Full Paper www.afm-journal.de Self-Assembly of Mechanoplasmonic Bacterial Cellulose– Metal Nanoparticle Composites Olof Eskilson, Stefan B. Lindström, Borja Sepulveda, Mohammad M. Shahjamali, Pau Güell-Grau, Petter Sivlér, Mårten Skog, Christopher Aronsson, Emma M. Björk, Niklas Nyberg, Hazem Khalaf, Torbjörn Bengtsson, Jeemol James, Marica B. Ericson, Erik Martinsson, Robert Selegård, and Daniel Aili* 1. Introduction Nanocomposites of metal nanoparticles (NPs) and bacterial nanocellulose (BC) enable fabrication of soft and biocompatible materials for optical, Templated synthesis or deposition of metal nanoparticles (NPs) in nanocellulose can catalytic, electronic, and biomedical applications. Current BC–NP nanocom- enable development of cellulose-based posites are typically prepared by in situ synthesis of the NPs or electrostatic nanocomposites with novel optical,[1,2] adsorption of surface functionalized NPs, which limits possibilities to control electronic,[3,4] catalytic,[5,6] and biophys- and tune NP size, shape, concentration, and surface chemistry and influ- ical properties.[7,8] Nanocellulose can be ences the properties and performance of the materials. Here a self-assembly derived from plant sources but can also be strategy is described for fabrication of complex and well-defined BC–NP produced by bacteria.[9] Bacterial nanocel- lulose (BC) is an exopolysaccharide chemi- composites using colloidal gold and silver NPs of different sizes, shapes, and cally identical to plant cellulose, produced concentrations. The self-assembly process results in nanocomposites with and secreted by aerobic Gram-negative distinct biophysical and optical properties. In addition to antibacterial mate- bacteria, such as Komagataeibacter xylinus rials and materials with excellent senor performance, materials with unique (formerly Gluconacetobacter xylinus).[10,11] mechanoplasmonic properties are developed. The homogenous incorpora- BC forms highly hydrated and entangled 3D networks of well-defined nanocel- tion of plasmonic gold NPs in the BC enables extensive modulation of the lulose fibrils that are about 40 to 60 nm optical properties by mechanical stimuli. Compression gives rise to near-field in diameter, 1 to 10 µm in length and a coupling between adsorbed NPs, resulting in tunable spectral variations and degree of crystallinity reaching 90%.[12] enhanced broadband absorption that amplify both nonlinear optical and ther- In contrast to plant-derived cellulose, moplasmonic effects and enables novel biosensing strategies. BC is not contaminated with lignin and hemicellulose[13,14] and can be utilized to O. Eskilson, Dr. M. M. Shahjamali, P. Sivlér, M. Skog, Dr. C. Aronsson, Dr. M. M. Shahjamali N. Nyberg, Dr. E. Martinsson, Dr. R. Selegård, Prof. D. Aili School of Engineering and Applied Sciences Laboratory of Molecular Materials Harvard University Division of Biophysics and Bioengineering 9 Oxford Street, Cambridge, MA 02138, USA Department of Physics, P. Güell-Grau Chemistry and Biology (IFM) Instituto de Microelectrónica de Barcelona (IMB-CNM, CSIC) Linköping University Campus UAB, Bellaterra, Barcelona 08193, Spain Linköping SE-581 83, Sweden Dr. E. M. Björk E-mail: daniel.aili@liu.se Nanostructured Materials Prof. S. B. Lindström Department of Physics, Division of Solid Mechanics Chemistry and Biology (IFM) Department of Management and Engineering (IEI) Linköping University Linköping University Linköping SE-58183, Sweden Linköping SE-581 83, Sweden Dr. H. Khalaf, Prof. T. Bengtsson Dr. B. Sepulveda Cardiovascular Research Centre Catalan Institute of Nanoscience and Nanotechnology (ICN2) School of Medical Sciences CSIC and BIST Örebro University Campus UAB, Bellaterra, Barcelona 08193, Spain Örebro SE-70362, Sweden The ORCID identification number(s) for the author(s) of this article J. James, Prof. M. B. Ericson can be found under https://doi.org/10.1002/adfm.202004766. Biomedical Photonics Group Department of Chemistry and Molecular biology © 2020 The Authors. Published by Wiley-VCH GmbH. This is an open University of Gothenburg access article under the terms of the Creative Commons Attribution Gothenburg SE-412 96, Sweden License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1002/adfm.202004766 Adv. Funct. Mater. 2020, 30, 2004766 2004766 (1 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de produce pure nanocellulose membranes with high mechanical face functionalization of the NPs, which reduces the versatility strength, large water-holding capacity and large specific surface of the materials. area.[9] In addition, BC is nontoxic, biodegradable, and inert Herein, we describe a generic self-assembly strategy for to human metabolism and demonstrates excellent biocompat- tunable functionalization of hydrated BC membranes with ibility.[15] BC is thus an attractive and versatile material for a presynthesized colloidal NPs (Figure 1a), that facilitates devel- wide range of applications and has, among others, been utilized opment of advanced BC-based nanocomposite materials. The as a scaffold for tissue engineering[16,13] and as a soft substrate self-assembly process was optimized to proceed under ambient for biosensors[2] and bioelectronics.[17] BC is also a material of conditions for both citrate- and surfactant-stabilized NPs of dif- great interest for wound care applications and BC-based wound ferent shapes and compositions. The interactions involved was dressings have shown promising results in the clinic for treat- investigated in detail and the resulting nanocomposite mate- ment of burns and nonhealing wounds.[18,19] rials could be tailored to demonstrate antimicrobial properties, Gold (AuNP) and silver (AgNP) nanoparticles demonstrate excellent refractive index sensing performance, and enhanced unique optical, antibacterial, and catalytic properties, and a thermoplasmonic and nonlinear optical effects. In addition, large number of methods have been developed for templated the homogenous incorporation of AuNPs in the BC allowed in situ synthesis of these NPs in BC for fabrication of func- for modulation of the plasmonic near-field coupling in three tional materials.[2,20] The porous structure of the fibrillar BC dimensions by mechanical deformation of the BC membranes. network enables diffusion of metal ions and reducing agents By compressing the hydrated BC–AuNP nanocomposites, the into the material where the ether oxygen and hydroxyl groups decrease in separation between the adsorbed NPs resulted in of cellulose can sequester the ions and stabilize the resulting plasmonic near-field coupling. The induced optical shifts and metal NPs.