Immobilization of E. coli bacteria in three-dimensional matrices for ISFET biosensor design

In recent years, cell-based biosensors (CBBs) have been very useful in biomedicine, food industry, environmental monitoring and pharmaceutical screening. They constitute an economical substitute for enzymatic biosensors, but cell immobilization remains a limitation in this technology. To investigate into the potential applications of cell-based biosensors, we describe an electrochemical system based on a microbial biosensor using an Escherichia coli K-12 derivative as a primary transducer to detect biologically active agents. pH variations were recorded by an ion-sensitive field effect transistor (ISFET) sensor on bacteria immobilized in agarose gels. The ISFET device was directly introduced in 100 ml of this mixture or in a miniaturized system using a dialysis membrane that contains 1 ml of the same mixture. The bacterial activity could be detected for several days. The extracellular acidification rate (ECAR) was analyzed with or without the addition of a culture medium or an antibiotic solution. At first, the microorganisms acidified their micro-environment and then they alkalinized it. These two phases were attributed to an apparent substrate preference of bacteria. Cell treatment with an inhibitor or an activator of their metabolism was then monitored and streptomycin effect was tested.     

Immobilization of the enzyme E. coli L-asparaginase on a water-soluble copolymer of vinylpyrrolidone and acrolein

A method for immobilizingE. coli L-asparaginase on a copolymer of vinylpyrrolidone and acrolein has been developed and optimized. The influence on the nature of the modification of the number of acrolein residues in the copolymer has been established. The enzymatic and some physicochemical properties of the immobilized forms of the enzyme obtained have been studied.A. Kirkhenshtein Institute of Microbiology, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 562–565, July–August, 1988.     

Immobilization of the enzyme E. coli L-asparaginase on a water-soluble copolymer of vinylpyrrolidone and acrolein

A method for immobilizingE. coli L-asparaginase on a copolymer of vinylpyrrolidone and acrolein has been developed and optimized. The influence on the nature of the modification of the number of acrolein residues in the copolymer has been established. The enzymatic and some physicochemical properties of the immobilized forms of the enzyme obtained have been studied. A. Kirkhenshtein Institute of Microbiology, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 562–565, July–August, 1988.     

Immobilization of the enzyme L-asparaginase from E. coli on polysaccharides I. Covalent binding with insoluble Sepharoses and CM-cellulose

Summary Immobilized L-asparaginase has been synthesized by various methods of covalent binding to insoluble Sepharoses and CM-cellulose.Some kinetic properties of the preparations obtained, the dependence of the activity on the pH of the medium, and their stability have been investigated.It has been established that the immobilized L-asparaginase possesses increased heat resistance, stability on storage, and stability to competing inhibitors.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 639–643, September–October, 1976.     

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.     

壳聚糖微球固定化L-天门冬酰胺酶研究

以反相悬浮交联法合成了壳聚糖微球,并将其应用于固定化L-天门冬酰胺酶。探讨了不同合成条件对成球情况的影响,研究了固定化酶的性质。结果表明,壳聚糖载体可以较好地吸附L-天门冬酰胺酶。固定化酶的活力回收率较高,且稳定性得到提高。     

Simple,inexpensive routes to E and Z zeatine ribosides and derivatives useful for immunoassay.

Allylic oxidation of 6-$\text{N}$-(3,3-dimethylallyl)adenosine $\text{1}$ gave $\text{trans}$ ($\text{E}$) zeatine riboside $\text{3}$, which was isomerized to the $\text{cis}$ ($\text{Z}$) isomer $\text{5}$ by u.v. irradiation. A method for the regiospecific 3′-$\text{O}$-succinoylation of these nucleosides is given.     

An anti E. coli O157:H7 antibody-immobilized microcantilever for the detection of Escherichia coli (E. coli).

