Hydrogels Incorporating Au@polydopamine Nanoparticles: Robust

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Hydrogels Incorporating Au@polydopamine Nanoparticles: Robust Performance for Optical Sensing Jiangjiang Zhang, Lei Mou, and Xingyu Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02459 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Analytical Chemistry

Hydrogels Incorporating Au@polydopamine Nanoparticles: Robust Performance for Optical Sensing Jiangjiang Zhang†, ‡, Lei Mou†, § and Xingyu Jiang *, †, ‡ †

Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Number 11 Zhongguancun Beiyitiao, Beijing 100190, China ‡ Sino-Danish College, University of Chinese Academy of Sciences, Beijing 100049, China § Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China ABSTRACT: Stimuli-responsive hydrogels (SRhG) that undergo response to physicochemical stimuli have been broadly applied in separation, biosensing, and drug delivery. Since, most of the SRhG are based on the structural behaviors (swelling or collapse). Herein, we describe a more simple and convenient colorimetric SRhG of polydopamine-coated gold nanoparticles (Au@PDA NPs) hydrogel. The newly developed SRhG is based on the in situ surface chemistry of Au@PDA NPs with core-shell structure embedding in agarose hydrogel. Silver ions can in situ form Ag NPs on surfaces of Au@PDA NPs (Ag_Au@PDA NPs with coresatellites like structure) at ambient conditions, which shift the localized surface plasmon resonance (LSPR) absorption peak and result in color change. The solid sensing phase of SRhG shows greatly improved stability and anti-interference ability comparing to that of solution phase sensing. With rational designs, Au@PDA NPs hydrogel shows great potential in optical sensing, for example biothiol detection and coupled with enzyme-cascade reaction for acetylcholinesterase activity detection and inhibitor assays with excellent sensitivity and selectivity.

Hydrogels, as soft material containing a mass of hydrate water, are characterized with crosslinked 3D frameworks or highly porous layered structure.1, 2 Taking advantage of merits such as biocompatibility, flexibility and mechanical stability, hydrogels especially stimuli-responsive hydrogels (SRhG), draw great interests in biological and biomedical fields.3-9 SRhG that undergo response to physicochemical stimuli have been broadly explored in separation, biosensing, and drug delivery.10-16 Literatures report bioresponsive hydrogels where peptides, antibody-antigen, host-guest molecules, DNADNAzyme or DNA-Aptamer and so on serve as functional crosslinkers.8, 17-28 As a result, target-induced break of the crosslinking pair will lead to the swelling or collapse of hydrogel, and thus the releasing of preloaded cargos like nanomaterial (e.g. gold nanoparticles) and protease. By recording the direct visual signal or coupling with enzymecascade reaction, SRhG-based sensors show great perspectives in optical sensing and point-of-care testing (POCT) fields.29-32 However, these smart designs involve expensive materials (such as protein and DNA), complicated synthesis and as well specific devices, which hinder their potential applications. Gold nanoparticles (Au NPs), that possess easy fabrication and functionalization, distance-dependent surface plasmon resonance (SPR) and multiple enzyme-mimic activities, are used for detection of ions, drugs, biomarkers, cancer cells and bacteria.33-46 Our group as well develops Au NPs-based material with excellent antibacterial activity.47-52 Considering their successful designs, while, these sensors yet face the drawbacks about poor-stability and anti-interference capability. We figure out combining mechanical stability of hydrogel and Au NPs to develop more simple and robust