[21] Although the cellulose can also contribute to the peak broadening can be directly correlated to the mechanical reduction of the metal ions,[2,22] reducing agents such as borhy- pressure and give rise to both amplified nonlinear optical dride, hydrazine, hydroxylamine, ascorbic acid,[23] and trietha- effects and enhanced thermoplasmonic heating. Moreover, nolamine[24] are widely used, as well as microwave[25] and UV- adsorption of proteins on the AuNPs introduces a steric barrier radiation.[26] In order to prevent NP aggregation after synthesis, that reduces the magnitude of the spectral changes, which ena- various polymers can be added to stabilize the NPs.[23] Possibili- bles a novel optical biosensing readout strategy using mechan- ties to chemically modify the BC with a reducing agent, such ical stimuli. This versatile strategy for functionalization of BC as amidoxime,[6] or by adsorption of poly(ethyleneimine)[22] with metal NPs can facilitate fabrication of multifunctional soft have also been explored. Modifying the BC or adding reducing nanomaterials for a wide range of optical and biomedical appli- agents or other reagents can influence the mechanical, struc- cations, including biosensing and advanced wound care. tural, and chemical properties of the BC.[26] In situ synthesis also occurs under conditions that are difficult to control and there are many aspects relating to the BC that can influence 2. Results and Discussion the outcome of the synthesis, including presence of unknown contaminating species remaining from the fermentation pro- 2.1. Nanoparticle Self-Assembly cess.[27] Consequently, there are limited possibilities to control NP properties, distribution, and concentration.[6] Preparation of Native BC is a nanofibrillar and translucent material (Figure 1b) composites comprising multiple types of nanoparticles can also with a porosity that typically exceeds 0.90,[37] which enables dif- be challenging since the sequential addition of different metal fusion of NPs and macromolecules into the material, although salts and reducing agents would likely result in nanoparticles with a reduced diffusivity. Noninteracting macromolecules (dex- that are undefined with respect to size and composition.[28] tran, 44–260 kDa) have been shown to experience a hindrance In contrast, a large number of strategies for solution- ratio of about 0.4 compared to the theoretical maximum diffu- based synthesis of high-quality colloidal metal NPs have been sion rate.[37] When immersed in an aqueous suspension of cit- developed and optimized to yield NPs of defined sizes,[29] rate-stabilized gold NPs (AuNPs) in the presence of moderate shapes,[30,31] and compositions[32] and with narrow size distri- NaCl concentrations (≈5 × 10−3 m), we observed that hydrated butions.[33] The possibility to use presynthesized colloidal NPs BC membranes over time adopted a pink hue, indicating that in the fabrication of BC-based nanocomposites can potentially AuNPs were adsorbing in the material. The concentration of enable fabrication of more well-defined and complex materials NPs in the membrane continued to increase until nearly all the with a wider range of optical and biophysical properties.[34] To AuNPs in the suspension had adsorbed in the BC, resulting in this end, composites of BC aerogels and presynthesized gold a bright red membrane (Figure 1c and Figure S1, Supporting nanorods (AuNRs) and spherical gold NPs (AuNPs) have been Information) with a well-defined localized surface plasmon res- generated by immersing freeze-dried BC in concentrated sus- onance (LSPR) band (Figure 1d) and an extinction maximum pensions of presynthesized colloidal NPs coated with cationic (λmax) at about 533 and 520 nm for AuNPs with a diameter of 50 surfactants (cetyl trimethylammonium bromide (CTAB))[1] and and 13 nm, respectively. The adsorption process follows pseudo- cationic thiols (4,6-diamino-2-pyrimidinethiol),[35] respectively. first-order kinetics with a rate constant of 0.013 s−1 for 50 nm Because of the capillary effects, the NPs were efficiently taken AuNPs (Figure 1e). After 14 d of incubation, about 90% of all the up in the BC aerogel network and were retained by electrostatic particles in the suspension (75 × 10−12 m) were adsorbed in the interactions between the NPs and the BC. In addition to the membrane. This corresponds to a concentration of 5 × 10−9 m need of drying the BC and the cytotoxicity of CTAB,[36] the pres- confined in the BC membrane. A similar trend with respect ence of surfactants or thiol monolayers complicates further sur- to the adsorption kinetics was also seen for 13 nm AuNPs, Adv. Funct. Mater. 2020, 30, 2004766 2004766 (2 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 1. a) Gold nanoparticles (AuNPs) were adsorbed in bacterial cellulose (BC) membranes to generate soft nanoplasmonic materials. b) SEM micrograph and photograph (inset) of native BC, scale bar: 3 µm. c) BC membranes (bottom of the cuvette) were immersed in aqueous suspensions of 50 nm AuNPs (left) and the adsorption of the AuNPs to the BC resulted in complete depletion of the AuNPs in suspension (right). d) Extinction spectrum and photo of an BC–AuNP composite membrane (ø 6 mm) prepared using 60 × 10−12 m 50 nm AuNP and 120 h incubation. e) Adsorption kinetics of 50 nm AuNPs (75 × 10−12 m) to BC membranes, 5 × 10−3 m NaCl, n ≥ 3, normalized loading is defined as the concentration of AuNP in BC normalized with respect to the initial AuNP concentration in suspension. Error bars show standard deviations. f) SEM of BC–AuNP composite after 120 h incubation and 75 × 10−12 m 50 nm AuNPs, scale bar: 3 µm, scale bar inset: 500 nm. g) SEM of a BC cross-section indicating homogenous adsorption of AuNPs in the BC, scale bar: 1 µm. h) SEM and photo (inset, left) of BC–AuNP composite (ø 6 mm) prepared using concentrated 13 nm AuNP suspension (14.6 × 10−9 m) and 14 d incubation, scale bar: 3 µm, scale bar inset: 500 nm. showing a rate constant of k = 0.008 s−1 (Figure S2, Supporting LPSR band (Figure 1d). Aggregation results in a larger redshift Information). Interestingly, for both sizes of NPs essentially all (>10 nm) and broadening of the LSPR band due to plasmonic available AuNPs were adsorbed in the BC membranes, resulting coupling,[40] which was not seen here. The specific surface area in an almost complete depletion of the AuNPs from the suspen- of the BC was about 75 m2 g−1 as determined using nitrogen sion (Figure 1c). Since the adsorption process was diffusion lim- adsorption (Figure S5, Supporting