Article pubs.acs.org/cm
Gold Nanorod-Mediated Photothermal Enhancement of the Biocatalytic Activity of a Polymer-Encapsulated Enzyme Sirimuvva Tadepalli,† Jieun Yim,† Keerthana Madireddi,† Jingyi Luan,† Rajesh R. Naik,*,‡ and Srikanth Singamaneni*,† †
Institute of Material Science and Engineering and Department of Mechanical Engineering and Material Science, Washington University in St. Louis, St. Louis, Missouri 63130-4899, United States ‡ 711th Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433, United States S Supporting Information *
ABSTRACT: The rational integration of biomolecules and functional nanostructures can enable remote-controlled biological processes such as molecular transport, catalysis, and molecular recognition. The photothermal ability of plasmonic nanostructures is highly attractive for optically modulating biomolecular processes such as biocatalysis. However, the studies pertaining to the photothermal enhancement of enzyme activity are mostly limited to thermophilic enzymes because of the thermal denaturation and loss of the activity of conventional enzymes at elevated temperatures. The lack of effective strategies for preserving the activity of immobilized enzymes at elevated temperatures hinders the potential use of plasmonic nanostructures as nanoheaters for the photothermal enhancement of enzyme activity. Here, we demonstrate a simple and highly effective strategy for stabilizing enzymes immobilized on plasmonic nanostructures by encapsulating them through in situ polymerization. Apart from enhanced thermal and biological stability, the encapsulation strategy provides enhancement of enzyme activity with an external optical trigger. The encapsulation strategy demonstrated here can be a highly attractive approach for designing remote-controlled biomolecular reactions.
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INTRODUCTION Interfacing biomolecules with functional synthetic materials provides the ability to remotely control the functionality of the biomolecules. Remote-controlled biocatalysis can potentially be very attractive to the chemical industry and a number of applications, including biosensing and imaging, cell-free biosynthesis, nanotherapeutics, and bioelectronics.1−3 While there have been reports of using plasmonic nanostructures as functional supports for enzymes, their multifunctional capabilities have not been fully exploited for enhancing enzyme catalysis.4,5 Apart from their ability to confine light to the nanoscale, plasmonic nanostructures also exhibit excellent photothermal properties involving the highly efficient conversion of light into heat, enabling them to act as nanoscale heating elements.6,7 Photothermal properties of plasmonic nanostructures are being widely investigated for their potential applications in locoregional cancer therapy.8−15 However, the use of a photothermal effect to enhance or modulate enzyme catalysis has been mostly limited to thermophilic enzymes.16,17 Photothermal heating of “normal” enzymes such as horseradish peroxidase will result in the loss of biocatalytic activity due to the thermal denaturation of the enzyme at elevated temperatures.18 The integration of enzymes with photothermal functional supports calls for innovative methods for stabilizing immobilized enzymes at elevated temperatures. Despite the growing demand for environmentally friendly, sustainable, and externally controllable biocatalysts, particularly in the chemical, pharmaceutical, food, textile, and paper © 2017 American Chemical Society
industries, the major bottleneck for the industrial applications of enzymes is their poor long-term operational stability and difficultly in the recovery and reusability of enzymes.19−24 Enzyme stabilization strategies that have been used can be broadly classified into three main categories: (i) design of intrinsically stable enzymes, (ii) chemical modification, and (iii) enzyme immobilization.25−27 Although intrinsically stable enzymes are applicable in a variety of harsh environments, the design of intrinsically stable enzyme is extremely laborious and time-consuming.28 The chemical modification of an enzyme is a tedious process and requires separate modification of each enzyme and cannot be used after irreversible denaturation.29 Enzyme immobilization can overcome these limitations by providing reusability, high enzyme recovery, and tailored enzyme activity.30,31 However, enzyme immobilization suffers from mass transfer limitations, leakage of enzymes, operational restraints, and the loss of enzyme activity. A novel approach for enzyme stabilization involves immobilization on a support (carrier) and entrapment (encapsulation) in a protective matrix.32,33 Enzymes are immobilized on supports through noncovalent interactions (e.g., hydrophobic and van der Waals) as covalent interactions render both the enzyme and the support unusable after the irreversible deactivation of the enzyme. On the other hand, physical interactions are generally Received: April 13, 2017 Revised: July 7, 2017 Published: July 10, 2017 6308
DOI: 10.1021/acs.chemmater.7b01527 Chem. Mater. 2017, 29, 6308−6314
Article
Chemistry of Materials
Figure 1. (A) Schematic illustration showing the polymer encapsulation strategy for enzyme stabilization. (B) Representative TEM image of AuNRs employed in this study. (C) Extinction spectrum of AuNRs showing a red shift in the LSPR wavelength after the adsorption of HRP. The inset shows dynamic light scattering measurements indicating an increase in the hydrodynamic size due to the adsorption of HRP on AuNRs. (D) Extinction spectra of AuNRs following each step in the polymer encapsulation process. (E) LSPR wavelength shift of each step during the polymer encapsulation process showing the release and recapture of the enzyme. (F) Atomic force microscopy image of the polymer-encapsulated enzyme adsorbed on the AuNRs. The Z scale is 50 nm. (G) Raman characterization of the AuNR−HRP conjugates before and after polymerization showing the characteristic peaks.
dependent adsorption of horseradish peroxidase (HRP) on gold nanoparticles modified with positively and negatively charged polyelectrolytes and found that positively charged nanostructures yield higher biocatalytic activity.38 On the basis of those findings, we have employed positively charged Au nanorods in this study. AuNRs with a length of 63.5 ± 1.5 nm and a diameter of 12.2 ± 0.5 nm are synthesized using a seedmediated approach (Figure 1B).39 The adsorption of HRP on the AuNR resulted in a red shift of ∼8 nm in the localized surface plasmon resonance (LSPR) wavelength, corresponding to an increase in the refractive index of the medium surrounding AuNR (Figure 1C). Dynamic light scattering measurements revealed that the hydrodynamic size of the AuNRs increased by ∼6 nm, suggesting the adsorption of a monolayer of HRP on AuNR (Figure 1C).40 The bionanoconjugate solution is filtered to remove the unconjugated HRP. The filtered bionanoconjugates are uniformly adsorbed on a glass substrate (Figure S1). The adsorbed AuNR−HRP substrates are catalytically active as verified by a colorimetric assay based on 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Figure S2). Figure 1A shows the various steps involved in the organosilica-based preservation of the biocatalytic activity of
too weak to keep the enzyme bound to the carrier under harsh industrial conditions that include high reactant and product concentrations. To prevent the leakage of enzymes, the entrapment or encapsulation of the enzyme in a polymeric network provides physical restraints. Typically, synthetic polymer matrices such as polyacrylamide, silica sol−gel, or biopolymers such as silk are used for enzyme encapsulation.34−37 Here, we demonstrate the photothermal enhancement of the biocatalytic activity of enzymes immobilized on plasmonic nanoheaters. The enhancement in the activity is enabled by the encapsulation of the immobilized enzyme by an in situpolymerized porous organosilica layer, which renders thermal and biological stability. We investigated the effect of the thickness of the organosilica layer on the thermal stability and catalytic activity of the immobilized enzyme. The in situpolymerized organosilica layer serves as a molecular imprint, enabling the release and recapture of the enzyme on demand.
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RESULTS AND DISCUSSION We used gold nanorods (AuNRs) as optically excitable nanoscale heating elements for the immobilization of enzymes. In our previous reports, we have investigated the charge6309
DOI: 10.1021/acs.chemmater.7b01527 Chem. Mater. 2017, 29, 6308−6314
Article
Chemistry of Materials
Figure 2. (A) LSPR wavelength shift upon the formation of a capping layer on the recaptured enzyme after different polymerization times. (B) Relative activity of the polymer-encapsulated enzyme after the formation of the capping layer for different polymerization times. Error bars indicate the standard deviation from three independent measurements. (C) Retained activity of the polymer-encapsulated enzyme after it had been subjected to heat treatment at 55 °C for 1 h. Error bars indicate the standard deviation from three independent measurements. (D) Retained activity of the polymer-encapsulated enzyme after it had been subjected to proteolytic degradation, suggesting that complete encapsulation prevents the protease from accessing the enzyme. Error bars indicate the standard deviation from three independent samples.
layer is formed on the surface of the enzyme to complete the encapsulation process. Each step in the polymer encapsulation procedure is monitored by the LSPR shift corresponding to the change in the refractive index around the plasmonic nanostructures. Extinction spectra of AuNR were obtained following each step in the encapsulation procedure: immobilization of AuNR−HRP conjugates on glass substrates (step 1), polymerization of the silane monomers (step 2), removal of the enzyme (step 3), and capture of fresh enzyme (step 4) (Figure 1D). The LSPR wavelength of the AuNR exhibited a red shift (∼8.5 nm) corresponding to the formation of an organosilica layer around the NR−HRP conjugate, a red shift (∼0.5 nm) due to the formation of a PEG layer, and a blue shift (∼5.5 nm) corresponding to the release of the enzyme. These steps are followed by rebinding of the enzyme (red shift of ∼5.5 nm) and the formation of a capping layer (red shift of ∼1.5 nm), which exhibit a progressive red shift due to the increase in the refractive index (Figure 1E). To confirm that the biomolecular imprints were specific to HRP, we measured the LSPR wavelength shift upon exposure of the HRP imprints to interfering proteins, bovine serum albumin (BSA), and glucose oxidase (GOx). We found that the LSPR wavelength shift corresponding to the exposure of the imprints to 0.1 mg/mL HRP (∼6 nm) is significantly larger than that obtained from exposure to interfering proteins BSA (0.25 mg/mL) and GOx (0.25 mg/mL), which show LSPR wavelength shifts of ∼1 and ∼0.5 nm, respectively (Figure S3). Apart from the comple-
bionanoconjugates. Following the immobilization of the bionanoconjugates on a glass substrate, an organosilica encapsulation layer is formed through copolymerization of trimethoxypropylsilane (TMPS) and (3-aminopropyl)trimethoxysilane (APTMS) around the AuNR−HRP surface. The ethoxy group of APTMS and the methoxy group of the TMPS undergo rapid hydrolysis to form ethanol, methanol, and trisilanols.41 Because of the subsequent condensation of the silanols, an aminopropyl functional amorphous polymer is formed with Si−O−Si bonds that results in a soft organosilica layer.34 The organosilica layer formed around the enzyme bears amine (-NH3+), hydroxyl (-OH), and methyl (-CH3) functional groups that provide concerted weak interactions, namely, electrostatic, hydrogen bonding, and hydrophobic interactions. The organosilica layer is PEGylated to ensure that there is no nonspecific binding during recapture of the enzyme (discussed below).42 The enzyme, which is bound to the surface of the AuNR through noncovalent interactions, is removed by exposure to sodium dodecyl sulfate (SDS). The removal of the enzyme results in cavities within the organosilica layer that are complementary in shape and chemical functionality to the enzymes. The polymer imprints are left to cure at room temperature to increase the porosity of the organosilica layer for an enhanced diffusion of the substrate molecules and to provide conformational flexibility to the enzyme critical for the catalytic activity.34 Subsequently, the enzyme is recaptured by virtue of the molecular recognition of the imprints within the organosilica layer. In the final step, a thin organosilica capping 6310
DOI: 10.1021/acs.chemmater.7b01527 Chem. Mater. 2017, 29, 6308−6314
Article
Chemistry of Materials
Figure 3. (A) Relative activity of immobilized and polymer-encapsulated HRP over multiple cycles of catalysis. Error bars indicate the standard deviation from three independent samples. (B) Relative activity of the polymer-encapsulated enzyme over multiple cycles of release and recapture. Error bars indicate the standard deviation from three independent measurements.
structure) and lose their bioactivity. In other words, the concerted nonspecific interactions (van der Waals, electrostatic, and hydrogen bonding) between the enzyme and the organosilica capping layer restrict the mobility of the biomolecule, impeding denaturation even under extreme conditions.34,37 Note that both bionanoconjugates and organosilica-encapsulated bionanoconjugates were exposed to a single round of catalysis before measuring the thermal stability to remove the effect of loss in enzymes during catalysis, as discussed below. We probed the biological stability of the organosilicastabilized enzyme by subjecting the encapsulated and uncapped enzyme to proteolytic degradation (see the Materials and Methods in the Supporting Information for details). The activity of the NR−HRP conjugate without an organosilica shell decreased to ∼5%, while the polymer-encapsulated enzyme retained ∼90% of the activity, suggesting that the organosilica shell hinders the accessibility of the immobilized enzyme to protease providing excellent biological stability toward proteolytic digestion (Figure 2D).43 Thus, the polymerencapsulated enzyme can be used in harsh industrial settings and can be used regardless of the storage condition. We also probed the reusability of the polymer-encapsulated enzyme over multiple rounds of catalytic reactions. The polymer-encapsulated enzyme (capping layer corresponding to a 5 min polymerization) retained ∼60% of the activity after 10 cycles of catalysis, whereas the enzyme without polymer encapsulation exhibited
