On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel

A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here.     

A convenient method to immobilize 4-dimethylaminopyridine on silica gel as a heterogeneous nucleophilic catalyst for acylation

A catalyst comprised of 4-dimethylaminopyridine (DMAP) functionalized silica gel (SG-DMAP) was prepared via a convenient N-alkylation of 4-methylaminopyridine (MAP) with γ-chloropropylated silica gel promoted by potassium iodide. FT-IR, TG, SEM results illustrated that the DMAP was successfully immobilized on silica gel and the BET result suggested the pore structure was not altered after immobilization process. The highest immobilization amount of 0.75 mmol/g was obtained at 130 °C for 12 h. Activity test demonstrated that silica gel immobilized DMAP could be serve as a heterogeneous catalyst for the acylation with significant activity and stability.     

Determination of phenol and catechol concentrations with oxygen probes coated with immobilized enzymes or immobilized cells

The enzyme phenol 2-hydroxylase was immobilized on Sepharose and used in conjunction with an O₂ electrode for quantitating phenol. Similarly, catechol 1,2-oxygenase was used for quantitating catechol. A third probe was prepared by immobilization ofTrichosporon cutaneum cells rather than purified phenol 2-hydroxylase for phenol quantitation. The whole cell system gave results comparable to the immobilized enzyme system.     

Atrazine degradation by Pseudomonas strain ADP entrapped in sol-gel glass

Sol-gel entrapment was evaluated as a method for immobilization of an atrazine degrading Pseudomonas. It was found that the bacterium lost much of its atrazine degrading activity upon immobilization. However, partial activity could have been restored by amendment of nutrients. Bacteria immobilized using a prehydrolysis technique for the preparation of the sol-gel, retained better activity in comparison to bacteria immobilized using a composite calcium alginate/sol-gel procedure. Further study is underway to improve the activity of sol-gel entrapped bacteria.     

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K_m of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.     

Screening of matrix suitable for immobilization of microbial cells

To find a suitable matrix for immobilization of microbial cells, synthetic and natural polymers were screened. As a result,kappa-carrageenan,iota-carrageenan, furcellaran, sodium alginate, ethyl succinylated cellulose, succinylated zein, and 2-methyl-5-vinyl-pyridine-methylacrylate-methacrylic acid copolymer were studied. These polymers were induced to gel under mild conditions.Streptomyces phaeochromogenes cells having glucose isomerase activity were successfully immobilized in these polymer matrices. If a gelinducing reagent were added to a substrate solution, these gel matrices could be stabilized. The microbial cells did not leak out from the gel lattice. When these immobilized cells were treated with hardening reagents such as glutaraldehyde or tannins, the gel matrices were strengthened, and the glucose isomerase activity became stable for a long period even in the absence of gel-inducing reagents. Among these polymer matrices tested,kappa -carrageenan was most suitable for immobilization of microbial cells.     

Crosslinked polyacrylamide-hydrazide (paah) as matrix for the gel entrapment of cells and enzymes - an overview

The development and use of a gel entrapment technique designed for the immobilization of viable, sensitive cells is described. The method is based on the controlled chemical cross-linking of prepolymerized, linear polyacrylamide partially substituted with acylhydrazide groups, by glyoxal. The methodology, applications and advantages of this technique for the immobilization of cells and enzymes by gel entrapment are reviewed.     

Entrap-immobilization of biocatalysts on cellulose acetate-inorganic composite gel fiber using a gel formation ofcellulose acetate–metal (Ti, Zr) alkoxide

We developed a new entrap-immobilization method, using a gel formationof cellulose acetate and metal (Ti, Zr) alkoxide. Several biocatalysts(-galactosidase, -chymotrypsine, invertase, urease, lipase andSaccharomyces cereviciae) were successfullyentrap-immobilized on this composite gel fiber. The immobilization process wassimple and the resultant immobilized biocatalysts on the gel fiber were easy tohandle. It is considered that the biocatalysts are physically entrapped amongthe gel networks and distribute throughout the gel fiber. The gel fiber wasstable in phosphate buffer solution, electrolyte solution and organic solvent,because the gel formation was due to coordination interaction between celluloseand transition metal. Therefore, it can be applicable as a support for abiotransformation in various reaction media. We examined some enzyme reactionsand biotransformation using the immobilized biocatalysts on this gel fiber andevaluated this immobilization matrix in the reactions compared to the resultsobtained by the other immobilization method. The immobilized biocatalyst showedstable activity for repeated cycles and over a long period of time. Moreover,continuous reaction could be carried out in a column reactor packed with thisimmobilized biocatalyst.     

Entrapment of living microbial cells in covalent polymeric networks

The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc _o/K _M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.     

Analysis of phenolic compounds catalyzed by immobilized horseradish peroxidase in silica glass

Horseradish peroxidases are entrapped into tetramethyl orthosilicate derived silicates by a mild sol–gel process. Compared with free enzymes in solution, silica immobilized Horseradish peroxidases are more robust, stable in a long-term and ease of recycle. Therefore, a phenolic compounds analysis method is established on the immobilized enzyme catalyzed oxidation reaction to produce intensely colored products for spectrophotometric analysis. The absorbance of colored product and analyte concentrations is linearly related. The similar methods also employ on analyzing 2-chlorophenol, 3-chlorophenol and 2,4-dichlorophenol. Furthermore, this method permits to reuse immobilized HRP to examine phenolic compounds. The thin silica film is used as an immobilization carrier. It has shorter pathlength for diffusion of analytes, which will lead to faster response times. Decomposition of quinone-imine colored product also has an effect on accuracy and precision of measurement. Decomposition rate constant is 5.549 × 10⁻⁴ min⁻¹. The interference substances have no obvious effect on the analytic results, except for formaldehyde and Pb²⁺. The proposed method can examine a real sample in waste water.     

一种简化的重氮化法制备固定化酶的载体合成方法

重氮化法是固定化酶时常用的一种方法。用多孔玻璃等无机物作载体时,一般是先用g-氨丙基三乙氧基硅烷与多孔玻璃等载体反应[1,2],生成烷基胺玻璃,然后与对硝基苯酰氯反应,产物经过还原,生成带有芳胺的衍生物,最后进行重氮化。本文通过烷基胺与对氨基苯甲酸反应,直接生成芳胺的衍生物,比常用的方法缩短了一步。通过在新合成的载体上对木瓜蛋白酶进行固定化,研究了固定化条件对酶活力回收的影响,最适固定化条件如下:pH为7.0,时间为6h,酶量为240mg/g载体,并比较了固定化酶和溶液酶的有关性质,考察了固定化酶的操作稳定性。结果表明,用这种方法合成的载体固定化酶,其对热稳定性、操作稳定性及产率都比较理想。     

Effect of sol–gel hydrophobicity on the distribution and structure of different proteins in organically modified sol–gel thin films

Although the use of silica sol–gels for protein entrapment has been studied extensively our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol–gel immobilization of biomolecules. This work describes the application of Fourier transform infrared (FTIR) microscopy to determine the influence of modifying the sol–gel hydrophobicity, by varying the content of the organically modified precursor propyltrimethoxysilane (PTMS), on the distribution and structure of three model proteins (lysozyme [EC 3.2.1.17], lipase [EC 3.1.1.3] and bovine serum albumin (BSA)) in silica sol–gel thin films. FTIR analysis of the overall immobilized protein positional distribution showed a Gaussian type distribution. FTIR microscopic mapping however, revealed that the spatial distribution of proteins was heterogeneous in the sol–gel thin films. When this positional information provided by FTIR microscopy was taken into account, areas of high protein concentration (clusters) were found and were not found to be homogeneously distributed. The shape of these clusters was found to depend on the type of protein entrapped, and in some cases on the composition of the sol–gel. Positional analysis of the distribution of the organically modified precursor PTMS in relation to the protein distribution was also conducted. The localized concentration of PTMS was found to positively correlate with the protein concentration in the case of lipase and negatively correlate in the case of lysozyme and BSA. These results indicate that lysozyme and BSA concentration was higher in areas of low hydrophobicity, while lipase concentration was higher in areas of high hydrophobicity within the sol–gel. Additionally, as determined by peak shape analysis of the amide I peak a higher PTMS content appeared to conserve protein structure in high concentration clusters for lipase. In contrast, lysozyme and BSA, appeared to retain their structure in high concentration clusters better at lower PTMS contents. A hypothesis speculating on the nature of the hydrophobic/hydrophilic interactions between the proteins and the sol–gel domains as the reason for these differences is presented.     

