Enhancement in enzymatic activity after attachment to nanoparticle surfaces has been observed in numerous enzyme systems, although the underlying mechanism for these enhancements remains largely unknown. This work explores the utility of a model based on a reaction scheme that takes into account some of the many interactions between substrate, product, and nanoparticle that can occur. This model was utilized to make predictions about the type of behavior that should manifest itself with quantum dots peripherally displayed around beta-galactosidase (&beta-gal) and confirmed empirically. &beta-gal is a homotetrameric enzyme which at ~465 kDa is significantly larger than the 4.2 nm diameter green emitting quantum dots utilized to decorate its periphery. Because &beta-gal operates near the diffusion limit, this provides an opportunity to selectively investigate certain aspects of enzyme enhancement when attached to a nanoparticle with minimal perturbation to the native enzyme structure. Enzymatic assays were performed with both free enzyme and quantum dot-decorated enzymes in a side-by-side format where kinetic processes were challenged by increasing viscosity with glycerol and competitive inhibitors such as lactose. The results from this model suggest it is possible to achieve significant enhancements in a diffusion limited enzyme’s catalytic rate (< i>k< sub>cat< sub>< i>) after NP attachment without substantial changes to the enzyme’s structure or function. Because cell free synthetic biology is gaining importance, this approach will yield insights on how enzymes can be utilized ex vivo and how being attached to NP scaffolds yields kinetic enhancement, possibly through enhanced product dissociation.
The development of light harvesting systems for directed, efficient control of energy transfer at the biomolecular level has generated considerable interest in the past decade. Molecular fluorophores provide a straightforward mechanism for determining nanoscale distance changes through Förster resonance energy transfer (FRET), and many systems seek to build off of this simple yet powerful principle to provide additional functionality. The use of DNA-based integrated biomolecular devices offer many unique advantages towards this end. DNA itself is an excellent engineering material – it is innately biocompatible, quickly and cheaply synthesized, and complex structures can be readily designed in silico. It also provides an excellent scaffold for the precise patterning of various biomolecules. Here, we discuss the systems that have been recently developed which add to this toolbox, including nanostructural dye patterning, photonic wires, and the incorporation of alternative energy propagation modalities, such as semiconductor quantum dots (QD) and the bioluminescent protein luciferase. In particular, we explore the incorporation of luciferase into various nanostructural conformations, providing the capability to efficiently control energy flow directionality. We discuss the nature of this system, including unexpected spectral complexities, in the context of the field.
KEYWORDS: Nanostructures, Luminescence, Molecules, Energy transfer, Energy harvesting, Resonance energy transfer, Fluorescence resonance energy transfer, Dendrimers, Light harvesting, Molecular photonics
DNA is a biocompatible scaffold that allows for the design of a variety of nanostructures, from straightforward double stranded DNA to more complex DNA origami and 3-D structures. By modifying the structures, with dyes, nanoparticles, or enzymes, they can be used to create light harvesting and energy transfer systems. We have focused on using Förster resonance energy transfer (FRET) between organic fluorophores separated with nanometer precision based on the DNAs defined positioning. Using FRET theory we can control the direction of the energy flow and optimize the design parameters to increase the systems efficiency. The design parameters include fluorophore selection, separation, number, and orientation among others. Additionally the use of bioluminescence resonance energy transfer (BRET) allowed the use of chemical energy, as opposed to photonic, to activate the systems. Here we discuss a variety of systems, such as the longest reported DNA-based molecular photonic wires (> 30 nm), dendrimeric light harvesting systems, and semiconductor nanocrystals integrated systems where they act as both scaffold and antennae for the original excitation. Using a variety of techniques, a comparison of different types of structures as well as heterogeneous vs. homogenous FRET was realized.
Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.
Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.