colorimetric SRhG-based sensor. Typically, the most commonly used strategy of target-induced aggregation/antiaggregation is hardly suitable for Au NPs-based hydrogel.53-55 Since the Brownian motion of Au NPs is restricted in hydrogel, and target-generated binding force can insufficiently drive the access and followed aggregation among Au NPs. In this study, we find core-shell structured polydopamine-coated Au NPs (Au@PDA NPs) in situ catalyze the reduction of silver ions, and followed by deposition of Ag NPs on surfaces of Au@PDA NPs. The produced Ag NPs-decorated Au@PDA NPs (Ag_Au@PDA NPs) with a core-satellites like structure shift the localized surface plasmon resonance (LSPR) absorption peak to shorter wavelength implementing with color transition from red to yellow. Owing to the in situ surface chemistry, Au@PDA NPs are workable in hydrogel for the further design of colorimetric SRhG-based sensors. Herein, we carry out the in situ surface chemistry of Au@PDA NPs and combine with agarose for Au@PDA NPs hydrogel to develop the SRhG-based colorimetric sensing platform. We use Ag+ as the stimulus trigger that can form Ag_Au@PDA NPs in hydrogel coupling with color change and spectral blue-shift. This Ag+-responsive SRhG of Au@PDA NPs hydrogel exhibits improved stability and antiinterference ability in hazardous sensing conditions. Ag+ shows strong chemical affinity to coordinate with biothiols (RSH, such as GSH and Cys). The coordination between RSH and Ag+ produces a stable metal-metal polymer that can prevent the polymerized Ag+ for next reactions. In the design, GSH/Cys coordinate with Ag+, and the produced Ag+-polymer prevents Ag+ of solution from diffusing to Au@PDA NPs hydrogel and accessing to Au@PDA NPs. As a result, Ag+-

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induced wavelength shift and color change of Au@PDA NPs hydrogel are inhibited, and thus the colorimetric sensing of biothiols is achieved with great simplicity using the SRhG of Au@PDA NPs hydrogel. For the application development, we further utilize acetylthiocholine (ATCh) as a substrate of acetylcholinesterase (AChE). The enzymatic hydrolysate thiocholine is similar to biothiol that can be involved to the Ag+-responsive Au@PDA NPs hydrogel. By recording the spectral blue-shift and color change, the Au@PDA NPs hydrogel is successfully applied to colorimetrically detect AChE activity with good performance on stability, selectivity and sensitivity. The calculated LOD value of 0.9 mU/mL is lower than many reports. Benefiting from the high sensitivity, inhibitor assays of AChE using huperzine A indicate the Au@PDA NPs hydrogel can also be an alternative efficient drug screening platform.

EXPERIMENTAL SECTION Materials and instruments. All commercially available reagents are purchased in analytical grade and used without further purification. Acetylcholinesterase (AChE, Electrophorus electricus Type V-S) is from Sigma–Aldrich (St. Louis, MO).We record the UV-vis absorption spectra of solution on a Shimadzu UV–2450 spectrophotometer with an optical path length of 10 mm at room temperature, and the UV-vis absorption spectra of hydrogel on a PerkinElmer EnspireTM multimode plate reader. Dynamic light scattering and Zeta-potential data are collected on a Zetasizer Nano ZS (Malvern) system. We perform the transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) spectrum measurements on a Tecnai G2 T20 ST (FEI) transmission electron microscope equipped with EDAX genesis 2000 XMS accessory operated at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) images are recorded by a Hitachi S4800 field emission scanning electron microscope.

RESULTS AND DISCUSSION Characterization of Au@PDA NPs hydrogel. We synthesize the Au NPs through a one-pot protocol under ambient conditions with ascorbic acid (AA) as a reductant and cetyltrimethyl ammonium bromide (CTAB) as a protecting agent. After centrifuging (6000 rpm, 10 min) and rinsing with deionized water, the obtained Au NPs are monodisperse with spherical shape and particle size of 39.8 ± 7.6 nm measured by transmission electron microscopy (TEM, Figure S1a and S1b). Polymerization of dopamine (DA) on surfaces of Au NPs is achieved by ultrasonic-assisted approach. We sonicate the mixture solution of Au NPs and DA (2 mg/mL, 10 mM TrisHCl, and pH 8.5) for 15 minutes (power, 200 W). The unreacted DA and individual polydopamine particles in the supernate are discarded by centrifugation (5000 rpm, 6 min). The residual pellet of polydopamine-coated Au NPs (Au@PDA NPs) is re-suspended in equivalent HEPES solution (1 mM, pH 7.0). Morphology of the product Au@PDA NPs from TEM image (Figure S1c and S1d) indicates that PDA coating appears homogeneous on surfaces of Au NPs. Au@PDA NPs produce a weak red-shift and slight broadening absorption peak compared to Au NPs (Figure S1e). Successful coating of PDA also results in the increase of hydrodynamic diameter to ~51 nm and slightly altered zetapotential (Figure S1f). The positive charge of Au@PDA NPs

