ABSTRACT
We demonstrate ultrasensitive fluorescence imaging of proteins on dot blots and western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CNPPV). We achieved a detection limit at the single-picogram level in dot blots; with conventional western blot, we detected 50 pg of transferrin and trypsin inhibitor after SDS-PAGE and transfer onto a PVDF membrane. In this protocol, we describe the preparation of Pdots and its streptavidin bioconjugate and the procedures of dot blot and western blots.
Subject(s)
Blotting, Western/methods , Immunoblotting/methods , Polymers/chemistry , Proteins/analysis , Quantum Dots/chemistry , Animals , Antibodies , Biotinylation , Electrophoresis, Polyacrylamide Gel/methods , Fluorescent Dyes/chemistry , Humans , Optical Imaging/methods , Semiconductors , Streptavidin/chemistry , Transferrin/analysisABSTRACT
Ultrasensitive fluorescence imaging of proteins on western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CN-PPV) is demonstrated. A detection limit at the single-picogram level in dot blots is achieved; with conventional western blotting, 50 pg of transferrin and trypsin inhibitor after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto a polyvinylidene fluoride membrane are detected. This method does not require any additional equipment or time compared with the conventional procedure with traditional fluorescent probes.
Subject(s)
Fluorescent Dyes/chemistry , Polymers/chemistry , Proteins/analysis , Semiconductors/standards , Blotting, Western/methods , Electrophoresis, Polyacrylamide Gel , Polyvinyls/chemistry , Sodium Dodecyl Sulfate/chemistryABSTRACT
Semiconducting polymer-based nanoparticles (Pdots) have recently emerged as a new class of ultrabright probes for biological detection and imaging. This paper describes the development of poly(2,5-di(3',7'-dimethyloctyl)phenylene-1,4-ethynylene) (PPE) Pdots as a platform for designing Förster resonance energy transfer (FRET)-based ratiometric pH nanoprobes. We describe and compare three routes for coupling the pH-sensitive dye, fluorescein, to PPE Pdots, which is a pH-insensitive semiconducting polymer. This approach offers a rapid and robust sensor for pH determination using the ratiometric methodology where excitation at a single wavelength results in two emission peaks, one that is pH sensitive and the other one that is pH insensitive for use as an internal reference. The linear range for pH sensing of the fluorescein-coupled Pdots is between pH 5.0 and 8.0, which is suitable for most cellular studies. The pH-sensitive Pdots show excellent reversibility and stability in pH measurements. In this paper, we use them to measure the intracellular pH in HeLa cells following their uptake by endocytosis, thus demonstrating their utility for use in cellular and imaging experiments.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Nanoparticles/chemistry , Polymers/chemistry , Semiconductors , Endocytosis , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Hydrogen-Ion Concentration , Intracellular Space/chemistry , Intracellular Space/metabolism , Linear Models , Molecular Imaging , Polymers/metabolismSubject(s)
Fluorescent Dyes/chemistry , Polymers/chemistry , 3T3 Cells , Alanine/analogs & derivatives , Alanine/chemistry , Animals , Click Chemistry , Fluorenes/chemistry , Fluorescent Dyes/chemical synthesis , Humans , Mice , Molecular Structure , Particle Size , Polymers/chemical synthesis , Proteins/analysis , Proteins/metabolism , Semiconductors , Staining and Labeling , Surface Properties , Tumor Cells, CulturedABSTRACT
This paper describes characterization of lyophilized lipid nanocapsules loaded with Alexa 488 by fluorescence correlation spectroscopy (FCS). Fluorimetry analysis of nanocapsules containing self-quenching concentrations of 5- and 6-carboxyfluorescein was performed to establish a point of reference for FCS. FCS results complemented the results obtained by fluorimetry for a bulk nanocapsule solution and provided additional information about the size and dye retention by individual nanocapsules. Using this method, we determined that nanocapsules composed of the thiol-functionalized lipids showed the best dye retention and the most consistent results. Dye retention, size, and photolysis efficiency of these thiol-functionalized nanocapsules doped with a far-red photosensitizer did not change substantially upon lyophilization and storage at -20 degrees C for up to 2 months, making lyophilization a suitable method for the long-term storage of nanocapsules with the appropriate lipid composition.