Ethanol Production Using Immobilized <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in Lyophilized Cellulose Gel

A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12–216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.     

基于中心辐射树枝状介孔二氧化硅构筑CPO固定化酶反应器及应用

首先制备粒径均匀的具有开放的三维中心辐射树枝状结构的介孔二氧化硅(DSP)粒子, 再通过静电相互作用在孔道内负载氯过氧化物酶(CPO)构筑了CPO@DSP固定化酶反应器. 通过改变硅源正硅酸乙酯(TEOS)和模板剂十六烷基三甲基氯化铵(CTAC)的浓度调控孔径大小, 研究了孔径对固定化酶反应器催化活性的影响; 同时基于酶促反应动力学分析探讨了孔道内酶催化反应的限域效应, 并进一步在CPO@DSP表面包覆海藻酸钠(SA)水凝胶薄膜以抑制酶反应器在使用过程中酶分子的泄露, 所得SA-CPO@DSP固定化酶反应器的重复使用性显著提高, 循环使用10次后, 仍能保持90%以上的催化活性. 将SA-CPO@DSP酶反应器用于环境水体中残留抗生素左氧氟沙星的降解, 对100 μg/mL的底物在25 min内降解率可达88%以上; 将该反应器用于苯酚的视觉比色检测, 裸眼可检测到5 μmol/L的苯酚, 表明SA-CPO@DSP酶反应器在环境保护方面具有良好的应用前景.     

Influence of Different Redox Conditions on the Biodegradation of the Pesticide Metabolites Phenol and Chlorophenols

Abstract

The fate and behaviour of phenol and monochlorophenols during bankfiltration and underground passage with variable redox conditions were investigated. A model ecosystem was used consisting in laboratory filter columns filled with natural underground material and operated with natural aerobic and anaerobic groundwater to create different redox situations.

The test substances (phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol) were added continuously to the infiltrating water and their concentration in the filter effluents determined. Beside the redox conditions other factors known to affect microbial degradation processes like the substrate concentration and the underground material were varied stepwise.

Phenol was degraded under both, aerobic and anaerobic conditions. The presence of oxygen is more favourable to degradation; no lag phase was observed under aerobic conditions. In a sulfate reducing environment, phenol could only be degraded after microbial adaptation. The length of the lag phase was strongly influenced by the substrate concentration and the undergroundmaterial. Prior contact with phenol resulted in a shorter lag phase.

Monochlorophenols behaved almost persistent in the model system. Degradation could only be observed in a test filter that provided a more active microbial population due to prior adaptation to phenol and a more favourable underground material.     

Stepwise controlled immobilization of colloidal crystals formed by polymer-grafted silica particles

Colloidal crystals formed by polymer-grafted silica particles were immobilized by a stepwise procedure consisting of gelation by radical copolymerization followed by solidification by ring-opening radical polymerization. In the first step, the poly(methyl methacrylate) (PMMA)-grafted silica colloidal crystal suspension was incorporated into the gel without altering the crystal structure by copolymerization of cross-linker, 1,2-dimethylacryloyloxyethane (DME) and methyl methacrylate (MMA). In the second step, ring-opening radical polymerization was performed after substituting the solvent with vinylidene-1,3-dioxolane. By this two-step procedure, the silica particle array of colloidal crystals was immobilized and made into durable material.     