Information). Based on ited, increasing nanoparticle concentration and applying gentle the average dry mass of the BC membranes (0.36 ± 0.05 mg, agitation significantly increased the rate of adsorption. The ø 6 mm), the surface concentration of AuNPs on the fibrils is composites used in subsequent experiments were prepared by estimated to 1.3 × 1012 m−2 after 120 h incubation with 50 nm incubating BC for about 12 h under conditions optimizing the AuNP at an initial concentration of 60 × 10−12 m, which cor- adsorption rate, as detailed in the experimental section. respond to a surface coverage of about 0.3% if assuming a Scanning electron microscopy (SEM) images of the homogenous distribution of AuNPs within the membranes. BC–AuNP composites show that the NPs were homogenously Hence, large amounts of AuNPs can be adsorbed in the mem- distributed in the membranes and also diffused into the interior branes without any risk of substantial plasmonic coupling due of the BC membranes (Figure 1f,g and Figure S3, Supporting to crowding. To investigate whether the amount of adsorbed Information). The SEM micrographs also indicate that the AuNPs could be further increased, BC membranes were incu- AuNPs adsorb to the nanocellulose fibrils rather than being bated in a more concentrated suspension of 13 nm diameter physically trapped in the pores. This was further confirmed AuNPs (14.6 × 10−9 m). After 14 d of incubation the AuNPs com- by the slight redshift of the LSPR band (Δλmax ≈ 1 nm) of the pletely covered the fibrils (Figure 1h). Because of the small sep- AuNPs after adsorption in the BC (Figure S4, Supporting Infor- aration between the AuNPs and the resulting near-field interac- mation). The scattering of the BC blueshifts the LSPR band by tions, the BC membranes show limited transmission in the vis- about 1 nm and the shift due to the substrate effect was conse- ible wavelength range and appear completely opaque (Figure 1h quently about 2 nm, which is similar to AuNPs immobilized inset and Figure S6, Supporting Information). on 2D polymer substrates.[38,39] This small shift cannot be dis- tinguished by eye and the perceived color of the membranes were hence very similar to that of the original suspension. 2.2. Modeling of Interactions Although a certain amount of lateral aggregation can be seen in the SEM micrographs, this is likely an effect of drying since the The AuNPs were strongly bound to the BC and only minor wet AuNP-functionalized BC membranes display a well-defined release, corresponding to less than 10% of the total amount of Adv. Funct. Mater. 2020, 30, 2004766 2004766 (3 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 2. a) The potential of mean force V(h) between the AuNPs and the BC was modeled by solving the Poisson–Boltzmann equation for a gold sphere interacting with a flat cellulose halfspace in a monovalent electrolyte. b) Simulation of interaction potential as a function of separation (h) between the AuNPs and the BC normalized by the Bjerrum length (λB) in presence of 0.1 × 10−3–10 × 10−3 m NaCl. c) UV–vis data and d) corresponding photographs showing the increase in AuNP adsorption with increasing concentration of NaCl. e) Simulation of the influence of PEG and NaCl concen- tration on the interaction potential and f) corresponding experimental UV–vis and g) photographs showing increased adsorption of PEG functionalized AuNPs with increasing NaCl concentration. AuNPs adsorbed in the BC, was observed even after extensive ΔV (Figure 2b). At a sufficiently high salt concentration, ΔV is washing in water or ethanol for 7 d (Figure S7a, Supporting reduced to the point where the AuNP adsorption process occurs Information). Moreover, only minor effects were observed on experimental time scales through an activated process. when the BC–AuNP composites were exposed to buffers with Under these conditions, the AuNPs can come into physical pH values ranging from 3 to 7.0, whereas at pH 8.5 some aggre- contact with the BC, where short-range van der Waals attrac- gation occurred (Figure S8, Supporting Information). To fur- tion dominates the adsorption process. These findings were ther understand the cause of this apparently strong interaction experimentally confirmed by monitoring the adsorption of between the AuNPs and the BC, the potential of mean force AuNPs to BC in the presence of 0.1 × 10−3 or 5 × 10−3 m NaCl V(h) was modeled, where h is the separation between the AuNP (Figure 2c,d), where the latter condition resulted in a drasti- and BC surfaces (Figure 2a and Figure S9, Supporting Informa- cally larger amount of adsorbed AuNPs. Due to the limited col- tion). The separation was normalized using the Bjerrum length loidal stability of citrate-stabilized NPs, higher concentrations λB ≈ 0.7 nm in water at room temperature (RT). The Poisson– of NaCl led to NP aggregation and could not be used. Hence, Boltzmann equation was solved for a gold sphere interacting optimizing conditions for the adsorption process involves bal- with a flat cellulose half-space in a monovalent electrolyte (for ancing colloidal stability and the thickness of the electric double details, see Supporting Information). Forces originating from layer by moderating the ionic strength of the solution. van der Waals interactions, osmotic pressure and electrostatic In an attempt to modify the short-range attraction between Maxwell stress were taken into account. In a range of low AuNPs and BC, the AuNPs were functionalized with carboxyl- ionic strength, electrostatic repulsion caused by double-layer terminated polyethylene glycol (PEG) disulfide with a molecular interactions of like-charged surfaces creates a potential barrier weight of 2.9 kDa. PEG-functionalization resulted in an LSPR ΔV = maxhV(h). Since the rate of adsorption is proportional to shift of Δλmax = 2.4 nm and an increase in hydrodynamic radius exp(-ΔV/kBT),[41,42] where kB is the Boltzmann constant and T (RH) from about 24.3 to about 27.4 nm (ΔRH = 3.1 nm), as well is the absolute temperature, this potential barrier essentially as a moderate change in zeta potential (ζ) from –66 to –48 mV, prevents adsorption of AuNPs onto the BC fibrils, which sta- confirming formation of a self-assembled monolayer of the bilizes the system. However, as the ionic strength increases, PEG on the AuNPs. Despite the less negative zeta potential, the thicknesses of the electric double-layers decrease, thus the PEG-functionalization completely prevented adsorption of reducing the repulsive electrostatic interaction as quantified by the AuNPs to BC at low salt concentrations (0.1 × 10−3 m NaCl). Adv. Funct. Mater. 