapment of horseradish peroxidase in sugar-modified silica monoliths: toward the development of a biocatalytic sensor

A miniaturized HRP-entrapped bioreactor was prepared by a one-step enzyme immobilization method using a biocompatible sol-gel processing method employing either diglycerylsilane (DGS) or sodium silicate (SS) as precursors and a covalently tethered sugar, N-(3-triethoxysilylpropyl)gluconamide (GLS) as a silica modifier. Factors such as leaching, catalytic efficiency and long-term stability were examined to assess the role of the precursor and modifier in influencing enzyme performance. The results showed that sodium silicate derived materials modified with covalently bound sugars at a level of 10 mol% were optically transparent and provided the highest catalytic turnover rate for entrapped HRP. The stability and reusability of the entrapped HRP was found to be satisfactory for at least 1 month in the GLS-doped SS materials, and the entrapped HRP was able to respond linearly to the presence of peroxide over the concentration range of 0-750 microM with a detection limit of 6 microM, demonstrating the potential of this material for the development of a reusable optical biosensor.     

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol-gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase-peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase-peroxidase system was immobilized in a single sol-gel matrix. The enzymatic activity of the encapsulated superoxide dismutase-peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol-gel encapsulation preserved the hierarchy of the enzyme's activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.     

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.     

Encapsulation in a sol–gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue

Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t _1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K _m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V _max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.     

Silica sol-gel composite film as an encapsulation matrix for the construction of an amperometric tyrosinase-based biosensor

An amperometric tyrosinase enzyme electrode for the determination of phenols was developed by a simple and effective immobilization method using sol-gel techniques. A grafting copolymer was introduced into sol-gel solution and the composition of the resultant organic-inorganic composite material was optimized, the tyrosinase retained its activity in the sol-gel thin film and its response to several phenol compounds was determined at 0 mV vs. Ag/AgCl (sat. KCl). The dependences of the current response on pH, oxygen level and temperature were studied, and the stability of the biosensor was also evaluated. The sensitivity of the biosensor for catechol, phenol and p-cresol was 59.6, 23.1 and 39.4 microA/mM, respectively. The enzyme electrode maintained 73% of its original activity after intermittent use for three weeks when storing in a dry state at 4 degrees C.     

Enzyme immobilization on electrospun polymer nanofibers: An overview

Enzyme immobilization has attracted continuous attention in the fields of fine chemistry, biomedicine, and biosensor. The performance of immobilized enzyme largely depends on the structure of supports. Nanostructured supports are believed to be able to retain the catalytic activity as well as ensure the immobilization efficiency of enzyme to a high extent. Electrospinning provides a simple and versatile method to fabricate nanofibrous supports. Compared with other nanostructured supports (e.g. mesoporous silica, nanoparticles), nanofibrous supports show many advantages for their high porosity and interconnectivity. This review mainly discusses the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation. Surface attachment refers to physical adsorption or covalent attachment of enzymes on pristine or modified nanofibrous supports, and encapsulation means electrospinning a mixture of enzyme and polymer. We make a detailed comparison between these two immobilization approaches and highlight their distinct characteristics. The prospective applications of enzyme immobilized electrospun nanofibers in the development of biosensors, biofuel cells and biocatalysts are also discussed.     

Encapsulation of <Emphasis Type="Italic">Pannonibacter phragmitetus</Emphasis> LSSE-09 in alginate–carboxymethyl cellulose capsules for reduction of hexavalent chromium under alkaline conditions

Cr(VI) was efficiently reduced to Cr(III) by Pannonibacter phragmitetus LSSE-09 encapsulated in liquid-core alginate–carboxymethyl cellulose capsules under alkaline conditions. Taking into account the physical properties of the capsules, the activity of encapsulated cells, and total Cr(III) concentration in the supernatant, optimal conditions (0.5% w/v sodium alginate; 2% w/v sodium carboxymethyl cellulose; 0.1 M CaCl₂; 30-min gelation time) for LSSE-09 encapsulation were determined. At optimal conditions, a relatively high reduction rate of 4.20 mg g_{(dry weight)}⁻¹  min⁻¹ was obtained. Total Cr(III) concentration in the supernatant was significantly decreased after reduction, because 63.7% of the formed soluble organo-Cr(III) compounds compared with those of free cells were captured by the relatively smaller porous structure of alginate capsules. The optimal pH value (9.0) for Cr(VI) reduction was not changed after encapsulation. In addition, encapsulated LSSE-09 showed no appreciable loss in activity after eight repeated cycles at 37°C, and 85.7% of its initial activity remained after 35-day storage at 4°C. The results suggest that encapsulated LSSE-09 in alginate–carboxymethyl cellulose capsules has potential biotechnological applications for the detoxification of Cr(VI)-contaminated wastewater.     

A reagent less fluorescent sol-gel biosensor for uric acid detection in biological fluids

The simultaneous encapsulation of a coupled uricase-peroxidase system and amplex red in a sol-gel matrix allows one to obtain a reagent-less and ready-to-use fluorescent biosensor for the accurate detection of uric acid in highly diluted biological fluids. The detection limit of the prepared biosensor was found to be 20 nM and was linear up to 1 microM. The high sensitivity found for the biosensor permitted a reliable determination of uric acid concentrations in the presence of interfering species (e.g., ascorbic acid) just by sample dilution (up to 50000 for urine and 10000 for serum and blood). The sol-gel encapsulation preserved the hierarchy of the enzyme activity as demonstrated by the performance of the fluorescent biosensor.     

