Combining biomolecules such as enzymes with nanoparticles (NPs) can provide higher activity and effectiveness creating novel functional bionanomaterials.  A particular design with extensive applicability as a sensor design are fluorescent NPs with conjugated enzyme substrates that quench the fluorescence. Upon modification of the substrate by a specific enzyme it no longer quenches the NP’s fluorescence, the change in fluorescence then becomes a simple readout of the enzymes activity. This form of assay is of great interest as it measures actual enzyme activity, as opposed to enzyme amount or mRNA precursor, and can be used in high throughput screens to test for enzyme co-factors or inhibitors. Yet very little is known about how the enzyme and NP interact at their interface. It is hypothesized that many of the positive interactions, as well as the negative ones, are due to the modified nanoenvironment in or near near the NP surface. These environments can have modified polarity, pH, or small molecule concentrations within the structures or near the NP surface as compared to the bulk solution. To this end we chose to examine a system where only the chemical nature of the surface functionalizing ligands of a CdSe/ZnS core shell quantum dot (QD) were modified, this changed the enzymes interaction with the NP resulting in modulated enzyme activity.<br/>The QD was conjugated with multiple copies of an organic dye (Cy3) labeled peptide that created a Förster resonance energy transfer (FRET) pair with the QD; the peptide also acted as a substrate for trypsin. The trypsin enzyme could cleave the peptide allowing the Cy3 to drift from the QD surface and diminish the FRET allowing for a ratiometric fluorescent signal. As mentioned the surface ligands of the QD were modified such that they could be positively or negatively charged, zwitterionic, neutral, and with differing lengths and hydrophobicity.<br/>Using a combination of experimental data and molecular dynamics simulations we found that key arrangements such as avoiding steric hindrance from longer surface ligands in combination with attractive forces (van der Waals and electrostatic) between the enzyme and the QD surface resulted in a 35-fold rise in enzymatic activity when compared to freely diffusing enzymes and substrates.

Controlling enzyme activity in enzyme-nanoparticle conjugates through selective ligand choice

254th National Meeting (2017)

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