Letter www.acsanm.org
Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
DNA−Nanoparticle Composites Synergistically Enhance Organophosphate Hydrolase Enzymatic Activity Anirban Samanta,†,⊥,∇ Joyce C. Breger,† Kimihiro Susumu,‡,∥ Eunkeu Oh,‡,∥ Scott A. Walper,† Nabil Bassim,§,○ and Igor L. Medintz*,† Center for Bio/Molecular Science and Engineering, Code 6900, ‡Optical Sciences Division, Code 5600, and §Materials Science and Technology Division, Code 6300, United States Naval Research Laboratory, Washington, D.C. 20375, United States ∥ KeyW Corporation, Hanover, Maryland 21076, United States ⊥ College of Science, George Mason University, Fairfax, Virginia 22030, United States Downloaded via 185.251.70.146 on July 23, 2018 at 02:07:12 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
S Supporting Information *
ABSTRACT: Cell-free synthetic biology relies on optimally exploiting enzymatic activity, and recent demonstrations that nanoparticle (NP) and DNA scaffolding can enhance enzyme activity suggest new avenues toward this. A modular architecture consisting of a DNA cage displaying semiconductor quantum dots (QDs) that, in turn, ratiometrically display the organophosphate hydrolase phosphotriesterase (PTE) was utilized as a model system. Increasing DNA cage concentration relative to QD-PTE and creating a dense composite enhanced PTE rates up to 12.5-fold, suggesting strong synergy between the NP and DNA components; this putatively arises from increased enzymatic stability and alleviation of its rate-limiting step. Such bioinorganic composites may offer new scaffolding approaches for synthetic biology. KEYWORDS: enzymes, DNA, phosphotriesterase, nanoparticle, quantum dot, cell free, synthetic biology, organophosphate hydrolase, bioremediation
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importantly, enhance their catalytic activity. Although the physical underpinning(s) behind this phenomenon are still largely unknown, mechanistic investigations show that the unique structured environment found at a NP−biomolecular interface is a major contributor.6−9 Representative examples of such enhancements in our studies include threefold increases in kcat for β-galactosidase and fourfold increases for the organophosphate hydrolase phosphotriesterase (PTE) when specifically displayed on semiconductor quantum dots (QDs).9,10 Contemporaneous with these studies, recent reports indicate that attachment to DNA scaffolds can similarly enhance enzyme activity. Here, the strongly charged layer surrounding the DNA is postulated to be a contributing factor.11−13 A preliminary investigation comparing PTE display on linear DNA, QDs, and a QD-DNA composite found threefold increases in PTE catalytic rates when attached to DNA and ∼10-fold increases in specific activity when assembled into a QD-DNA composite.14 Here, we seek to extend on these preliminary results and probe whether more complex NP-DNA-enzyme composite architectures can synergistically enhance enzymatic activity to an even greater extent than that previously noted.
t the most fundamental level, success in all aspects of synthetic biology is predicated on the function of enzymes. As this is still a nascent field, much of the current effort is focused on the development of model prokaryotic organisms and preliminary biocatalytic pathway engineering within them.1,2 Cell-free synthetic biology is a complementary approach toward the same goals, with most of the effort centered around so-called transcription-translation (TX-TL) systems.3 Although clearly effective, TX-TL approaches also require significant metabolic engineering and reaction optimization for each new targeted application, while overall efficiency can still be plagued by the presence of competing pathways.3,4 One alternative approach that can address these latter issues is to effectively reduce the desired biocatalytic pathway to a minimal version, where only the required enzymes, cofactors, and substrates are present; this is sometimes referred to as synthetic biochemistry.5 Here, the challenging issues now include overcoming diffusion limitations, which increase with reaction volume, and loss of longterm enzymatic stability. Paradoxically, attempting to address this by attaching the enzymes to macroscopic surfaces can increase enzyme stability, but this is very often at the cost of a loss of kinetic activity.6,7 It is here that nanoparticle (NP) display of an enzyme potentially has much to offer. A growing body of work now confirms that displaying enzymes on NPs can both increase their stability and, more © XXXX American Chemical Society
Received: June 4, 2018 Accepted: July 9, 2018
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DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
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ACS Applied Nano Materials
Figure 1. Assembly of the DNA cage displaying QDs and PTE. (A) The three-way DNA junction displaying pendant His5 is hybridized into a cage, which is then self-assembled with QDs followed by PTE. Each side of the cage is ∼17 nm long. (B) Structure of the QD stabilizing DHLA-CL4 surface ligand and paraoxon conversion into pNP by PTE. Blue sphere around QDs represents the DHLA-CL4.