2020, 30, 2004766 2004766 (4 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de However, the presence of PEG improved the colloidal stability pension, the ratio of adsorbed NPs could be controlled. Altering of the AuNPs and effects of higher ionic strengths could be the ratio of the two types of NPs changed the relative intensi- explored. Increasing the NaCl concentration to 30 × 10−3 m ties of the AgNP and AuNP LSPR bands, resulting in a transi- NaCl resulted in a decrease in hydrodynamic radius to 24.6 nm, tion in color of the BC membranes from yellow to red/orange likely due a compression of both the PEG layer and the elec- (Figure 3d,e). The two types of NPs were homogenously distrib- tric double layer, but the particles remained dispersed. Interest- uted in the materials and could not be distinguished in SEM ingly, at this ionic strength, the PEG–AuNPs clearly adsorbed micrographs (Figure 3f) but energy-dispersive X-ray spectros- to the BC (Figure 2f,g), albeit to a slightly lower extent than copy (EDS) confirmed the presence of both silver and gold in the non-functionalized AuNPs at 5 × 10−3 m NaCl. These find- the composites (Figure 3e). ings were further confirmed by modeling. The adsorbed PEG In addition to NP size and composition, shape is an impor- layer was modeled as a spherical dielectric shell on the AuNPs tant factor for tuning the optical properties of colloidal NPs with the same properties as water with the charged groups (Figure S10, Supporting Information). Fabrication of BC com- of the polymer located on the outer surface of this shell. The posites with nonspherical anisotropic NPs can further expand thickness of the shell was taken as the difference between the the range of nanoplasmonic properties of the materials. Most hydrodynamic radius of functionalized and nonfunctionalized anisotropic colloidal NPs, such as AuNRs, are synthesized in AuNPs. The simulated potential of mean force was not much the presence of surfactants, most notably CTAB. The pres- affected by the presence of the polymeric brush, and at high ence of CTAB can complicate both surface immobilization and ionic strength the polymers appears not to provide sufficient functionalization,[45] and previous attempts to impregnate BC steric repulsion to prevent adsorption (Figure 2e). with CTAB-stabilized NPs exploited freeze-dried BC aerogels where capillary effects could accelerate the adsorption pro- cess.[1] To facilitate adsorption of CTAB-stabilized AuNRs in 2.3. Self-Assembly of BC–AgNP, BC–AgNP/AuNP, hydrated BC, excess CTAB was first carefully removed and the and BC–AuNR Composites NRs were redispersed in water. In the absence of excess CTAB, AuNRs adsorbed readily in the hydrated BC membranes The strategy developed for the self-assembly of metal NPs in (Figure 3h, inset). The BC–AuNR composites show a signifi- hydrated BC was not restricted to spherical AuNPs. To verify the cant broadening of the long wavelength resonance in the LSPR versatility of the approach, we also investigated adsorption of spectrum compared to the dispersed rods (Figure 3h and Fig- silver NPs (AgNPs) for generating of BC–AgNP composites and ures S10 and S11, Supporting Information). SEM micrographs BC–AgNP/AuNP composites, as well as adsorption of various indicate that AuNRs align with the cellulose nanofibrils, anisotropic CTAB-stabilized gold NPs, such as gold nanorods resulting in formation of both parallel bundles and AuNRs (AuNRs). BC–AgNP composites have a distinct yellow color arranged in a tail-to-tail orientation (Figure 3i). The ordering and show a well-defined LSPR band at about λmax = 400 nm of the AuNRs along the cellulose nanofibrils result in defined (Figure 3a). The SEM micrographs show that the AgNPs are near-field interaction between the rods. Simulations show well-dispersed and adsorbed to the fibrils (Figure 3b). Silver- that the tail-to-tail arrangement of the AuNRs produces a pro- impregnated wound dressings are widely used in treatment of nounced redshift whereas the formation of parallel bundles infected wounds and BC–AgNP composites can thus further induces a blueshift of the long wavelength resonance corre- enhance the performance of BC as a wound care material.[43] sponding to the long axis of the rods (Figure S10, Supporting To also investigate whether this fabrication strategy result in Information), which explains the observed broadening of the materials with antimicrobial properties, the BC–AgNP com- LSPR band. For the short wavelength resonance, this near-field posites were immersed in suspension of Staphylococcus aureus effect is much weaker for both tail-to-tail and parallel arrange- (107 CFU) and the bacteriolytic effect was assessed using the ments of the rods and, consequently, no shift of this plasmon fluorescent nucleic acid stain Sytox Green. The native BC did band was seen. Combined, the interactions between the BC not show any antibacterial effects, but the viability of the bac- and the AuNRs result in a quasi-ordered nanocomposite with teria was markedly reduced in the vicinity of the BC–AgNP very distinct optical properties. This assembly strategy could composite membranes (Figure 3c). Since the AgNPs appear to also be extended to other anisotropic NPs, including longer be as strongly bound to the BC as the AuNPs, the antibacterial AuNRs (Figure 3j and Figure S10b,e, Supporting Information) effect is likely due to a gradual dissolution of the nanoparticles and gold nanoprisms (Figure 3j and Figure S10c,f, Supporting which generates Ag+ ions.[44] Information), which further indicate the extensive possibili- Whereas in situ metal NP synthesis can only yield compos- ties to produce BC–metal NP composites with tailored optical ites with a single type of NPs, this is not a restriction when properties. using self-assembly. We demonstrate this by incubating BC membranes in a suspension containing both AgNPs and AuNPs, resulting in a mixed BC–AgNP/AuNP composite mate- 2.4. Mechanoplasmonic Properties rial (Figure 3d–g). The BC–AgNP/AuNP composite membranes show two distinct LSPR bands at λmax ≈ 398 nm and at λmax ≈ As indicated by the SEM cross section of the BC–AuNP 532 nm originating from the immobilized AgNPs and AuNPs, (Figure 1g), the AuNPs readily diffuse into the bulk of the respectively (Figure 3d). By varying the concentration of AgNPs nanocellulose network. Since the BC membranes are highly between 38 × 10−12 m and 0.48 × 10−9 m and the concentration hydrated, individual fibrils are typically well separated in the wet of AuNPs between 17 × 10−12 and 47 × 10−12 m in the initial sus- state. When compressing the material, the separation between Adv. Funct. Mater. 