Kinetic enhancement of the diffusion-limited enzyme beta-galactosidase when displayed with quantum dots

Brown, Carl W.; Oh, Eunkeu; Hastman, David A.; Walper, Scott A.; Susumu, Kimihiro; Stewart, Michael H.; Deschamps, Jeffrey R.; Medintz, Igor L.

doi:10.1039/c5ra21187e

Cited by 35 publications

(56 citation statements)

References 43 publications

(78 reference statements)

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“…Supporting this notion, a recent report confirms that structural rearrangement of the localized environment around colloidal NPs is a universal phenomenon regardless of what the actual solvent is [16]. We have even found a significant enhancement when working with the large multimeric β-galactosidase enzyme and QDs [17]. Given the large size of this tetrameric enzyme (465 kD), the QDs were functionally decorated around the enzyme instead of the converse configuration.…”

supporting

confidence: 63%

Interesting Developments at the nanoparticle–protein interface: Implications for Next Generation Drug Delivery

Medintz

2016

Ther. Deliv.

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supporting

confidence: 63%

Interesting Developments at the nanoparticle–protein interface: Implications for Next Generation Drug Delivery

Medintz

2016

Ther. Deliv.

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“…The origin of this pattern is not yet understood; however, we hypothesize that it is associated with enzyme packing and fitting on the NPs which could be far more constrained for smaller materials in the former experimental format. Moreover, investigation of beta-galactosidase activity when assembled on QDs also suggests the possibility of substrate accumulation or sequestration in the NP interface, 53 and this, too, would be constrained or limiting for smaller materials in the first experimental format by the increasing amount of enzyme present. Lastly, it is again worth pointing out the complexities of this interfacial environment and how much still remains unknown about it.…”

Section: Discussionmentioning

confidence: 99%

Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

Hondred

Breger

Garland

et al. 2017

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The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and potential chemical warfare agent use. Herein, we improve the enzymatic activity of a phosphotriesterase trimer (PTE), an enzyme that selectively recognizes OPs directly, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm diameter) gold nanoparticles (AuNPs). The number of enzymes immobilized on the AuNP was controlled by conjugating increasing molar ratios of PTE onto the AuNP surface via metal affinity coordination. This occurs between the PTE-His termini and the AuNP-displayed Ni-nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE-AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE with the following PTE-AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE displayed around the AuNP and (2) fixed concentration of PTE while increasing the bioconjugate molar ratio of PTE-AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE hydrolyzed the substrate paraoxon, a commercial insecticide and OP nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all experiments, a maximum enzymatic velocity (V) increased by 17-fold over free enzyme for the lowest PTE-AuNP ratio and the largest AuNP (i.e., ratio of 1 : 1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme-NP bioconjugates.

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“…The E. coli β-gal was first synthesized and cloned as described previously [36]. Briefly, the β-gal gene from E. coli K12 was synthesized by Genscript (Piscataway, NJ, USA) to include a 5’ NotI site and a 3’ XhoI site.…”

Section: Methodsmentioning

confidence: 99%

Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase

et al. 2018

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The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with β-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.

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Kinetic enhancement of the diffusion-limited enzyme beta-galactosidase when displayed with quantum dots

Abstract: Schematic of a tetrameric β-galactosidase enzyme attached to and displaying 625 nm emitting QDs coated with a CL4 ligand via each of the 4 pendent His6 tags.

Cited by 35 publications

References 43 publications

Interesting Developments at the nanoparticle–protein interface: Implications for Next Generation Drug Delivery

Interesting Developments at the nanoparticle–protein interface: Implications for Next Generation Drug Delivery

Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase

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