Figure 2. Cage assembly with QDs. (A, left) 10% PAGE separation of assembled DNA cage-C vs 50 bp DNA marker-M. (A, right) Cage assembled on QD at indicated ratios and separated on a 1% agarose gel. (B) Representative TEMs of DNA-cage/QD 1:4 structures and higher-resolution images (assembly ratio = 4 QDs/cage; size bar = 50/10 nm). Visual analysis indicates assemblies consisted of a distribution estimated at more than four QD/cluster or cross-linked structures (∼10%), four QD/cluster (∼40%), three QDs/cluster (∼40%), and two QDs/cluster (∼10%). Note, 625 nm QDs utilized in these data.
the junction and then subsequently form a cage using an extended 24 h annealing protocol. The cage displays four open faces between each of its crossed-over parallel duplicated ds vertices with three His5-peptidyl moieties surrounding each opening. This is intended to allow QDs to attach to each face via metal-affinity coordination between the QD ZnS shell and the pendant His5 motifs. Use of 3×-His5 at each face is meant
For the current effort, a previously designed DNA tetrahedral assembly was modified.15 As shown by the schematic in Figure 1, the cage begins with a three-way double-stranded (ds) DNA junction. The junction’s termini display a pentahistidine (His5) peptide via incorporation of a chimeric peptide−DNA conjugate. Supporting Information Table S1 lists the three DNA sequences that self-assemble into B
DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
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Figure 3. DNA cage-QD-PTE catalytic activity. Initial rates of pNP formation for (A) 540 and (B) 625 nm QDs assembled with 8 PTE/QD and increasing ratios of 4, 8, 16, and 32 cages/QD. (C) Comparison of PTE activity progress curves shown in enzyme time for eight PTE, 540 QDs assembled with eight PTE (QD-PTE), and then 8 cage/QD-PTE. (D) Specific activity (μmol paraoxon hydrolyzed min−1 mg−1) vs PTE concentration for approximately eight PTE, eight PTE as assembled to 540 QDs (QD-PTE), four cage per eight PTE, and four cage per eight PTE as assembled to 540 QDs; the highest concentration of the latter sample was not included, since this assay proceeded too rapidly to yield reliable results. PTE added to preassembled DNA cage or DNA cage-QD. Lines of best fit shown.
relies on the same metal-affinity interaction between the QD’s available ZnS shell (i.e., not blocked by the previous DNA attachment) and the PTE’s terminal His6.9 PTE is capable of hydrolyzing a variety of organophosphate esters including nerve agents such as sarin and tabun, which makes it useful in several developing biodefense applications.18 PTE’s ability to hydrolyze the commercial pesticide O,O-diethyl O-(4-nitrophenyl) phosphate (paraoxon) to p-nitrophenol (pNP) with a strong 405 nm absorbance (ε ≈ 18.4 mM−1 cm−1) readily lends itself to experimental assaying as utilized previously.9,14,18,20 Figure 2A-left shows a representative polyacrylamide gel electrophoresis (PAGE) separation of the DNA cage with pendant His5 incorporated (estimated size 534 bp) next to a DNA ladder. The three-dimensional (3-D) nature of the hollow