2020, 30, 2004766 2004766 (5 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 3. a) UV–vis spectrum and photo of BC–AgNPs (ø 20 nm). b) SEM of BC–AgNP membranes (ø 40 nm) incubated in 3.75 × 10−12 m AgNPs for 7 d, scale bar: 200 nm. c) Sytox Green staining of BC membranes with and without adsorbed AgNPs (ø 40 nm) immersed in Staphylococcus aureus (107 CFU) suspension, scale bar: 500 µm. d) UV–vis spectra of BC membranes incubated in a mixture of AgNP (ø 20 nm) and AuNP (ø 50 nm) suspensions with varying particle concentration ratios. e) photos of membranes incubated in different ratios of AgNPs (ø 20 nm) and AuNPs (ø 50 nm), from top to bottom [AgNP]:[AuNP]: 6:1; 10:1; 17:1; 28:1. f) SEM micrograph of BC membrane incubated in a mixture of AgNP (ø 20 nm, 25 × 10−12 m) and AuNP (ø 50 nm, 46 × 10−12 m) suspensions for 7 d, scale bar: 200 nm. g) EDX measurement of the NP functionalized BC membranes. h) UV–vis spectrum, photo, and i) SEM micrograph of AuNRs (58 × 13 nm) adsorbed in BC, scale bar: 200 nm. j) UV–vis spectra and photo of BC–AuNRs (50 × 10 nm) and gold BC-nanoprisms (70 nm). individual fibrils will decrease, increasing the chances of near- LPSR band was seen when pressure was applied (Figure 4b,c field interactions between adsorbed AuNPs (Figure 4a). A sim- and Figure S13, Supporting Information) and that increased ilar phenomenon has been described by Park et al.,[46] where as a function of pressure (Figure 4d). Part of the redshift can BC–AuNP composites subject to drying showed amplified be attributed to an increase in effective refractive index (RI) surface-enhanced Raman scattering (SERS) signals due to defor- in the vicinity of the AuNPs upon compression as a result of mation of the BC and formation of hotspot. To investigate this decreasing separation between the individual nanocellulose effect further, a defined pressure was applied on the BC–AuNP fibrils. However, the magnitude of the LSPR shift, including membranes using a rheometer with an optically transparent the broadening of the LSPR band, clearly indicate that the com- plate and a reflecting geometry. The optical response was meas- pression reduces the separation between a large fraction of the ured in real-time using a fiber-optical spectrophotometric setup immobilized AuNPs enough to give rise to plasmonic near-field (Figure S12, Supporting Information). Interestingly, a distinct coupling. The broadening, manifested as the appearance of and instantaneous redshift and a pronounced broadening of the a distinct shoulder in the wavelength range between 600 and Adv. Funct. Mater. 2020, 30, 2004766 2004766 (6 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 4. a) Compression of the water swollen BC–AuNP composites reduces separation between adsorbed AuNPs resulting in plasmonic coupling and a distinct redshift of the LSPR band. b) LSPR peak shift when pressure is applied to a BC–AuNP composite. c) UV–vis spectra obtained at timepoint t = 0 s and t = 900 s. d) LSPR peak shift as a function of pressure during compression in (b) indicated in red. e) Real-time LSPR peak shift and broad- ening of a BC–AuNP composite upon compression. f) UV–vis spectra of BC–AuNP when pressure is increased in distinct steps, and g) corresponding real-time data of the LSPR peak shift. h) Photo of BC–AuNP composites after compression in a cross-pattern, resulting in a substantial colorimetric shift in the compressed regions. The noncompressed parts of the BC–AuNP composite retained the original red color. i) UV–vis spectra obtained from the compressed and non-compressed parts of the BC–AuNP composite shown in (h). 800 nm, was quantified as the ratio of the intensity at 665 nm experiencing significant near-field interactions increased with and the LSPR peak maximum. increasing pressure. A stepwise increase in the pressure con- BC–AuNP composites subject to moderate compression sequently resulted in a concomitant stepwise redshift in the (<500 kPa) show a partial reversion of the redshift when peak position and broadening of the LSPR band (Figure 4f,g). releasing the pressure, whereas the broadening of the LSPR The inelastic response of the material further allows for con- band was retained (Figure 4e). The blueshift of the LSPR trolling the nanoplasmonic properties with spatial resolution peak position when releasing the pressure indicate a partial by applying different pressure in different parts of the com- recovery and restoration of the local RI in the membranes. The posites. Pressure applied in a cross-pattern result in a spa- relaxation of the BC was, however, not sufficient to separate tially distinct mechanoplasmonic shift that could be clearly AuNPs forced into close proximity, as indicated by the irre- distinguished both spectrophotometrically and by the naked versible broadening of the LSPR band. The fraction of AuNPs eye (Figure 4h,i). Noncompressed regions of the membrane Adv. Funct. Mater. 2020, 30, 2004766 2004766 (7 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 5. a) Influence of BSA adsorption (10 mg mL−1) on the mechanoplasmonic response and b) real-time LSPR peak shift and broadening during compression of BC–AuNP modified with BSA. c) Corresponding photographs of BC–AuNP composite without BSA (i) before and (ii) after compres- sion, and (iii) when incubated with BSA before compression. d) Influence of BSA concentration on the mechanoplasmonic response. Error bars show standard deviations. remained unaffected and retained the bright red color indica- bioanalytical assays and sensors that enables optical biodetec- tive of well-separated AuNPs. tion by means of mechanical stimuli. To further confirm that compression leads to an increase in the near-field electromagnetic interaction between the immo- bilized AuNPs, bovine serum albumin (BSA, 10 mg mL−1) 2.5. Improving Signal-to-Noise Ratio was adsorbed on the AuNPs prior compression. The addition of BSA resulted in a small shift in the LSPR band (Δλmax = The 3D architecture of BC result in a high local concentration 3.7 nm) indicative of adsorption of the protein on the NPs of AuNPs in the BC–AuNP composites and consequently a (Figure S14a, Supporting Information). Although a significant high absolute LSPR signal intensity, which facilitate sensitive transient redshift was seen upon compression, the adsorbed detection of spectral changes. Whereas the mechanoplasmonic BSA completely inhibited the broadening of the LSPR band response was very distinct, LSPR shifts caused by the local (Figure 5a–c). Photographs of the BC–AuNP composites before RI changes caused by protein adsorption are much less pro- and after compression highlights the distinct color change nounced (Figure S14, Supporting Information). However, the that occurs in the absence of BSA whereas the corresponding possibility to detect small refractometric changes in combina- photo­graphs of BC–AuNP composites exposed to BSA before tion with high surface sensitivity make LSPR in general an compression show a similar color as the noncompressed attractive and widely used transduction method for RI-based bio- composites (Figure 5c). For the latter, the perceived color can sensing.[8,48] Despite their rather low RI sensitivity (η < 100 nm be slightly different as compared to the noncompressed per RIU)[38] spherical AuNPs are often used in biosensing appli- samples due to the differences caused by the RI shift upon cations because of their high figure of merit (FOM) defined as BSA-adsorption as well as the thinning of the membrane upon η divided by the resonance linewidth (full width at half compression. The striking differences in the LSPR band and maximum, FWHM).[49] In conventional LSPR-based refracto- color between samples with and without BSA after compres- metric biosensors, the AuNPs are typically immobilized on a sion, however, clearly indicate that the adsorbed BSA can pro- 2D glass or polymer substrate.[50–52] For the BC–AuNP com- vide a steric barrier that prevents close contact between the posite, the LSPR peak position was identical to AuNPs adsorbed AuNPs. This drastically reduces the near-field coupling and to a conventional polymeric 2D surface but signal intensity was consequently the magnitude of the optical response when about a factor of 6 higher (Figure 6a). In contrast to a trans- compressed. The BSA-dependent mechanoplasmonic response parent glass/plastic substrate the BC is translucent, but since showed a clear concentration dependence. The lower the con- the BC membranes used were very thin (<0.5 mm), the effects centration of BSA the larger the mechanoplasmonic response, of scattering on the sensitive was negligible but resulted in a with a dynamic range that spans over five orders of magnitude slight blueshift (Δλmax ≈ 1 nm) of the LSPR band compared to (Figure 5d and Figure S14d, Supporting Information). The AuNPs in suspension (Figure S4b,c, Supporting Information). spectral variations induced in the mechanoplasmonic response The RI sensitivity of the immobilized AuNPs was very similar (peak position and broadening) in the presence and absence for the two substrates, 68 nm/RIU and 69 nm/RIU for the 2D of BSA are on par with the distinct colorimetric response of surface and BC, respectively (Figure 6b). In addition, the FOM aggregating AuNPs in suspension, which have been widely for the AuNPs immobilized on the 2D surface and in BC was explored for bioanalytical applications.[47] However, since the identical, 1.2 RIU−1, indicating that the BC–AuNP should be AuNPs are adsorbed on the BC, the difficulties with controlling well suited also for RI sensing. In addition, the higher signal- NP colloidal stability, especially in complex media (e.g., physi- to-noise ratio (S/N) of the BC–AuNP composite (≈450) as com- ological fluids), are completely avoided, which can facilitate pared to the corresponding 2D substrate (≈250) can have a development of innovative colorimetric mechanoplasmonic dramatic positive effect on sensor performance. Simulating the Adv. Funct. Mater. 2020, 30, 2004766 2004766 (8 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 6. a) UV–vis spectra and b) corresponding refractive index sensitivity of AuNPs immobilized in BC (3D) and on a polymeric 2D substrate. Error bars show standard deviation. Inset in a) shows normalized UV–vis spectra of AuNPs in 3D (BC–AuNP) and 2D, respectively. c) Precision in LSPR peak determination as a function of signal-to-noise ratio as determined experimentally and by simulations. d) LSPR peak shift upon BSA adsorption to BC–AuNP (red) and AuNPs on a polymeric 2D substrate (blue). Error bars show standard deviations. effects of increasing S/N on the accuracy in detecting the true 2.6. Nonlinear Optical Effects and Plasmonic Heating LSPR peak position show that a sensor using the BC–AuNP composite should be able to discriminate a shift in the LSPR The possibility to dynamically change the plasmonic proper- band that is about a factor of 2.3 smaller than when using a con- ties of the BC–AuNP composites by applying pressure further ventional 2D substrate (Figure 6c). These findings were experi- enables exploration of nonlinear optical effects.[54] The redshift mental verified by acquiring 10 consecutive UV–vis spectra at of the LSPR band upon compression of the BC–AuNP mem- different concentration of AuNPs (ø 50 nm) to achieve an esti- branes enhances the absorption cross-section in the near- mate of the variation in the LSPR peak position at different S/N infrared (NIR) range. This in turn increases the probability of ratios (Figure 6c). The improvement in precision in the readout a sequential two-photon absorption process when illuminated of the response upon BSA adsorption (Figure 6d) further using focused NIR light from femto-second-pulsed lasers, shows that the BC–AuNP composite can outperform conven- operating in the tissue transparent wavelength region.[55,56] tional 2D-LSPR biosensor configurations and facilitate develop- The resulting multiphoton-induced luminescence (MIL) of ment of novel BC–AuNP based sensor strategies. In addition, the compressed BC–AuNP membranes could be clearly visual- because of the intense color of the membranes, even relatively ized in far-field multiphoton laser scanning microscopy (MPM) small LSPR shifts can be potentially visualized by the naked (Figure 7a,b and Figure S15, Supporting Information). In con- eye (Figure S14b,c, Supporting Information). This effect can trast, the noncompressed membranes showed no MIL signal likely be pronounced using nanoparticles with an LSPR band (Figure 7a, inset). We have recently demonstrated that aggre- in a wavelength range better suited for visualization of color gation of spherical AuNPs in suspension results in MIL as a changes, which we have shown in previous work can enable result of specific biorecognition,[55] but to our knowledge, a detection of peak shifts as small as 2–3 nm.[53] mechanically induced MIL signal has never previously been Adv. Funct. Mater. 