is owing to the presence of CTAB, since PDA is negative at netural condition. We fabricate Au@PDA NPs hydrogel by microwaveassisted heating of Au@PDA NPs mixture solutions containing certain amount of agarose. Agarose (gel point at wt 1.5 %, 36 oC) is selected to build the hydrogel frameworks carrying Au@PDA NPs because of its broad use and cheap. After cooling down to room temperature, the solidified neartransparent gel product presents reddish color deriving from the dispersive Au@PDA NPs (Figure 1a). Due to the mechanical stability and easy manufacture properties, we manufacture the as-prepared Au@PDA NPs hydrogel to the 96-arrays simply using the commercial 96-well cell plate (Figure 1b). The hydrogel arrays contribute to the high throughput screening assays. Absorption spectrum of Au@PDA NPs hydrogel (Figure 1c) shows a strong absorption peak at λ = 526 nm, due to the Au@PDA NPs. For the individual agarose gel, we observe no distinct absorption peak besides the gradual increased absorbance in the full scan range (800 ~ 400 nm). To further understand the structural information, we carry out scanning electron microscopy (SEM) measurements with low-temperature brittle fracture of Au@PDA NPs hydrogel after freeze-dried in vacuum. We observe porous layered structures and highly branched frameworks of Au@PDA NPs hydrogel (Figure 1d ~ f). At high magnification, the branch of Au@PDA NPs hydrogel shows bright spots with sizes between 50 nm and 80 nm (marked with red cycles in Figure 1g). These spots are similar to Au@PDA NPs in size, and are identified as Au@PDA NPs which well dispersed in the hydrogel. Ag+ responses of Au@PDA NPs hydrogel. As a proof of concept, we test the response to Ag+ using the above described Au@PDA NPs hydrogel. After incubation with Ag+ (200 µM), Au@PDA NPs hydrogel produces a chromatic change from red to yellow (Figure 1h). The individual agarose gel remains colorless without any change in the absence and presence of Ag+. Absorption peak (λ = 526 nm) of Au@PDA NPs hydrogel undergoes a discernible blue-shift supplementing with increased absorbance almost in the full scanning range. For agarose gel, we observe no recognizable absorption peak or absorbance increase compared to the non-incubated agarose gel. Both the color change and spectral shift derive from the interaction between Ag+ and Au@PDA NPs. Using the Au@PDA NPs, we indeed observe the same spectral blue-shift and color change to yellow in the presence of Ag+ under ambient condition (Figure 2b). To precisely understand the response, we also synthesize the citrate-capped Au NPs (Cit-Au NPs, 13 nm, Figure S2) and study their response to Ag+ or Ag NPs. Absorption spectra show that simple mixing of Cit-Au NPs and Ag NPs (Figure S3) produce a double-peak curve in which the peaks contribute to individual Cit-Au NPs (λ = 520 nm) and Ag NPs (λ = 400 nm, Figure S4). We do not observe any spectral shifts due to the mixing of Au NPs and Ag NPs. Introducing Ag+ and reducing agent AA into Cit-Au NPs solution generates a broad and flattened absorption peak at λ = 420 nm (Figure S4). While the absorption peak of Cit-Au NPs (λ = 520 nm) disappears. TEM characterization of the product shows a coreshell structure of Ag@Cit-Au NPs (Figure S5). These observations are significantly different from the finding of Ag+-induced spectral blue-shift of Au@PDA NPs, indicating a different structure of Au@PDA NPs after incubation with


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Figure 1. Characterization of Au@PDA NPs hydrogel. (a) Photograph of Au@PDA NPs hydrogel vialled in a glass bottle. (b) