Effect of Cationic Surfactants on Enhancement of Firefly Bioluminescence in the Presence of Liposomes

Firefly bioluminescence (BL) was greatly affected by cationic surfactants coexisting with liposomes containing phosphatidylcholine and cholesterol. In this study, the effects of the type and concentration of cationic surfactants on BL were studied in the presence of the liposomes. Three types of cationic surfactant: benzalkonium chloride (BAC), n-dodecyltrimethylammonium bromide (DTAB), and benzethonium chloride (BZC), were used. As a common effect in these surfactants, BL intensity was increased and then drastically decreased with increasing surfactant concentration. This can be explained by the formation of cationic liposomes as BL enhancers at low concentration of the surfactant, and by the transformation into cationic (mixed) micelles as inhibitors at high concentration. The maximal BL intensity and the concentration for the maximal BL were dependent on the type of the surfactants. To explain the differences in these parameters in the enhanced BL, we determined the distribution coefficient, K, of the surfactants to the liposomal membrane. The result indicated that the surfactant with higher K value gives the maximal BL intensity at lower concentration

Application of Horseradish Peroxidase-Encapsulated Liposomes as Labels for Immunodotblotting

Liposomes are spherical vesicles consisting of phospholipid bilayers surrounding an aqueous volume. Recently, attention has focused on liposomes as a signal-enhancement agent, since a number of marker molecules can be encapsulated in their aqueous interior. An antibody labeled with liposome encapsulating enzyme, such as glucose oxidase, has been employed for immunoassays. On the other hand, HRP-conjugated liposomes were prepared by covalently attaching HRP to the outside of the lipid bilayer of liposome. However, the preparation of HRP-conjugated liposomes was tedious and time-consuming, since several reaction steps are required for linking HRP covalently to the surface of liposomes. In order to hold simply HRP in liposomes, we examined how to encapsulate HRP into the aqueous interior of liposomes prepared by an extrusion technique (VETs).4 In addition, the HRP-encapsulated VETs were coupled covalently to anti-rabbit IgG(antibodytagged VETs).5 the detection of HRP encapsulated in the antibody-tagged VETs was made by luminol chemiluminescent (CL) method. On the other hand, biotin-tagged molecules are currently more universal as a marker in immunoassays compared to antibody-tagged marker moleucles. In the present study, the HRP-encapsulated liposomes containing biotinylated dipalmitoylphosphatidylethanolamin were prepared by an extrusion technique (biotin-tagged VETs). In addition, biotin-tagged VETs were applied to labels in immunodotblotting of rabbit IgG

On-chip genotoxic bioassay based on bioluminescence reporter system using three-dimensional microfluidic network

Microchip-based genotoxic bioassay using sensing Escherichia coli strains has been performed. In this method, the assay was conducted in three-dimensional microfluidic network constructed by a silicon perforated microwell array chip and two poly(dimethylsiloxane) (PDMS) multi-microchannel chips. The sensing strains having firefly luciferase reporter gene under transcriptional control of umuD as an SOS promoter were put into the channels on one of the PDMS chips and immobilized in the silicon microwells. Samples containing genotoxic substances and substrates for luciferase were into the channels on the other PDMS chip. The optimum conditions of the assay in the on-chip format have been investigated using mitomycin C (MMC) as a genotoxic substance. As a result, the dose-dependence of bioluminescence intensity was obtained at once on the chip. Additionally, the response ratios of the bioluminescence between mutagen- and non-induced strains were successfully enhanced by improving the on-chip assay methods and conditions. Several well-known genotoxic substances were subjected to the on-chip assay, and were detected with the detection limits comparable to those in the conventional method with reduced time

Aqueous Micellar Two-Phase Systems for Protein Separation

The extractive technique for protein purification based on two-phase separation in aqueous micellar solutions (aqueous micellar two-phase system (AMTPS)) is reviewed. The micellar solution of a nonionic surfactant, such as polyoxyethyl-ene alkyl ether, which is most frequently used for protein extraction, separates into two phases upon heating above its cloud point. The two phases consist of a surfactant-depleted phase (aqueous phase) and a surfactant-rich phase. Hydrophilic proteins are partitioned to the aqueous phase and hydrophobic membrane proteins are extracted into the sur-factant-rich phase. Because of the methodological simplicity and rapidity, this technique has become an effective means, and thus has been widely used for the purification and characterization of proteins. In contrast to polyoxyethylene alkyl ether, micellar solutions of a zwitterionic surfactant, such as alkylammoniopropyl sulfate, separate below the critical temperature. Alkylglucosides can also separate into two phases upon adding water-soluble polymers. Recently, these two-phase systems have been exploited for protein separation. Additionally, hydrophobic affinity ligands, charged polymers, and ionic surfactants have been successfully used for controlling the extractability of proteins in AMTPS

Uptake of Transition Metal Ions Using Liposomes Containing Dicetylphosphate as a Ligand

The uptake of Cu2+ was investigated using various types of liposomes composed of phosphatidylcholine (PC), cholesterol (Chol) and dicethylphosphate (DCP). DCP played a role as a ligand for Cu2+. Multilamellar vesicles (MLVs) were more effective for the uptake of Cu2+ compared to unilamellar vesicles prepared by the extrusion technique. The uptake efficiency of MLVs for Cu2+ was dependent on the molar ratio of DCP in MLVs. The uptake percent of Cu2+ was 92% using MLVs having a PC:DCP:Chol molar ratio of 4:3:3; 95% of the total vesicle Cu2+ was bound to DCP of the outer membrane surface of the MLVs, and the remaining 5% of the total Cu2+ was distributed into the interior side of the MLVs. MLVs having a PC:DCP:Chol molar ratio of 4:3:3 were also effective as separation media for Mn2+, Co2+, Ni2+ and Zn2+. The uptake efficiency of the MLVs for the transition-metal ions increased in the order Co2+< Zn2+ < Ni2+ < Mn2+ < Cu2+

Determination of Peroxidase Encapsulated in Liposomes Using Homogentisic Acid .GAMMA.-Lactone Chemiluminescence

Homogentisic acid γ-lactone (HAL) chemiluminescence (CL) was applied to the determination of horseradish peroxidase (HRP) encapsulated in liposomes. HRP was detected after the lysis of HRP-trapped liposomes with Triton X-100. CL response rate, detection limit and linear range of calibration curve for HRP in HAL CL were compared with those in p-iodophenol (p-IP)-enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in p-IP enhanced luminol CL, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in p-IP-enhanced luminol CL. The linear range of calibration curve for HRP in HAL CL was improved by a factor of 50 compared with that in p-IP-enhanced luminol CL. From these results, it was found that HAL CL were superior to p-IP-enhanced luminol CL for the determination of HRP encapsulated in liposomes

Enhancement of Firefly Bioluminescence Using Liposomes Containing Cationic Components

The effect of cationic components in liposome on the bioluminescence (BL) intensity from the firefly luciferin-luciferase reaction with ATP was investigated by use of vesicles formed by the extrusion technique (VET). Stearyltrimethyl-ammonium chloride (STAC), stearylamine (SA) and tetracaine (TC) were used as cationic components. The VET containing STAC and SA enhanced the maximum BL intensity. In contrast, a lowering of the maximum BL intensity was observed in the presence of the VET containing TC. The sensitivity for ATP in the presence of the VET containing STAC was improved by a factor of 2 and 10 times compared to that in the presence of the VET containing SA and that in water alone, respectively. The differences in the BL enhancement between cationic components could be explained in terms of different membrane surface potentials of the VET containing these cationc components

Cationic Liposomes Enhanced Firefly Bioluminescent Assay of Bacterial ATP in the Presence of an ATP Extractant

Cationic liposomes composed of two components, diethylaminoethyl-carbamoyl cholesterol and phosphatidylcholine, were applied to an enhancer for a firefly bioluminescent (BL) assay of bacterial ATP in the presence of an ATP extractant. Trichloroacetic acid (TCA), which inhibits the activity of luciferase, was used as an ATP extractant. Cationic liposomes enhanced the BL intensity as long as luciferase was active. The detection limits for cell numbers of Escherichia coli extracts in the presence of cationic liposomes and in water alone were 199 and 897 colony forming units ml-1, respectively. The sensitivity for bacterial ATP in the presence of cationic liposomes was improved by a factor of 2.5 times compared to that in the presence of diethylaminoethyl-dextran

Direct Determination of Horseradish Peroxidase Encapsulated in Liposomes by Using Luminol Chemiluminescence

Horseradish peroxidase (HRP) encapsulated in liposomes was directly detected by using luminol chemiluminescence (CL) with H2O2 without lysis of liposomes. At a low concentration of H2O2, the initial rate of HRP-catalyzed luminol CL in liposomes was slower than that of HRP-catalyzed luminol CL in a lipid-free bulk solution. The decrease in the initial rate of the CL reaction in liposomes was due to the membrane permeation of luminol and H2O2. At a high concentration of H2O2, the initial rate of the CL reaction in liposomes was the same as that in a lipid-free bulk solution. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H2O2 by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalyzed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The CL intensity was dependent on the amount of HRP-encapsulated liposomes used. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalyzed luminol CL in a lipid-free bulk solution

Effect of Mixing Modes on Chemiluminescent Detection of Epinephrine with Lucigenin by an FIA System Fabricated on a Microchip

The chemiluminescent (CL) detection of epinephrine (EP) with lucigenin (Luc) was performed using a micro flow cell fabricated on a silicon chip. A solution of EP was injected into the Luc carrier stream. The Luc solution containing EP and an alkaline solution were successively poured into the flow cell by a pressure-driven flow system. Two types of flow cells were fabricated for estimating the effect of the mixing modes in the flow cells on the intensity of light emission. In flow cell 1, two streams entered through separate inlet ports and merged to flow adjacently. In flow cell 2, a Luc solution containing EP was split up to 36 partial flows by passage through the nozzles, and was injected into the alkaline solution. The intensity of light emission in flow cell 2 increased markedly compared to that in flow cell 1. The detection limit of 8.0 × 10-7 M for EP in flow cell 2 was a factor of six-times better than that in flow cell 1. The improvement in the sensitivity for EP could be explained in terms of the distortion of laminar flow in flow cell 2