Use of immobilized cells of the strainCorynebacterium sp. for 4-nitrophenol degradation

4-Nitrophenol degrading bacterial strainCorynebacterium sp. 8/3 was isolated from chemically polluted soil. The product of cometabolic transformation of 4-nitrophenol was identified as 4-nitrocatechol., Effect of immobilization (encapsulation in calcium alginate) ofCorynebacterium sp. cells on the process of 4-nitrophenol transformation was investigated. 4-Nitrophenol was converted by encapsulated cells and encapsulation had a protective effect, on 4-nitrophenol degrading bacteria in repeated cycles of incubation. Transformation of 4-nitrophenol to 4-nitrocatechol by encapsulated cells was influenced by pH of medium but was not influenced by concentration of alginate and CaCl₂. The count of viable cells in alginate beads declined approximately by one order of magnitude after 10 d of incubation. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Physicochemical characterization of stealth liposomes encapsulating an organophosphate hydrolyzing enzyme

The present studies were focused on the preparation and characterization of stericaly stabilized liposomes (SLs) encapsulating a recombinant organophosphorus hydrolyzing phosphotriesterase (OPH) enzyme for the antagonism of organophosphorus intoxication. Earlier results indicate that the liposomal carrier system provides an enhanced protective effect against the organophosphorus molecule paraoxon, presenting a more effective therapy with less toxicity than the most commonly used antidotes. Physicochemical characterization of the liposomal OPH delivery system is essential in order to get information on its in vitro stability and in vivo fate. Osmolarity, pH, viscosity, and encapsulation efficiency of the SL preparation and the surface potential of the vesicles were determined. The membrane rigidity and the impact of OPH enzyme on it was studied by electron-paramagnetic resonance spectroscopy, using spin probes. The in vitro stability of the liposomal preparations, the vesicle size distribution, and its alteration during a 3-week storage were followed by dynamic light-scattering measurements. Further, the stability of encapsulated and nonencapsulated OPH was compared in puffer and plasma.     

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions

An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased. Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concentrations was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.     

Classy non-wovens based on animate L. gasseri-inanimate poly(vinyl alcohol): upstream application in food engineering

We explored electrospinning as a feasible and practicable mode for encapsulation and stabilization of Lactobacillus gasseri. The utilized nanocomposite was prepared using sol-gel composed of animate L. gasseri and inanimate PVA. The objective was to examine the ability of electrospinning method to protect functional properties of probiotic L. gasseri. The PVA was used as an encapsulation matrix as it is biocompatible and hydrophilic in nature thus facilitate an easy revival of bacteria. The characterization of as-spun bioproduct was done by energy-dispersive X-ray spectrometer, SEM, and TEM, whereas thermal behavior was analyzed by thermogravimetry. The viability was confirmed by traditional pour plate method and fluorescence microscopy. Furthermore, to test whether the functionality of L. gasseri was affected, the encapsulated L. gasseri were fed to mouse for colonization. Our results pointed out that encapsulated bacteria were viable for months, and their metabolism was not affected by immobilization; thus, they could be used in food engineering and trade.     

Immobilization in polyelectrolyte complex capsules: Encapsulation of a gluconate-oxidizing Serratia marcescens strain

In previous papers, it was shown that eukaryotic microbial systems can be encapsulated in polyelectrolyte complexes (PEC) prepared from sodium cellulose sulfate and poly(dimethyldiallylammonium chloride) with maintainance of vitality. In the present study, prokaryotic cells were successfully encapsulated in these PEC. Serratia marcescens B345 (IMET 11312) was chosen as a model organism. This strain converts gluconic acid to 2-ketogluconic acid. Since the 2-ketogluconic acid produced has very strong complexing properties, the number of applicable immobilization methods is restricted. Due to the high stability of PEC towards complexing agents, these problems can be overcome by the described method.

As already described in previous papers, a preimmobilization of cells in a PEC coprecipitate prior to capsule formation proved to be advantageous also for encapsulation of bacilli. The mean productivity of the encapsulated S. marcescens cells was 1–4.4 g l⁻¹ h⁻¹ in comparison to 5 g l⁻¹ h⁻¹ for free cells. The productivity was highly dependent on the flow rate of the reactor. The encapsulated cells were used for 1,200 h in a continuous biotransformation process for the production of 2-ketogluconic acid.     

Encapsulation of rat hepatocyte spheroids for the development of artificial liver

Hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, there was a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid might be applied to the development of a bioartificial liver.     

Array biosensor based on enzyme kinetics monitoring by fluorescence spectroscopy: application for neurotoxins detection

The aim of the present work is to develop an evanescence wave array biosensor exploiting the “kinetic” approach of enzymatic reaction and further detection of the reaction products via pH sensitive fluorophore reporter. To demonstrate the feasibility of this approach, we have developed a biosensor array with the potential for direct detection of organophosphates using as a biorecognition element, an enzyme organophosphorus hydrolase (OPH), conjugated with a pH-sensitive fluorophore, carboxynaphthofluorescein (CNF). The presence of reference spots allows the discrimination of the enzymatic and non-enzymatic based pH changes; bovine serum albumin (BSA) was used as a non-enzymatic scaffold protein for CNF attachment at the reference spots. An array biosensor unit developed at the Naval Research Laboratories (NRL) was adopted as the detection platform and appropriately modified for enzyme-based measurements. A planar multi-mode waveguide was covered with an optically transparent TiO₂ layer to increase the surface area available for immobilization.

The biosensor enabled the detection of 2.5 μM paraoxon, and 10 μM DFP and parathion, respectively. Very short response time of 30 s can be achieved with a total analysis time of less than 2 min. When operated at room temperature and stored at 4 °C, the waveguide retained reasonable activity for greater than 45 days.