Abstract
We wish to report the successful assembly of a nanoparticle silica with proteinase K enzyme using a PEG bridge. The bridge is a hetero-bi-functional activated material which reacts with free amino groups on one end (SVA) and Sulfydryl groups on the other (MAL). This 20,000 molecular weight cross linker was selected to allow for the retention of enzymatic activity.
The nanoparticles that were created are intended for use in clinical diagnostic devices, such that the enzyme is active on large substrates and can be immobilized according to device architecture. We report here that the enzymatic activity of this nanoparticle silica conjugate directed against intact hemoglobin incubated at 42° C for 15 minutes – allows for complete digestion of hemoglobin molecule we have detected free tyrosine using the Folin-Ciocalteu's phenol reagent and according to the Enzymatic Assay of Proteinase K.
The use of confocal microscopy and flow cytometry emphasized as a technique to validate the attachment of the enzyme PEG +Silica. This was accomplished through the fluorescent label multiplexing. The silica itself was doped with fluorescent dylight 405 and proteinase K was labelled with dylight 594.
We can successfully report that distinct encapsulated silica –dylight 405 as well as a proteinase K+ PEG + Silica conjugate was confirmed by both flow cytometry as well as confocal microscopy. The technique whereby these particles were created has widespread application in the pharmaceutical and biomedical device industry.
Faculty Sponsors
Terry McCaffrey, Ph.D.
Project Type
Event
Location
Alvin Sherman Library
Start Date
4-10-2015 1:00 PM
End Date
4-10-2015 5:30 PM
Retention of Enzymatic Activity of a Proteinase K-PEG Silica Nanoparticle Conjugate for Use on a Biomedical Point of Care Device
Alvin Sherman Library
We wish to report the successful assembly of a nanoparticle silica with proteinase K enzyme using a PEG bridge. The bridge is a hetero-bi-functional activated material which reacts with free amino groups on one end (SVA) and Sulfydryl groups on the other (MAL). This 20,000 molecular weight cross linker was selected to allow for the retention of enzymatic activity.
The nanoparticles that were created are intended for use in clinical diagnostic devices, such that the enzyme is active on large substrates and can be immobilized according to device architecture. We report here that the enzymatic activity of this nanoparticle silica conjugate directed against intact hemoglobin incubated at 42° C for 15 minutes – allows for complete digestion of hemoglobin molecule we have detected free tyrosine using the Folin-Ciocalteu's phenol reagent and according to the Enzymatic Assay of Proteinase K.
The use of confocal microscopy and flow cytometry emphasized as a technique to validate the attachment of the enzyme PEG +Silica. This was accomplished through the fluorescent label multiplexing. The silica itself was doped with fluorescent dylight 405 and proteinase K was labelled with dylight 594.
We can successfully report that distinct encapsulated silica –dylight 405 as well as a proteinase K+ PEG + Silica conjugate was confirmed by both flow cytometry as well as confocal microscopy. The technique whereby these particles were created has widespread application in the pharmaceutical and biomedical device industry.
