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Synthesis, characterization, and applications of strongly fluorescent, multicolored protein nanoparticles (GlowDots) are reported here. Bovine serum albumin was cross-linked under controlled conditions to form nanoparticles, where particle size was controlled from 20 to 100 ± 10 nm by choosing appropriate reaction conditions. The absorption as well as the emission wavelengths were controlled without changing the particle size, unlike quantum dots. Each GlowDot was loaded with up to 214 ± 50 chromophores, and hence, the particles have high molar absorptivities (106 M–1 cm–1) as well as high brightness (105 to 106 M–1 cm–1). A large number of functional groups cover the particle surface and these are further functionalized to enhance cellular uptake. GlowDots that were labeled with fluorescein and functionalized with taurine, for example, were quickly taken up by HeLa, MDA-MB-231, PC3, and L6 myoblast cells, as interrogated by fluorescence imaging studies. GlowDots were biocompatible, size tunable, biodegra...

A powerful strategy to photocleave the protein backbone at a single site, developed over nearly two decades, is described here. The remarkable ability to target one or even a couple of sites on a large protein with a small molecule, under photochemical control, is a considerable challenge. Systematic modification of the structure of the photoreagent provides valuable information on the binding site recognition as well as the mechanism of the photocleavage reaction. Some factors that impact the photocleavage reaction include the exact location of the probe binding site on the protein, conformations of the bound probe, protein size, functional groups present on the probe that interact with the protein surroundings either in a favorable or unfavorable manner, overall charge on the photoreagent, and photophysical/photochemical properties of the probe. The protein photocleavage studies were preceded by detailed binding studies by a variety of spectroscopic methods. Methods as simple as absorption and fluorescence spectroscopies or more sophisticated circular dichroism spectroscopy were used. Conclusions that are most consistent with the binding data indicated a single binding site on most proteins, irrespective of the probe or the protein, with only one exception noted so far. Such high selectivity for targeting a binding site on a biomolecule by a small molecule is remarkable. Photocleavage studies and laser flash photolysis studies were used to probe the photochemical peptide bond cleavage reactions, which indicated the important role of aromatic cation radicals in the mechanistic path. Our work focused mainly on few generic proteins serum albumin, lysozyme, and avidin; but may be extended to other proteins with appropriate modifications. This article strives to provide some guidance on such design strategies for other proteins or other photoreagents. Molecular modeling studies clearly supported our conclusions and the methods described here could be useful in a rational design of novel photoreagents to target desired sites on proteins in future studies.

A simple method for interlocking glucose oxidase and horseradish peroxidase in a network of cellulose fibers coated with bovine serum albumin (BSA)-exfoliated graphene (biographene) is reported here. The resulting paper reactor is inexpensive and stable. Biographene is expected to function as an electron shuttle, making the reaction between the enzyme and the substrate more efficient, and this hypothesis is examined here. The BSA used to separate the sheets of graphene provides extra carboxylic acid groups and primary amines to help interlock the enzymes and the graphene in between the fibers. The decrease in entropy associated with interlocking the enzymes on a solid support is likely responsible for the increase in enzymatic stability/activity observed. Each cellulose disk contained 5.2 mg of enzyme per gram of paper and 93% of the enzyme is retained after washing for 0.5–2 h. This simple methodology provides a low cost, effective approach for achieving high enzymatic activity and good loadings on a benign, versatile support.