cage means that it migrates at a different rate than the purely dsDNA ladder at slightly less than 500 bp.15 From this, and visual analysis of similar gel results, we estimate that 30% forms correctly, 20% as a larger dimer, with the remaining 50% of structure as concatenated multimers and other forms that did not enter the gel under the conditions used here. We do note that the dimeric and concatenated multimeric structures could still function as intended to some extent, as they would still incorporate the QDs and PTE. Alternatively, the
to increase localized avidity and the probability of binding along with minimizing cross-linking. A growing body of work has confirmed that this high affinity (equilibrium binding constant Keq ≈ 1 × 10−9 M) interaction can attach multiple biologicals including appropriately modified DNA to QDs in a range of buffers and even remain bound in challenging in vivo biological environments.16,17 Assembly is nearly spontaneous yielding ratiometrically controlled conjugates while requiring only mixing of the components. This QD-bioconjugation strategy is amenable to any protein displaying an available polyhistidine sequence, and currently no evidence of conjugate dissociation over time has been found in the large number of assemblies formed using it.16 540 nm emitting CdSe/CdZnS/ZnS core/shell/shell (diameter 4.6 ± 0.4 nm) and 625 nm emitting CdSe/ZnS (diameter 9.3 ± 0.8 nm) QDs surface functionalized with DHLA-CL4 ligands for aqueous colloidal stability were utilized; their application in other experimental formats has been previously described.9,14,17,19 Following self-assembly of the DNA-cage QD structure (details in the Supporting Information), the QDs were, in turn, self-assembled with the dimeric enzyme PTE (monomer molecular weight (MW) ≈ 37 kDa), used again as a model system here; this assembly order was used throughout. Ratiometric PTE display on the QDs C
DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
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ACS Applied Nano Materials Table 1. Apparent Kinetic Parametersa Vmax (μM s−1) PTE only control (∼8) QD-PTE (8 PTE/QD) 4 cage/QD-PTE (8 PTE/QD) 8 cage/QD-PTE (8 PTE/QD) 16 cage/QD-PTE (8 PTE/QD) 32 cage/QD-PTE (8 PTE/QD)
0.15 0.30 1.01 1.00 1.32 1.86
± ± ± ± ± ±
0.00 0.01 0.00 0.05 0.16 0.1
QD-PTE (8 PTE/QD) 4 cage/QD-PTE (8 PTE/QD) 8 cage/QD-PTE (8 PTE/QD) 16 cage/QD-PTE (8 PTE/QD) 32 cage/QD-PTE (8 PTE/QD)
0.34 0.53 0.55 0.69 1.11
± ± ± ± ±
0.06 0.03 0.02 0.01 0.14
kcat (s−1)
KM (μM)
540 nm QD 41 ± 0 49 ± 0 129 ± 10 101 ± 3 219 ± 33 335 ± 1 242 ± 30 333 ± 17 239 ± 83 440 ± 53 522 ± 70 620 ± 56 625 nm QD 149 ± 6 112 ± 21 76 ± 1 176 ± 9 76 ± 8 182 ± 4 121 ± 11 231 ± 3 173 ± 20 370 ± 47
kcat/KM (μM−1 s−1)
SA (μmol min−1mg−1)
1.21 0.79 1.55 1.38 1.92 1.19
± ± ± ± ± ±
0.01 0.04 0.23 0.18 0.44 0.05
40.9 ± 0.1 82.1 ± 2.5 276.9 ± 0.1 275.6 ± 13.8 394.5 ± 47.3 479.2 ± 43.1
0.76 2.33 2.38 1.91 2.17
± ± ± ± ±
0.17 0.09 0.26 0.15 0.52
80.3 ± 14.5 151.0 ± 7.7 151.6 ± 3.3 188.8 ± 2.5 333.4 ± 42.4
Note: PTE added to preassembled DNA cage-QD. Cage varied. QD concentration fixed at 0.375 nM and PTE at 8 nM.
