ABSTRACT
Colloidal particles with fluorescence read-out are commonly used as sensors for the quantitative determination of ions. Calcium, for example, is a biologically highly relevant ion in signaling, and thus knowledge of its spatio-temporal distribution inside cells would offer important experimental data. However, the use of particle-based intracellular sensors for ion detection is not straightforward. Important associated problems involve delivery and intracellular location of particle-based fluorophores, crosstalk of the fluorescence read-out with pH, and spectral overlap of the emission spectra of different fluorophores. These potential problems are outlined and discussed here with selected experimental examples. Potential solutions are discussed and form a guideline for particle-based intracellular imaging of ions.
Subject(s)
Biosensing Techniques , Calcium/chemistry , Nanotechnology/methods , Optics and Photonics , Benzoxazines/chemistry , Endocytosis , Fluorescent Dyes/chemistry , Gold/chemistry , HeLa Cells , Humans , Hydrogen-Ion Concentration , Ions , Metal Nanoparticles/chemistry , Microscopy, Fluorescence , Particle Size , Peptides/chemistry , Polymers/chemistryABSTRACT
The concept of enzyme-assisted substrate sensing based on use of fluorescent markers to detect the products of enzymatic reaction has been investigated by fabrication of micron-scale polyelectrolyte capsules containing enzymes and dyes in one entity. Microcapsules approximately 5 µm in size entrap glucose oxidase or lactate oxidase, with peroxidase, together with the corresponding markers Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)) complex and dihydrorhodamine 123 (DHR123), which are sensitive to oxygen and hydrogen peroxide, respectively. These capsules are produced by co-precipitation of calcium carbonate particles with the enzyme followed by layer-by-layer assembly of polyelectrolytes over the surface of the particles and incorporation of the dye in the capsule interior or in the multilayer shell. After dissolution of the calcium carbonate the enzymes and dyes remain in the multilayer capsules. In this study we produced enzyme-containing microcapsules sensitive to glucose and lactate. Calibration curves based on fluorescence intensity of Ru(dpp) and DHR123 were linearly dependent on substrate concentration, enabling reliable sensing in the millimolar range. The main advantages of using these capsules with optical recording is the possibility of building single capsule-based sensors. The response from individual capsules was observed by confocal microscopy as increasing fluorescence intensity of the capsule on addition of lactate at millimolar concentrations. Because internalization of the micron-sized multi-component capsules was feasible, they could be further optimized for in-situ intracellular sensing and metabolite monitoring on the basis of fluorescence reporting.
Subject(s)
Biosensing Techniques/methods , Capsules/chemistry , Enzymes, Immobilized/metabolism , Fluorescent Dyes/chemistry , Glucose Oxidase/metabolism , Mixed Function Oxygenases/metabolism , Peroxidase/metabolism , Armoracia/enzymology , Aspergillus niger/enzymology , Calcium Carbonate/chemistry , Coordination Complexes/chemistry , Enzymes, Immobilized/chemistry , Glucose/analysis , Glucose Oxidase/chemistry , Lactic Acid/analysis , Mixed Function Oxygenases/chemistry , Pediococcus/enzymology , Peroxidase/chemistry , Phenanthrolines/chemistry , Rhodamines/chemistry , Ruthenium/chemistryABSTRACT
Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARF-1 into polyelectrolyte multilayer capsules. Co-precipitation of calcium carbonate, urease and dextran followed up by multilayer film coating and Ca-extracting by EDTA resulted in the formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10(-6) to 10(-1) M. The presence of urea can be monitored on a single capsule level as illustrated by confocal fluorescent microscopy. Variations in urease:dye ratio in capsules, applicability and limits of use of that type multi-component microencapsulated sensors are discussed.
