Fluorescence urease

Evidence for conformers of urease has been presented by two groups of investigators. Chernitskii et al. (51) noted fluorescence maxima at pH 3-4, 5.5-6.5, 7.5-9, and >10.5 when urease solutions were irradiated with 280 or 296 nm radiation. Addition of urea increased the fluorescence... 

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARE-1 into polyelectrolyte multilayer capsules. The co-precipitation of calcium caibonate, urease, and dextran followed up by multilayer film coating and Ca- extracting by EDTA resulted in formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10 to 10 M. Sensitivity to urea in concentration range of 10 to 10 M was monitored on capsule assemblies (suspension) and on single capsule measurements. Urea presence can be monitored on single capsule level as illustrated by confocal fluorescent microscopy. 

Aim of this worit was to demonstrate a particular example of a sensor system, which combines catalytic activity for urea and at the same time, enabling monitoring enzymatic reaction by optical recording. The proposed sensor system is based on multilayer polyelectrolyte microcapsules containing urease and a pH-sensitive fluorescent dye, which translates the enzymatic reaction into a fluorescendy registered signal. 

Confocal images were obtained by Leica Confocal Laser Scanning Microscope TCS SR For capsules visualization 100 x oil inunersion objective was used throughout. 10 pi of the SNARF-1 dextran/urease capsules suspension was placed on a covershp. To this suspension 10 pi of 0.1 mol/L urea is added. After about 20 min confocal images were obtained. The red fluorescence emission was accumulated at 600-680 mn after excitation by the FITC-TRIC-TRANS laser at 543 mn. 

In order to verify a feasibility of fluorescence based nrea sensing on two component co-encapsulation we fabricated two polyelectrolyte capsnle samples of the (PSS/ PAHj PSS shell architecture with different content of dye and mease. The first sample Sample I) contained in average 0.6 pg SNARF-1 dextran per capsule, while the content of the SNARF-1 dextran in the other sample was 0.2 pg per capsifle Sample II). The concentration of active urease in samples was opposite 0.2 and 0.6 pg/capsule respectively what gives an average SNARF-1 dextran/urease ratio of 3 1 and 1 3 in these investigated samples. 

Spectrofluoremetric studies were carried out to determine the correlation between the fluorescence intensity of the SNARF-1 dextran/urease capsules and the pH of the medium. Both the capsule samples were stored for 10 min in the 0.05 M TRIS-maleate buffer at pH in the range 5.5-9. The excitation wavelength was 540 nm. The capsules fluorescence spectra of the first sample are shown in Figure 2. The spectra obtained for both samples were similar. The encapsulated dye is capable to provide information of the medium acidity in a reasonably wide range of pH. It is seen that fluorescence... 

FIGURE 2 Fluorescence spectra of the SNARF-1 dextran/urease capsules in the 0.05 M... 

FIGURE 5 SNARF-1 dextran/urease capsule fluorescence spectra in water in the presence of urea from 1 O to 0.1 M. 

FIGURE 6 Kinetics of the change of the SNARF-1 dextran/urease capsule fluorescence intensity e q)ressed through the fluorescence intensity ratio R at 580 and 640 nm in the presence of 10 M (curve 1 - sample 1, curve 2 - sample II) and 10 M urea (curve 3 - sample I, curve... 

To calculate the apparent pH caused by urea concentration the values of R., R pK and Ig (B)/ l54o (A) were used. R was determined as a fluorescence intensity ratio at 580 nm and 640 nm in the capsules stored in bidistilled water (pH = 6.4). is the ratio of the fluorescence intensity spectrum related to the minimal R value at 580 nm and 640 nm in the SNARF-1 dextran/urease capsules when high concentration of urea (0.1 M) was added and 30 min after the enzymatic reaction begun. The pK value were assumed to be = 7.15 and 7.25 for sample 1 and 2 respectively, that was in accord with the experiment with buffer solutions (Figure 3,4/ From the values obtained a calibration curve of the pH dependence inside the capsules on the urea concentration present in the solution was plotted. The calibration curve is presented in Figure 7. 

The feasibility studies on single capsule detection of urea presence were carried out using confocal fluorescent microscopy. CLSM image of SNARF-1 dextran/urease capsules in absence and at O.IM urea added to the same capsules are presented on Figure 8. Small distinctions in the form and the sizes of capsule population are connected with non-uniformity of SNARF-1 dextran/urease CaCO particles received in co-precipitation process what is rather often observed for calcium carbonate templated capsules containing proteins [20]. 

FIGURE 8 Confocal fluorescence microscopy images of (PSS/EAH) PSS capsules loaded with SNARF-1 dextran (MW = 70kDa) and urease enzyme in water (image A) and 0.1 M urea concentrations (image B). The Table 1 shows the increase of mean energy of individual capsules before (Fj ) and after the addition 0.1 M urea solution to water capsule suspension. The red fluorescence emission was accumulated at 600-680 run after excitation by the FITC-TRIC-TRANS laser at 543 nm. 

In this study we have demonstrated a particular example of a sensor system, which combines catalytic activity for the substrate (urea) and at the same time enabling to monitor the enzymatic reaction by co-encapsulated pH sensitive dye. Substrate sensitive enzyme urease was co-encapsulated together with SNARF-1 coupled to dextran in multilayer tnicrocapsules. Enzymatic activity was recorded by fluorescent changes caused by increasing of pH in course of enzymatic cleavage of urea as measured on... 

FIGURE 20.3 Change of fluorescence intensity ratio -R at 580 and 640 nm for curve 1- SNARF-1 dextran water solution curve 2 containing SNARF-1 dextran capsules curve 3 SNARF-1 dextran/urease capsules (sample I, 0.6 pg dye/capsule curve 4 SNARF-1 dextran/ urease capsules (sample II, 0.2 pg dye/capsule) in 0.05 M TRIS-maleate buffer at pH in range 5.5-9. 

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