a
Menten descriptive process in these types of configurations.6−10,14,19,20 This means that as kcat increases in magnitude, the enzyme will manifest a concomitant apparent loss in affinity (i.e., increase in KM), since they are both not expected to improve simultaneously. This same mechanism will also manifest as the apparent kcat/KM not rising in tandem with the same magnitude as kcat alone due to the increase in KM; see, for example, Table 1. Initial experiments incorporated cages assembled with designated ratios of 4-QD/cage and a further 8-PTE/QD did not show any significant increases in kcat in comparison to just the 8-PTE/QD without the DNA cage present (data not shown). Further, increasing either the ratio of PTE per QD or QD-PTE per cage in these constructs did not significantly alter any of these observed apparent rates. Subsequent experiments increased just the ratio of DNA cage relative to the QD-PTE component during the assembly phase. DNA cage-540 QD-PTE composites, where the ratio of cage to QD-PTE was increased from 4 QD per cage with 8 PTE/QD to 4 cage/QD with 8 PTE/QD, demonstrated significant improvements in kinetic activity as compared to that seen for QD-PTE alone (Table 1). For example, Vmax increased ca. threefold from 0.3 to 1 μM/s, while kcat similarly increased from 101 to 355 s−1. With added DNA, the cage-QD-PTE KM value doubled from 129 to 219 μM (poorer affinity again), enzyme efficiency (kcat/KM) doubled from ∼0.8 to 1.6 μM−1 s−1, and PTE specific activity (SA) increased ca. 3.5-fold. We qualify all kinetic descriptors as apparent values, since it remains unclear if enzymatic activity at an NP interface can be fully described with the Michaelis−Menten model.6−10,14,19 The Vmax and kcat increases noted for constructs with the larger 625 QDs treated similarly with 4× cage DNA were more modest at ∼55%. Interestingly, KM was improved twofold (decreased) in this scenario, which led to an overall threefold increase in kcat/KM from 0.76 to 2.33 μM−1 s−1; the latter value is almost twofold better than the native enzyme alone. Changes to the SA were also modest for the 625 QD with increased cage presence at approximately twofold. Continuing by increasing DNA cage ratios to 8, 16, and 32× that of the 540 QD-PTE during self-assembly resulted in an overall order of magnitude increase in Vmax and a more than 12.5-fold increase in kcat (Table 1). In conjunction KM also increased more than 10-fold as did the SA. Similar increases were also observed with the 625 QDs. These were again more modest and, interestingly, showed a biphasic profile with the results for 4, 8, and 16× DNA appearing somewhat intermediate to that
monomeric structures could be purified by gel or using dialysis, although this would tend to be at the cost of overall yield. The agarose gel in Figure 2A-right confirms via changes in QD migration that cages incorporating the His5 do indeed attach to QDs; cages lacking the His5 did not alter the QD migration rate (Supporting Information Figure S2). The structural and steric constraints of the DNA cage design (Figure 1A) suggest that it should ideally bind and display four QDs on its periphery, while each QD can at most bind to two cages and potentially link them together. It is unclear if such binding of two cages to a given QD would preclude its nearest-neighbor QDs from also binding and cross-linking to multiple cages. It is suggested that these constraints, and the fact that both the QD and the DNA are strongly negatively charged, along with the complexity of such hybrid organic−inorganic composites, are the reason for the small changes in QD migration noted when interacting with the DNA cages in Figure 2. Atomic force microscopy (AFM) imaging of cages with/without QDs present also confirmed assembly (Supporting Information Figure S3). Transmission electron microscopy (TEM) micrographs collected from 1:4 cage/625 QD structures (Figure 2B) reveal QD assemblies with what appear to be predominantly three or four QDs per cluster as expected. The lack of DNA contrast and flattening of the assemblies with concomitant shape distortion that follows drying on the TEM grids makes it hard to ascertain exact formation statistics for these structures beyond visual estimates. Ratiometric PTE self-assembly to the preformed DNA-QD structures by metal-affinity coordination was verified by monitoring migration changes during agarose gel electrophoresis (Supporting Information Figure S2). No evidence of PTE displacing cage from the QDs was observed. Simulations estimated maximum PTE packing densities of ∼13 and ∼28 PTE for the 540 (surface area ∼66 nm2) and 625 QDs (∼270 nm2), respectively.9,14 With the ability to self-assemble the three-component DNA cage-QD-PTE composite structures, we next investigated their catalytic properties using paraoxon as substrate. As shown in Figure 3A,B (black and green symbols) and Table 1, assaying both 540 and 625 nm QDs assembled with eight PTE versus the PTE alone confirmed that the initial rate and kcat doubled and mirrored previous enhancement results for similar QDs assembled with the same ratio of PTE.9 As before, the corresponding KM values increased (i.e., decreased affinity) by approximately three- to fourfold. This is believed to arise since kcat and KM are linked mechanistically as part of the Michaelis− D
DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
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Figure 4. TEM micrographs of DNA cage-QD-PTE structures assembled with 16-fold excess DNA cage per 625 nm QD. DNA was counterstained with uranyl acetate for contrast as described (Supporting Information) and is visible as the gray shadow around the QDs. (A, B) Clusters with correct stoichiometry or slight excess of QD and/or DNA (incorporating ∼15% of the total QDs present in sample). (C, D) Higher-order clusters with more QD or DNA present (∼25% of sample). (E, F) Larger clusters−lower resolution (∼60% of sample).
