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1.
Microfluidic devices are increasingly used to perform biological experiments on a single-cell basis. However, long-term stability of cell positions is still an issue. A novel biocompatible method for cell entrapment and release on a microchip is presented. It is based on the controlled formation of an alginate hydrogel by bringing two laminar flows of alginate and calcium ions in the range of 2 mM to 40 mM into contact. The resulting growth of a gel bar is used to enclose and immobilize yeast cells. Adding ethylenediaminetetraacetic acid (EDTA) to the alginate solution allows for control of the hydrogel growth, and by varying the ratio of Ca(2+) to EDTA concentrations gel growth or gel shrinkage can be induced at will. Trapped cells are released during shrinkage of the gel. The trapping efficiency for different cell speeds is investigated and the properties of gel growth are discussed using a diffusion model. Precise positioning of a single cell is demonstrated. The technique presented allows not only the reversible immobilization of cells under gentle conditions but also offers the potential of long-term cell cultures as shown by on-chip incubation of yeast cells. The procedure may provide a simple and fully biocompatible technique for a multitude of innovative experiments on cells in microsystems. 相似文献
2.
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. 相似文献
3.
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. 相似文献
4.
5.
Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases
V. Ivanova M. Stefanova A. Tonkova E. Dobreva D. Spassova 《Applied biochemistry and biotechnology》1995,50(3):305-316
Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques
were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide,
and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and
biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts
with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated
batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase
yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells. 相似文献
6.
M. Rietti-Shati D. Ronen R. T. Mandelbaum 《Journal of Sol-Gel Science and Technology》1996,7(1-2):77-79
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. 相似文献
7.
Eleonora Winkelhausen Elena Velickova Samuel A. Amartey Slobodanka Kuzmanova 《Applied biochemistry and biotechnology》2010,162(8):2214-2220
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. 相似文献
8.
Joachim Klein 《Macromolecular Symposia》1988,19(1):93-105
The immobilization of whole microbial cells has become an important tool in the development of biocatalytic processes in the pharmaceutical and food industry. Not only dead, i.e. non growing cells, but recently with higher priority living and growing cells are the biological species, for which simple and efficient polymeric carriers had to be found. In comparison to other methods, like adsorption or encapsulation, entrapment into a polymer network is the most widely used technique. The network can be formed on the basis of a)ionic interactions (ionotropic gelation of polyelectrolytes), b) of polycondensation reactions (epoxides, polyurethanes, silicones) or c) of polymerization reactions (crosslinking polymerization of vinylic monomers, oligomers or polymers). The characteristic features and the efficiency-controlling parameters of some immobilized cells systems are discussed as illustrative examples. 相似文献
9.
溶胶凝胶技术在化学及生物传感器领域中的应用 总被引:4,自引:0,他引:4
本文介绍了一种新的用于化学或生物传感器固定敏感试剂的溶胶凝胶技术,用此技术制成的传感器基质具有优异的光学特性和热力学,机械稳定性,且它形成的化学条件温和,尤其适用于包埋生物大分子。 相似文献
10.
Research into paper-based sensors or functional materials that can perform analytical functions with active recognition capabilities
is rapidly expanding, and significant research effort has been made into the design and fabrication of bioactive paper at
the biosensor level to detect potential health hazards. A key step in the fabrication of bioactive paper is the design of
the experimental and operational procedures for the immobilization of biomolecules such as antibodies, enzymes, phages, cells,
proteins, synthetic polymers and DNA aptamers on a suitably prepared paper membrane. The immobilization methods are concisely
categorized into physical absorption, bioactive ink entrapment, bioaffinity attachment and covalent chemical bonding immobilization.
Each method has individual immobilization characteristics. Although every biomolecule–paper combination has to be optimized
before use, the bioactive ink entrapment method is the most commonly used approach owing to its general applicability and
biocompatibility. Currently, there are four common applications of bioactive paper: (1) paper-based bioassay or paper-based
analytical devices for sample conditioning; (2) counterfeiting and countertempering in the packaging and construction industries;
(3) pathogen detection for food and water quality monitoring; and (4) deactivation of pathogenic bacteria using antimicrobial
paper. This article reviews and compares the different biomolecule immobilization techniques and discusses current trends.
Current, emerging and future applications of bioactive paper are also discussed. 相似文献
11.
Santa GL Bernardino SM Magalhães S Mendes V Marques MP Fonseca LP Fernandes P 《Applied biochemistry and biotechnology》2011,165(1):1-12
The present work aims to provide the basic characterization of sol–gel immobilized inulinase, a biocatalyst configuration
yet unexploited, using as model system the hydrolysis of inulin to fructose. Porous xerogel particles with dimensions in slight
excess of 10 μm were obtained, yielding an immobilization efficiency of roughly 80%. The temperature– and pH–activity profiles
displayed a broader bell-shaped pattern as a result of immobilization. In the latter case, a shift of the optimal pH of 0.5
pH units was observed towards a less acidic environment. The kinetic parameters estimated from the typical Michaelis–Menten
kinetics suggest that immobilization in sol–gel did not tamper with the native enzyme conformation, but on the other hand,
entrapment brought along mass transfer limitations. The sol–gel biocatalyst displayed a promising operational stability, since
it was used in more than 20 consecutive 24-hour batch runs without noticeable decay in product yield. The performance of sol–gel
biocatalyst particles doped with magnetite roughly matched the performance of simple sol–gel particles in a single batch run.
However, the operational stability of the former proved poorer, since activity decay was evident after four consecutive 24-hour
batch runs. 相似文献
12.
Navid Amini Saeedeh Mazinani Seyed-Omid Ranaei-Siadat Mohammad Reza Kalaee Saeed Hormozi Kaveh Niknam Nasrin Firouzian 《Applied biochemistry and biotechnology》2013,170(1):91-104
In this work, polyacrylamide/multi-walled carbon nanotubes (MWCNT) solution is electrospun to nanocomposite nanofibrous membranes for acetylcholinesterase enzyme immobilization. A new method for enzyme immobilization is proposed, and the results of analysis show successful covalent bonding of enzymes on electrospun membrane surface besides their non-covalent entrapment. Fourier transform infrared spectroscopy, mechanical and thermal investigations of nanofibrous membrane approve successful cross-linking and enzyme immobilization. The enzyme relative activity and kinetic on both pure and nanocomposite membranes is investigated, and the results show proper performance of designed membrane to even improve the enzyme activity followed by immobilization compared to free enzyme. Scanning electron microscopy images show nanofibrous web of 3D structure with a low shrinkage and hydrogel structure followed by enzyme immobilization and cross-linking. Moreover, the important role of functionalized carbon nanotubes on final nanofibrous membrane functionality as a media for enzyme immobilization is investigated. The results show that MWCNT could act effectively for enzyme immobilization improvement via both physical (enhanced fibers’ morphology and conductivity) and chemical (enzyme entrapment) methods. Figure
Mechanism for APTS surface modification of nanofibrous nanoweb for enzyme immobilization 相似文献
13.
《Electroanalysis》2004,16(18):1542-1549
The immobilization of enzymes on an electrode surface is of great importance in bioelectrochemistry. The entrapment of enzymes into a polymer matrix is simple and a speedy technique for the production of biosensors. This procedure of enzyme immobilization by electropolymerization has a great significance in fabrication of micro sensors in the preparation of multiplayer devices. In current study, glucose oxidase enzyme that is specific for the glucose determination was entrapped into polypyrrole matrix containing p‐benzoquinone in PIPES buffer and glucose sensitivity of the biosensor was investigated. Then, artificial neural network analysis was done for the nonlinear calibration plot. This implementation can be used for the sensor failure detection, as well. The estimation power of the neural network used in the direct and inverse calibration modelling was examined by statistical methods. It presented the good performance for the estimation power. 相似文献
14.
A. P. V. Gonçalves J. M. S. Cabral M. R. Aires-Barros 《Applied biochemistry and biotechnology》1996,60(3):217-228
Fusarium solani pisi recombinant cutinase, immobilized by entrapment in calcium alginate and by covalent binding on porous silica, was used to
catalyze the hydrolysis of tricaprylin. The influence of relevant parameters on the catalytic activity such as pH, temperature,
and the substrate concentration were studied. Cutinase immobilized by entrapment presented a Michaelis-Menten kinetics for
tricaprylin concentrations up to 200 mM. At higher concentrations of substrate, inhibition was observed. For covalent binding immobilization, diffusional limitations
were observed at low substrate concentrations and substrate inhibition occurred for concentrations higher than 150 mM. The stability of immobilized cutinase was also evaluated. The enzyme immobilized by entrapment showed a high stability,
in contrast to the immobilization on porous silica. 相似文献
15.
Whole cells ofBrevibacterium flavum having high fumarase activity were immobilized using K-carrageenan. The reason for the high stability of fumarase activity
of immobilized cells was investigated.
One of main reasons for stabilizing fumarase activity by immobilization using K-carrageenan against organic solvents such
as ethanol and acetone was the lower concentration of these solvents in the carrageenan gel compared with that in outer bulk
solution. The stabilization of fumarase activity in the immobilized cells against protein-denaturing reagents was found to
be related to rheological properties of K-carrageenan gel. Another reason for stabilizing fumarase activity by immobilization
with K-carrageenan was to protect the cells from lysis.
When immobilized cells were freeze-thawed, their fumarase activity increased and operation stability decreased. Therefore,
one reason for the high decay of fumarase activity caused by the freeze-thawing may be a change in the pore size of the K-carrageenan
gel.
Fumarase activity and the operational stability of immobilized cells was found to depend on gelling conditions. Therefore,
the steric structure of the K-carrageenan gel may be related to the decay of fumarase activity. 相似文献
16.
Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and
crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme
and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon
immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole
cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term
storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells. 相似文献
17.
M. Portaccio B. Della Ventura D. G. Mita N. Manolova O. Stoilova I. Rashkov M. Lepore 《Journal of Sol-Gel Science and Technology》2011,57(2):204-211
Micro-Attenuated Total Reflection (ATR) Fourier Transform Infrared spectroscopy was used to investigate sol–gel layers for
biosensing applications prior and after glucose oxidase (GOD) immobilization. The changes occurring in sol–gel infrared spectrum
after GOD immobilization were clearly evidenced confirming the retaining of the enzyme activity. Moreover, micro-ATR experimental
technique allowed us to investigate the spatial distribution of enzyme concentration. The non-destructive nature of our approach
also enabled to monitor the time stability of sol–gel layers and of embedded GOD. The temporal evolution of some peaks in
infrared spectra of these sol–gel layers was compared with absorption and steady-state fluorescence measurements. The results
reported here confirm that micro-ATR infrared spectroscopy can be usefully employed for a non- or minimally invasive detailed
characterization of supports for enzyme immobilization. 相似文献
18.
Brányik Tomáš Kuncová Gabriela Páca Jan Demnerová Kateřina 《Journal of Sol-Gel Science and Technology》1998,13(1-3):283-287
This work deals with changes in microbial phenol degradation and cell proliferation caused by immobilization into silica gel. Mixed microbial culture and the yeast Candida tropicalis were immobilized in silica layers and pieces prepared by mixing of prepolymerized tetraethoxysilane with cell suspension. The phenol degradation rate of cells entrapped in silica gel was compared with those immobilized into an organic polymer-polyurethane. The phenol degradation efficiency decreased in the following order: free cell suspension > cells entrapped into polyurethane foam > cells entrapped into prepolymerized TEOS. Inside the silica there was no growth observed by optical microscope. The immobilization of bacterium Pseudomonas species 2 into silica gel, cells which co-metabolize PCBs with biphenyl, did not result in substantial change of intermediate concentration. 相似文献
19.
For continuous production of cephalexin, whole cells ofXanthomonas citri were immobilized by entrapment in polyacrylamide gel and kappa-carrageenan gel. It wasfound that cells immobilized with kappa-carrageenan
showed better thermal stability compared to those immobilized by polyacrylamide gel. The cells immobilized with kappa-carrageenan
were treated with glutaraldehyde and hexamethylenediamine to prevent gel destruction during prolonged operation. By immobilizing
intact cells, the optimal temperature for the synthetic enzyme reaction shifted higher by 8°C and the optimal pH became broader
around 6.2 In continuous operation, the immobilized cells retained better operational stability at 25 than at 37°C, and also
showed maximal conversion up to 83% at 25°C. 相似文献
20.
A novel and versatile processing method was developed for the formation of gel scaffolds with in situ AChE-AuNPs immobilization for biosensing of organophosphorus compounds. The biosensor designed by our new approach shows high sensitivity, selectivity and reactivation efficiency. This flow-induced immobilization technique opens up new pathways for designing a simple, fast, biocompatible, and cost-effective process for enhanced sensor performance and on-site monitoring of a variety of toxic organophosphorus compounds. 相似文献