2020, 30, 2004766 2004766 (9 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de Figure 7. a) Far-field multiphoton laser scanning microscopy (MPM) image of BC–AuNP composite (AuNP ø 13 nm) after compression and before compression (inset), scale bars: 100 µm. b) MPM intensity distribution before (red) and after (blue) compression. c) Photothermal heating of com- pressed (C) and uncompressed (U) BC–AuNP composite with medium (MC/U) and low (LC/U) concentration of AuNPs, respectively, when exposed to laser light. d) Transmission spectroscopy of BC–AuNP composites mentioned in (c). reported. Triggering nonlinear optical effects by mechanical of the materials, enabling new applications based on nonlinear stimuli represents a new possibility for modulating the optical optical effects and enhanced thermoplasmonic heat generation. properties of soft nanoplasmonic materials. The versatility of this self-assembly strategy, further demonstrated In addition, the large absorption cross section of AuNPs can by generating antibacterial BC–AgNP composites, composites result in significant heat generation when irradiated at the LSPR containing both AgNPs and AuNPs, as well as BC with aniso- frequency as a result of electron–phonon and phonon–phonon tropic NPs, opens the path for BC–metal NP composites as mul- processes.[57] A weak plasmonic heating in the uncompressed tifunctional soft materials for emerging applications, spanning BC–AuNP composites was obtained by irradiation with NIR laser from wound care and biosensing to optics and electronics. diodes showing emission in the first and second optical tissue windows (808 and 1064 nm), which slightly increased when increasing the concentration of the AuNPs (Figure 7c and Figure 4. Experimental Section S16, Supporting Information). In contrast, compression of the BC–AuNP composites resulted in a large decrease in transmittance General: BC produced from Komagataeibacter xylinus was obtained from S2Medical AB (Linköping, Sweden). The BC membranes were (Figure 7d) and a drastic increase in heat generation leading to a about 0.5 mm in thickness and 6 mm disks were cut out using a biopsy fourfold amplification of the light-induced temperature increment. punch. All chemicals were obtained from Merck KGaA (Darmstadt, The possibility to tune the plasmonic properties by mechanical Germany) and used without further purification unless otherwise stimuli can thus be utilized to enhance the thermoplasmonic per- noted. AuNPs (ø 50 nm) and AgNPs (ø 40 nm) were obtained from formance of the BC–AuNP composites, which could be exploited BBI Solutions, (Crumlin, UK) and AgNPs (ø 20 nm) were obtained from to develop very efficient, biocompatible and highly stable implant- Cline Scientific AB (Gothenburg, Sweden). Synthesis procedures for all other NPs are described in the Supporting Information. able heat generators controlled by high penetration NIR light. NP Immobilization in BC: For immobilization of AuNPs and AgNPs, BC membranes were rinsed in MQ water and incubated from 12 h up to 30 d in an aqueous suspension of the NPs at RT on a rocking table. The concentration of AuNPs was varied between 60 × 10−12 m and 3. Conclusions 14.6 × 10−9 m, and the concentration of AgNPs was 3.75 × 10−12 m. The A novel and versatile method for fabrication of nanocomposites NaCl concentration was varied between 0.11 × 10−3 and 5 × 10−3 m. When coimmobilizing AuNPs (ø 50 nm) and AgNPs (ø 20 nm), AgNPs based on bacterial cellulose (BC) and metal NPs based on self- (0.77 × 10−9 m) and AuNPs (46 × 10−12 m) were mixed in varying ratios assembly is described. In contrast to methods relying on in situ ([AgNP]:[AuNP]; 1:0.9; 1.9:1; 2.9:1; 4.2:1) and incubated for 7 d at RT on synthesis, self-assembly enables the use of well-defined colloidal a rocking table followed by rinsing in MQ water. Extinction spectra of NPs of different sizes, shapes and composition, resulting in NP–BC composites were recorded using a microplate reader (Tecan nanocomposites with distinct and tunable optical and biophysical Infinite M1000 Pro, Tecan Austria GmbH, Grödig/Salzburg, Austria). properties, including mechanoplasmonic effects. Compression For adsorption kinetics experiments, a BC membrane was placed at the bottom of a cuvette and 1 mL AuNP (ø 50 nm) suspension of BC–AuNP composites leads to controlled near-field coupling (75 × 10−12 m, 5 × 10−3 m NaCl) was added on top. UV–vis spectra of between the NPs, which is translated to large controlled spectral the suspensions were measured at several time points with a benchtop variations, including resonance redshifts and large increase in the UV–vis spectrophotometer (UV-2450, Shimadzu). Samples were kept on absorption wavelength ranges. Importantly, the adsorption of bio- a rocking table between measurements. For refractive index sensitivity molecules on the NPs can totally screen their near-field interac- measurements and BSA adsorption measurements, BC membranes were tion upon compression, thereby offering a route for development placed in a 96-well microplate and 300 µL AuNP (ø 50 nm) suspension of colorimetric mechanoplasmonic biosensors. The 3D structure (60 × 10−12 m, 4 × 10−3 m NaCl) was added on top. Incubation time was 5 d after which the BC–AuNP composites were rinsed with MQ water. of the BC–AuNP composites further provide a drastic increase Adsorption of anisotropic NPs to BC was done by carefully heating in the signal-to-noise ratio of the spectral measurements. The a suspension of gold AuNR suspension (≈0.5 mL in an Eppendorf mechanically induced broadening of the plasmonic absorption tube) until all CTAB crystals were dissolved. The suspension was then band can also be used to accurately amplify the optical properties centrifuged at 8000 rpm for 6.5 min and the supernatant removed. The Adv. Funct. Mater. 2020, 30, 2004766 2004766 (10 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de NPs were then resuspended in 500 µL MQ water and a BC membrane [12] K.-Y. Lee, G. Buldum, A. Mantalaris, A. Bismarck, Macromol. Biosci. was added to the tube. The sample was left on a rocking table and 2014, 14, 10. incubated for 48 h before being extracted from the suspension and [13] D. Klemm, D. Schumann, U. Udhardt, S. Marsch, Prog. Polym. Sci. rinsed with MQ water. Experimental details for analysis of optical and 2001, 26, 1561. biophysical properties are available in Supporting Information. [14] M. M. Rahman, A. N. Netravali, ACS Macro Lett. 2016, 5, 1070. [15] R. Portela, C. R. Leal, P. L. Almeida, R. G. Sobral, Microb. Biotechnol. 