Immobilization of Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate

A new Acidithiobacillus ferrooxidans cell immobilization technique utilizing the complex of PVA solution and sodium alginate solution crosslinked by Ca(NO₃)₂ as entrapment medium is reported. The mixture of A. ferrooxidans suspension and the entrapment complex were extruded into a solution of Ca(NO₃)₂ (1-5%) to form beads, then the beads were frozen at −20 °C for 1-2 days and thawed at room temperature. The forming mechanism, characteristic of this immobilized beads and the factors affecting activity of immobilized cells were also discussed. A maximum oxidation rate of 4.6 g Fe²⁺/(L h) was achieved in batch cultures by these immobilized cells. Precipitation formed during culture process was analyzed. The forming mechanism of this precipitation and how this precipitation affects the whole system were also discussed. In addition, the immobilization technique is operated simply, and the gel beads have high stability even under non-sterile conditions. So its application on an industrial scale would be more practicable.     

Encapsulation of Bromothymol Blue pH‐Indicator into a Sol‐Gel Matrix

Transparent monolithic silica doping with bromothymol blue has been prepared by the acid catalyzed sol‐gel reaction of tetraethylorthosilicate in the presence of bromothymol blue. The immobilized bromothymol blue shows behavior similar to its solution counterpart. It retains its structure during the sol‐gel reactions in terms of response to pH. Polarized light microscopy has indicated that the bromothymol blue molecules are strongly interacted within the host silica network. The immobilization of bromothymol blue into sol‐gel matrix could be used as a solid indicator.     

Amperometric 2,4‐Dichlorophenoxyacetate Biosensor System Based on a Microbial Reactor and a Tyrosinase‐Modified Electrode

Abstract

A novel 2,4‐dichlorophenoxyacetate (2,4‐D) biosensor system was constructed with a reactor for microbial degradation and a flow cell based on a tyrosinase‐modified graphite electrode for product detection. The microorganism, isolated from the agricultural soil collected at northern Kyusyu Island and identified as Ralstonia sp. was employed as the 2,4‐D degrader. Immobilization was performed with a glass column packed with silica gel particles by circulating Luria‐Bertani medium containing 2,4‐D inoculated with the bacteria. The degradation capability of the immobilized cells packed in the reactor was confirmed by circulating a mineral salt medium containing 2,4‐D and monitoring the decrease in 2,4‐D content. The tyrosinase electrode was employed to monitor phenolic and catecholic compounds, since it could be presumed that 2,4‐dichlorophenol and 3,5‐dichlorocatechol could be produced as intermediates in the degradation of 2,4‐D by Ralstonia sp. The flow cell of three electrodes configuration was assembled by using the enzyme electrode as a working electrode. Consequently, amperometric response current could be observed by injecting 2,4‐D solution with phosphate buffer as the mobile phase at the applied potential of 0.5 V vs. Ag/AgCl. The sensitivity of the system was shown to depend on the composition of the mobile phase by comparing the sensitivities obtained with phosphate buffer and mineral salt medium as the mobile phase.     

Biotransformation from geraniol to nerol by immobilized grapevine cells (V. vinifera)

The abilities of two grapevine cell suspensions (Vitis vinifera L. cv. Gamay Fréaux andVitis vinifera L. cv. Monastrell) to biotransform geraniol into nerol in a biphasic system based on the culture medium and Miglyol 812 were compared. The Gamay grape cell suspension was able to transform higher concentrations of geraniol into nerol than the Monastrell one. Gamay grape cells were immobilized in both calcium alginate beads and polyurethane foams. The cytotoxic effect of increasing concentrations of geraniol, as well as the ability of the immobilized cells to biotransform geraniol into nerol, was checked. Immobilization proved to be advantageous in protecting cells against the toxicity of the substrate. Furthermore, immobilization also seemed to have an effect on the secondary metabolism, the cells immobilized in polyurethane foams being more efficient at performing the isomerization process (40% conversion of geraniol into nerol) than both the freely suspended and calcium alginate immobilized cells (20% conversion).