increase the rate of product formation over time becomes readily apparent here. As before, increasing the PTE ratio per QD beyond 8 in these composites actually lowered catalytic activity toward the solution phase values (Supporting Information Figure S4). We suggest this to arise from an increasing component of free solution-phase PTE dominating the assay’s catalytic profile as noted previously.9,10,14,19,20 Previous mechanistic studies into how PTE activity is enhanced when displayed at a QD interface revealed that a major contributor was alleviation of the enzyme’s rate-limiting step, that is, the rate of enzyme−product release.9,10 This presumably arises as a function of the NP’s universal ability to structure its surrounding environment.6,8,19,24 Although still mostly uncharacterized, it is postulated that such structuring, in conjunction with the influence of the NP-solution interface, gives rise to complex physicochemical phenomena such as pH and ionic gradients, changes in viscosity and density, phase separation, and strong counter charge layers.6,8,19,24 Moreover, these may extend to a distance twice the NP diameter or more.6,8,19,24 Elucidating how the presence of excess DNA cages serves to further augment enzymatic enhancement in these multicomponent biological-inorganic composites is clearly a nontrivial undertaking. Some insight, however, can
of the 32× cage construct (Figure 3B); this is also similar to resulting patterns observed when increasing the ratio of PTE to QD without DNA present.9 Moreover, the values observed with 4, 8, and 16× excess DNA cage present were not radically different. We did note that the SA for the QD-PTE constructs were essentially the same for the 540 and 625 QD samples at 82.1 ± 2.5 and 80.3 ± 14.5 μmol min−1 mg−1, respectively. However, the addition of approximately four DNA cage to the sample assembly increased the SA ca. 3.3 times to 276.9 ± 0.1 μmol min−1 mg−1 for the 540 QDs and 1.9 times to 151.0 ± 7.7 μmol min−1 mg−1 for the 625 QDs. The origin of this difference is currently unknown, but we speculate it to arise from the complex interplay of QD size and curvature on the displayed PTE activity in conjunction with chemical effects from the surrounding DNA. This also suggests that the synergistic enhancement of PTE activity may be influenced in many still-to-be resolved ways. Figure 3C presents some representative data in the form of a progress curve plot of enzyme time. Raw progress curve data versus time were converted into enzyme time ([E] × t) with units of nanomolar seconds as described.21−23 Here, enzyme time for PTE alone, 540 QD-PTE, and 540 QD-PTE in the presence of 8× cage are plotted versus the concentration of pNP product formed. The ability of the latter to dramatically E
DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
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alone only produced the previously reported and more modest increases.9,14 Arrhenius analysis of PTE activity in these structures again confirmed that the enhancement did not arise from a change to the enzyme’s energy of activation (Supporting Information Figure S6).9 As before, the greatest enhancements in kinetic activity are more often noted for the smaller-sized QD materials.9,10,14,28 This presumably arises from the interweaving of the unique structured environments found around the participating DNA and NP materials. In the case of β-galactosidase displaying multiple QDs around itself due to its large tetrameric size, it was initially postulated that such environments can promote substrate sequestration and lead to high local concentrations of substrate around the NP complex.29 However, more detailed studies and a preliminary kinetic model of that activity suggested that such substrate accumulation effects would be minimal and only transiently enhance the very initial rates.10 In conjunction with the previous literature, the current results suggest a very complex interplay of processes whereby the enzyme is both stabilized along with having its rate-limiting step alleviated. As witnessed here, the complex nature of these composite materials does not lend itself to easy physicochemical characterization; the same will probably be true for elucidating the kinetic underpinnings of this enhancement. Future work will focus on trying to elucidate a cage ratio/structure that maximizes enzyme performance. Despite this, the ability to stabilize and increase an enzyme’s activity by an order of magnitude just by altering its local presentation environment (with added DNA in this case) is certainly worth pursuing. PTE is undergoing concerted development for potential use in bioremediation of nerve agents,18,30 and this is directly dependent upon the ability to both produce it in mass quantities and stabilize and store it long-term, all while enhancing its relative activity. The current results suggest that some type of NP-polymer hybrid that is structurally and functionally analogous to that described here may certainly be worth further investigation. Further, the assembly chemistry demonstrated here is amenable to testing with many other recombinantly expressed enzymes. Another potential payoff that may be especially relevant is one where multiple enzymes can be brought together in this manner to engage in enhanced multistep biocatalysis.5,19,31