2019, 12, 586. [16] A. Svensson, E. Nicklasson, T. Harrah, B. Panilaitis, D. L. Kaplan, Supporting Information M. Brittberg, P. Gatenholm, Biomaterials 2005, 26, 419. Supporting Information is available from the Wiley Online Library or [17] J. Gutierrez, S. C. M. Fernandes, I. Mondragon, A. Tercjak, ChemSu- from the author. sChem 2012, 5, 2323. [18] A. Aboelnaga, M. Elmasry, O. A. Adly, M. A. Elbadawy, A. H. Abbas, I. Abdelrahman, O. Salah, I. Steinvall, Burns 2018, 44, 1982. [19] T. Sivlér, P. Sivlér, M. Skog, L. Conti, D. Aili, Adv. Skin Wound Care Acknowledgements 2018, 31, 306. This work was supported by the Swedish Foundation for Strategic [20] T. Maneerung, S. Tokura, R. Rujiravanit, Carbohydr. Polym. 2008, Research (SFF) grant no. FFL15-0026 and framework grant RMX18-0039 72, 43. (HEALiX), the Swedish Government Strategic Research Area in Materials [21] J. He, T. Kunitake, A. Nakao, Chem. Mater. 2003, 15, 4401. Science on Functional Materials at Linköping University (Faculty Grant [22] T. Zhang, W. Wang, D. Zhang, X. Zhang, M. Yurong, Y. Zhou, L. Qi, SFO-Mat-LiU no. 2009-00971), the competence center FunMat-II that Adv. Funct. Mater. 2010, 20, 1152. is financially supported by Vinnova (grant no. 2016-05156), and the [23] L. C. de Santa Maria, A. L. C. Santos, P. C. Oliveira, H. S. Barud, Knut and Alice Wallenberg Foundation (grant no. KAW 2016.0231), the Y. Messaddeq, S. J. L. Ribeiro, Mater. Lett. 2009, 63, 797. Swedish Research Council (grant Nos. 2017-05178 and 2015-05002) [24] H. S. Barud, T. Regiani, R. F. C. Marques, W. R. Lustri, Y. Messaddeq, and the Spanish Ministerio de Ciencia, Innovación y Universidades S. J. L. Ribeiro, J. Nanomater. 2011, 2011, 721631. (MICINN) through the MAT2016-77391-R. The ICN2 was supported [25] S. Roig-Sanchez, E. Jungstedt, I. Anton-Sales, D. C. Malaspina, by the Severo Ochoa Centres of Excellence programme, funded by the J. Faraudo, L. A. Berglund, A. Laromaine, A. Roig, Nanoscale Horiz. Spanish Research Agency (AEI, grant no. SEV-2017-0706). The authors 2019, 4, 634. thank Bela Nagy for input on modeling and S/N simulation and M. [26] S. Pal, R. Nisi, M. Stoppa, A. Licciulli, ACS Omega 2017, 2, 3632. Magnusson at Linköping University for valuable input on signal theory. [27] S. Gea, C. T. Reynolds, N. Roohpour, B. Wirjosentono, N. Soykeabkaew, E. Bilotti, T. Peijs, Bioresour. Technol. 2011, 102, 9105. [28] L. Francisco-Aldana, E. Morales-Narváez, J. Phys.: Photonics 2019, 1, Conflict of Interest 04LT01. The authors declare no conflict of interest. [29] N. G. Bastús, J. Comenge, V. Puntes, Langmuir 2011, 27, 11098. [30] M. R. Langille, M. L. Personick, J. Zhang, C. A. Mirkin, J. Am. Chem. Soc. 2012, 134, 14542. [31] M. L. Personick, C. A. Mirkin, J. Am. Chem. Soc. 2013, 135, 18238. Keywords [32] M. M. Shahjamali, M. Bosman, S. Cao, X. Huang, S. Saadat, E. Martinsson, D. Aili, Y. Y. Tay, B. Liedberg, S. C. J. Loo, H. Zhang, antimicrobials, bacterial cellulose, gold nanoparticles, nanocomposite, F. Boey, C. Xue, Adv. Funct. Mater. 2012, 22, 849. sensors [33] C. Daruich De Souza, B. Ribeiro Nogueira, M. E. C. M. Rostelato, J. Alloys Compd. 2019, 798, 714. Received: June 4, 2020 [34] I. Pastoriza-Santos, C. Kinnear, J. Pérez-Juste, P. Mulvaney, Revised: June 16, 2020 L. M. Liz-Marzán, Nat. Rev. Mater. 2018, 3, 375. Published online: August 9, 2020 [35] Y. Li, Y. Tian, W. Zheng, Y. Feng, R. Huang, J. Shao, R. Tang, P. Wang, Y. Jia, J. Zhang, W. Zheng, G. Yang, X. Jiang, Small 2017, 13, 1700130. [36] Y. Wang, D. Aili, R. Selegård, Y. Tay, L. Baltzer, H. Zhang, [1] L. Tian, J. Luan, K. K. Liu, Q. Jiang, S. Tadepalli, M. K. Gupta, B. Liedberg, J. Mater. Chem. 2012, 22, 20368. R. R. Naik, S. Singamaneni, Nano Lett. 2016, 16, 609. [37] A. M. Sokolnicki, R. J. Fisher, T. P. Harrah, D. L. Kaplan, J. Membr. [2] E. Morales-Narváez, H. Golmohammadi, T. Naghdi, H. Yousefi, Sci. 2006, 272, 15. U. Kostiv, D. Horák, N. Pourreza, A. Merkoçi, ACS Nano 2015, 9, [38] E. Martinsson, B. Sepulveda, P. Chen, A. Elfwing, B. Liedberg, 7296. D. Aili, Plasmonics 2014, 9, 773. [3] X. Du, Z. Zhang, W. Liu, Y. Deng, Nano Energy 2017, 35, 299. [39] E. Martinsson, M. A. Otte, M. M. Shahjamali, B. Sepulveda, D. Aili, [4] Z. Shi, G. O. Phillips, G. Yang, Nanoscale 2013, 5, 3194. J. Phys. Chem. C 2014, 118, 24680. [5] M. Kaushik, A. Moores, Green Chem. 2016, 18, 622. [40] D. Aili, P. Gryko, B. Sepulveda, J. A. G. Dick, N. Kirby, R. Heenan, [6] M. Chen, H. Kang, Y. Gong, J. Guo, H. Zhang, R. Liu, ACS Appl. L. Baltzer, B. Liedberg, M. P. Ryan, M. M. Stevens, Nano Lett. 2011, Mater. Interfaces 2015, 7, 21717. 11, 5564. [7] E. Lam, K. B. Male, J. H. Chong, A. C. W. Leung, J. H. T. Luong, [41] D. F. Evans, H. Wennerström, The Colloidal Domain: Where Physics, Trends Biotechnol. 2012, 30, 283. Chemistry, Biology, and Technology Meet, 2nd ed., John Wiley & Sons, [8] Y. Chen, S. Han, S. Yang, Q. Pu, Dyes Pigm. 2017, 142, 126. Ltd, New York 1999. [9] D. Klemm, F. Kramer, S. Moritz, T. Lindström, M. Ankerfors, [42] H. A. Kramers, Physica 1940, 7, 284. D. Gray, A. Dorris, Angew. Chem., Int. Ed. 2011, 50, 5438. [43] J. Wu, Y. Zheng, X. Wen, Q. Lin, X. Chen, Z. Wu, Biomed. Mater. [10] A. J. Brown, J. Chem. Soc., Trans. 1886, 49, 432. 2014, 9, 035005. [11] U. Römling, M. Y. Galperin, Trends Microbiol. 2015, 23, 545. [44] Z. M. Xiu, J. Ma, P. J. J. Alvarez, Environ. Sci. Technol. 2011, 45, 9003. Adv. Funct. Mater. 2020, 30, 2004766 2004766 (11 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH www.advancedsciencenews.com www.afm-journal.de [45] C. Andrésen, S. Jalal, D. Aili, Y. Wang, S. Islam, A. Jarl, B. Liedberg, [51] X. Y. Zhang, A. Hu, T. Zhang, W. Lei, X. J. Xue, Y. Zhou, W. W. Duley, B. Wretlind, L.-G. Mårtensson, M. Sunnerhagen, Protein Sci. 2010, ACS Nano 2011, 5, 9082. 19, 680. [52] N. Nath, A. Chilkoti, Anal. Chem. 2004, 76, 5370. [46] M. Park, H. Chang, D. H. Jeong, J. Hyun, BioChip J. 2013, 7, 234. [53] P. Chen, X. Liu, G. Goyal, N. T. Tran, J. C. Shing Ho, Y. Wang, D. Aili, [47] D. Aili, R. Selegård, L. Baltzer, K. Enander, B. Liedberg, Small 2009, B. Liedberg, Anal. Chem. 2018, 90, 4916. 5, 2445. [54] M. Kauranen, A. V Zayats, Nat. Photonics 2012, 6, 737. [48] Y.-J. Lai, W.-L. Tseng, Analyst 2011, 136, 2712. [55] J. Borglin, R. Selegård, D. Aili, M. B. Ericson, Nano Lett. 2017, 17, 2102. [49] L. J. Sherry, R. Jin, C. A. Mirkin, G. C. Schatz, R. P. Van Duyne, Nano [56] J. Borglin, S. Guldbrand, H. Evenbratt, V. Kirejev, H. Grönbeck, Lett. 2006, 6, 2060. M. B. Ericson, Appl. Phys. Lett. 2015, 107, 234101. [50] C. Xue, Z. Li, C. A. Mirkin, Small 2005, 1, 513. [57] S. Link, M. A. El-Sayed, Int. Rev. Phys. Chem. 2000, 19, 409. Adv. Funct. Mater. 2020, 30, 2004766 2004766 (12 of 12) © 2020 The Authors. Published by Wiley-VCH GmbH