A silicon microcantilever sensor was developed for the detection of Escherichia coli O157:H7. The microcantilever was modified by anti-E. coli O157:H7 antibodies on the silicon surface of the cantilever. When the aquaria E. coli O157:H7 positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the E. coli O157:H7 antigen by the antibodies on the surface of the microcantilever. A negative control sample that does not contain E. coli O157:H7 antigen did not cause any bending of the microcantilever. The detection limit of the sensor was 1 x 10(6) cfu/mL when the assay time was < 2 h.     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides IV. Covalent binding with dialdehyde-dextran

The synthesis has been effected of immobilizedE. coli L-asparaginase on medical dextran — poliglyukin. The influence of the bound polymer on some physicochemical properties of the final products have been studied. An increased resistance to heat and stability on storage of the immobilized forms of L-asparagine in comparison with a native enzyme has been found. It has been shown that the polymer modification of L-asparaginase leads to a decrease in the antigenic affinity of the immobilized enzyme as compared with the native enzyme.     

Quantification of E. coli adhesion to polyamides and polystyrene with atomic force microscopy

Atomic force microscopy (AFM) was used to measure adhesion forces between E. coli bacteria and surfaces consisting of a series of polyamides and polystyrene, materials that are prominent in carpeting, upholstery, and other indoor surfaces. Bioparticle adhesion to such surfaces in air is poorly understood, yet these interactions are thought to play a key role in their accumulation and release as indoor air pollutants. The polymers employed were polyamide 6 (PA6), polyamide 6,6 (PA66), polyamide 12 (PA12) and polystyrene (PS). We report the interaction forces between immobilized E. coli and AFM tips coated with each polymer. The adhesion forces for the tip-bacterial interactions were in the range between 2.9 and 6.7 nN, which is of the same magnitude as the polymer-polymer interactions for the same series of polymers. Polystyrene had stronger adhesion with E. coli than any of the three polyamides, by an average factor of 1.4. The work of adhesion and Hamaker constants of the probe-surface interactions were calculated using a square-pyramid flat-surface model developed previously. A drag-force analysis suggests that model spheres with the same adhesion force as E. coli-poly(amide) (F approximately 4 nN) will remain adherent under normal foot traffic (F approximately 0.2 nN), but will release during vacuum cleaning (F>or=30 nN).     

Immobilization of the enzyme L-asparaginase fromE. coli on polysaccharides. II. Covalent binding with soluble CM-cellulose

The covalent binding of L-asparaginase to soluble CM-cellulose has been carried out by the azide method, and some physicochemical properties of the preparation obtained have been studied. It has been established that the modified L-asparaginase possesses a higher heat stability than the native enzyme and also a greater resistance to proteolytic enzymes.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 383–388, May–June, 1979.     

A new enzymatic method for determination of E. coli

Inthispaper,arapid,sensitiveandsimpleenzymaticmethodfordetectingtheE.coltcellshasbeenestablished.P-GalactosidaseisakindofendoenzymeexistingintheE.coltcells.Itcanbereleasedfromthecellsbythepore-formingactionofcolicinEI,andcanbedetectedrapidlybyenZymatic..thodl.Colici.,areplasmid-encodedproteins,bindspecificallytoparticularreceptorsontheoutermembraneofsensitivecellsan.dactmainlythroughtwopathways'.Beca.s.theycanformporesinthemembrane,colicinshavebeenwidelyusedastoolstostudythebiologicalandphy…     

Laser trap studies of end-on E. coli adhesion to glass

Rod-shaped Escherichia coli K12:D21 bacteria were previously found to adhere by their ends (poles) [J.F. Jones, J.D. Feick, D. Imoudu, N. Chukwumah, M. Vigeant, D. Velegol, Appl. Environ. Microbiol. 69 (2003) 6515.]. In the current study we used a Nd:YAG 1064 nm laser trap to quantify the fraction of adherent bacteria and the time scale for the adhesion to occur. For the E. coli studied, 15.9+/-3.4% of the bacteria adhered when presented end-on for 15s to a cleaned glass surface that was not treated for specific interactions. These bacteria were found to adhere either instantaneously (approximately <1s) or not at all, and the adhesion was shown to be independent of power (force) of the laser trap. Additionally, for a given bacterium, either 0 or 1 ends were adhesive, never both ends. It is hypothesized that the end-on adhesion of D21 is related to bacterial polarity that dynamically results from the division process. We studied the reattachment of cells after adhesion and subsequent removal, finding that most bacteria reattach, some at least five times. However, a small fraction of D21 did not reattach after the first removal. Bacterial cells with observable division planes were tested for end-on adhesion; none of the 18 cells studied adhered by either end. On the other hand, we examined 50 daughter cells immediately after division, and four of the cells were adhesive. End-on adhesion is shown to be an important initial adhesion strategy for the E. coli strain via a single end with adhesion occurring instantaneously. Knowledge about adherent nanodomains (here, on one end) on bacteria will lead to better predictions of sticking coefficients and bacteria transport through porous media.     

Targeting of liposomes surface-modified with glycyrrhizin to the liver. I. Preparation and biological disposition

We consider glycyrrhizin to be a new ligand for liposomes to the liver because it is known that about 80% of glycyrrhizin is excreted into the bile after intravenous administration in rats. In order to modify the liposomal surface with glycyrrhizin, 30-stearyl glycyrrhizin (GLOSt), one of the lipophilic glycyrrhizin derivatives, was synthesized. The structure of this new compound was identified by nuclear magnetic resonance (NMR), infrared (IR) and mass spectra (MS). Sonicated liposomes were prepared from hydrogenated egg phosphatidylcholine-cholesterol-GLOSt or dicetyl phosphate (DCP) (4:4:1) and were labelled with [3H]inulin as an aqueous marker. It was confirmed by measuring the encapsulation efficiencies and the mean diameters that GLOSt-containing sonicated liposomes (GLOSt-SUV) were SUV-type as well as DCP-containing control liposomes (control-SUV). Four hours after intravenous injection into rats at a dose of 90 mumol as total lipid per kg of rat body weight, GLOSt-SUV showed 4-fold more accumulation (42.4%) in the liver than control-SUV. Therefore, glycyrrhizin is considered to be a useful new ligand on liposomes for targeting to the liver.     

Expression of a partial synthetic human TNF cDNA in E. coli.

A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exon codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fermentator was used to produce rhTNF. About 20 g (wet weight) of bacterial pellet per liter medium and 10(6)-10(7) units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer. rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.     

Adaptation to UVA radiation of E. coli growing in continuous culture

Adaptive responses of bacteria to physical or chemical stresses in the laboratory or in the environment are of great interest. Here we investigated the ability of Escherichia coli growing in continuous culture to adapt to UVA radiation. It was shown that E. coli indeed expressed an adaptive response to UVA irradiation at an intensity of 50W/m(2). Cells grown in continuous culture with complex medium (diluted Luria Bertani broth) at dilution rates of 0.7h(-1), 0.5h(-1) and 0.3h(-1) were able to maintain growth under UVA irradiation after a transient reduction of specific growth rate and recovery. In contrast, slow-growing cells (D=0.05h(-1)) were unable to induce enough protection capacity to maintain growth under UVA irradiation. We propose that faster growing E. coli cells have a higher adaptive flexibility to UVA light-stress than slow-growing cells. Furthermore it was shown with flow cytometry and viability stains that at a dilution rate of 0.3h(-1) only a small fraction (1%) of the initial cell population survived UVA light-stress. Adapted cells were significantly larger (30%) than unstressed cells and had a lower growth yield. Furthermore, efflux pump activity was diminished in adapted cells. In a second irradiation period (after omitting UVA irradiation for 70h) adapted cells were able to trigger the adaptive response twice as fast. Additionally, this study shows that continuous cultivation with direct stress application allows reproducible investigation of the physiological and possibly also molecular mechanisms during adaptation of E. coli populations to UVA light.     

E. coli adhesion to silica in the presence of humic acid

The influence of humic acid on the adhesion of Escherichia coli to silica particles or glass surfaces was investigated. After adsorbing various amounts of humic acid to the particles or surfaces, bacteria were added to the sample and allowed to adhere. For the silica particles the number of bacteria-particle couplets formed were counted from video microscopy images. For the glass surfaces, a differential electrophoresis force was applied, and the force required to detach the bacteria was quantified. These experiments showed a slight increase in the number of couplets formed in the presence of humic acid, and also showed a slight increase in the force required for detachment of the bacteria. Although an increase in adhesion number and strength was measured, the magnitude of the increase was small, indicating that humic acid plays a small role in bacterial adhesion to silica or glass surfaces.