be drawn both from the literature and from some initial investigatory experiments. Comparative analysis among various examples of enzymatic enhancement when attached to DNA scaffolds suggests that the nucleic acid chemistry structures its surrounding environment in a manner similar to that postulated for NPs. In particular, it has been argued that the pH near the negatively charged DNA nanostructures is lower than that found in the surrounding bulk solution, and this creates a more optimal pH environment for enzymes that are proximal to or anchored to the DNA scaffold.25,26 It is possible that a similar phenomenon is at work here or contributing to some extent. However, the complexity of the NP interface, and its exceedingly small physical presence, does not readily lend itself to direct interfacial pH measurements.27 For experimental insight into the underlying enhancement, we looked at the SA of selected constructs as the concentration of each assembly was serially diluted from stock toward the final assay concentrations; see Figure 3D. Comparing the activity of decreasing concentrations of enzyme in this format can provide information on whether they are being destabilized/dissociating and start to lose activity at low concentrations, which is typically seen with multimeric enzymes.21,22 The free PTE, 540 QD-PTE, and 540 QDPTE in the presence of 4× cage samples show very little loss in their SA (average of 23 ± 2.6%) while being diluted over this range. More interesting are the results from the free PTE with just DNA cage, which shows an ∼72% loss in SA. Overall, this plot clearly shows that the combination of PTE attachment to QD enhances the enzyme’s SA, and the addition of excess DNA cage in this assembly enhances that even further in a synergistic manner. The ability of the DNA alone to enhance PTE activity is not as profound nor as stable over this concentration range, although it is clearly trending upward at the highest concentrations of 7.5 and 15 nM. Higher concentrations could not be reliably measured to follow this trend, as the assay activity proceeded far too rapidly to provide meaningful results. Nevertheless, it does suggest an avenue for further investigation into the subtleties that surround enhancement and DNA concentration. DNA cage-QD-PTE structures assembled with excess cage were next interrogated by dynamic light scattering (DLS); however, the resulting data indicated a complex wide-ranging mixture of conglomerate sizes and provided no useful data. Direct TEM analysis of QD structures assembled in the presence of 16× cage with the DNA counterstained proved to be more informative. As shown by the representative micrographs in Figure 4, a range of QD-DNA cluster structures are visible including those with four or slightly more QDs (top) along with higher order structures having seven or more QDs present (middle) or much higher numbers of QDs (bottom). More importantly, the close-up images show the presence of a significant amount of excess stained DNA surrounding each QD cluster. This indicates that the excess DNA is both acting to cross-link individual cage-QD assemblies and also forming a mesh that surrounds each of the resulting QD clusters. This excess DNA mesh was not seen for structures having the original stoichiometry of 4QD per DNA cage; see Figure 2B. Overall, these results confirm the NP and DNA can act synergistically to enhance enzyme activity. Excess DNA alone did not result in any significant PTE enhancement over the relevant concentrations tested here, while attachment to QD
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00933. Detailed experimental information and additional
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supporting data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kimihiro Susumu: 0000-0003-4389-2574 Eunkeu Oh: 0000-0003-1641-522X Scott A. Walper: 0000-0002-9436-3456 Igor L. Medintz: 0000-0002-8902-4687 Present Addresses ∇
(A.S.) Ramakrishna Mission Vidyamandira, Belur Math, Howrah, WB-711202, India.
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DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
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(16) Blanco-Canosa, J.; Wu, M.; Susumu, K.; Petryayeva, E.; Jennings, T. L.; Dawson, P. E.; Algar, W. R.; Medintz, I. L. Recent Progress in the Bioconjugation of Quantum Dots. Coord. Chem. Rev. 2014, 263−264, 101−137. (17) Hess, K. L.; Tostanoski, L. H.; Andorko, J. I.; Susumu, K.; Deschamps, J. R.; Medintz, I. L.; Jewell, C. M.; Oh, E. Engineering Immunological Tolerance Using Quantum Dots to Tune the Density of Self-Antigen Display. Adv. Funct. Mater. 2017, 27, 1700290. (18) Tsai, P. C.; Fox, N.; Bigley, A. N.; Harvey, S. P.; Barondeau, D. P.; Raushel, F. M. Enzymes for the Homeland Defense: Optimizing Phosphotriesterase for the Hydrolysis of Organophosphate Nerve Agents. Biochemistry 2012, 51, 6463−6475. (19) Vranish, J. N.; Ancona, M. G.; Walper, S. A.; Medintz, I. L. Pursuing the Promise of Enzymatic Enhancement with Nanoparticle Assemblies. Langmuir 2018, 34, 2901−2925. (20) Hondred, J. A.; Breger, J. C.; Garland, N. T.; Oh, E.; Susumu, K.; Walper, S. A.; Medintz, I. L.; Claussen, J. C. Enhanced Enzyme Activity of Phosphotriesterase Trimer Conjugated on Gold Nanoparticles for Pesticide Detection. Analyst 2017, 142, 3261−3271. (21) Selwyn, M. J. A Simple Test for Inactivation of an Enzyme During Assay. Biochim. Biophys. Acta, Enzymol. Biol. Oxid. 1965, 105, 193−195. (22) Cornish-Bowden, A. Fundamentals of Enzyme Kinetics, 4th ed.; John Wiley & Sons: Hoboken, NJ, 2013. (23) Diaz, S. A.; Sen, S.; Boeneman Gemmill, K.; Brown, C. W., III.; Oh, E.; Susumu, K.; Stewart, M. H.; Breger, J. C.; Aragones, G. L.; Field, L. D.; Deschamps, J. R.; Kral, P.; Medintz, I. L. Elucidating Surface Ligand-Dependent Kinetic Enhancement of Proteolytic Activity at Surface-Modified Quantum Dots. ACS Nano 2017, 11, 5884−5896. (24) Zobel, M.; Neder, R. B.; Kimber, S. A. Universal Solvent Restructuring Induced By Colloidal Nanoparticles. Science 2015, 347, 292−294. (25) Rabe, K. S.; Muller, J.; Skoupi, M.; Niemeyer, C. M. Cascades in Compartments: En Route to Machine-Assisted Biotechnology. Angew. Chem., Int. Ed. 2017, 56, 13574−13589. (26) Zhang, Y.; Tsitkov, S.; Hess, H. Proximity Does Not Contribute to Activity Enhancement in the Glucose Oxidase-Horse Radish Peroxidase Cascade. Nat. Commun. 2016, 7, 13982. (27) Sapsford, K. E.; Tyner, K. M.; Dair, J. B.; Deschamps, J. R.; Medintz, I. L. Analyzing Nanomaterial Bioconjugates: A Review of Current and Emerging Purification and Characterization Techniques. Anal. Chem. 2011, 83, 4453−4488. (28) Claussen, J. C.; Malanoski, A.; Breger, J. C.; Oh, E.; Walper, S. A.; Susumu, K.; Goswami, R.; Deschamps, J. R.; Medintz, I. L. Probing the Enzymatic Activity of Alkaline Phosphatase Within Quantum Dot Bioconjugates. J. Phys. Chem. C 2015, 119, 2208−2221. (29) Brown, C. W., III; Oh, E.; Hastman, D. A., Jr.; Walper, S. A.; Susumu, K.; Stewart, M. H.; Deschamps, J. R.; Medintz, I. L. Kinetic Enhancement of the Diffusion-Limited Enzyme Beta-Galactosidase When Displayed with Quantum Dots. RSC Adv. 2015, 5, 93089− 93094. (30) Alves, N. J.; Moore, M.; Johnson, B. J.; Dean, S. N.; Turner, K. B.; Medintz, I. L.; Walper, S. W. Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase. ACS Appl. Mater. Interfaces 2018, 10, 15712−15719. (31) Vranish, J. N.; Ancona, M. G.; Oh, E.; Susumu, K.; Medintz, I. L. Enhancing Coupled Enzymatic Activity by Conjugating One Enzyme to a Nanoparticle. Nanoscale 2017, 9, 5172−5187.
(N.B.) Department of Materials Science and Engineering, McMaster University, Hamilton, Ontario, Canada. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Office of Naval Research (ONR), Naval Research Laboratory (NRL), and the NRL-Nanosciences Institute for financial support. I.L.M. acknowledges United States Department of Agruculture Grant No. 201667021-25038.
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DOI: 10.1021/acsanm.8b00933 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX
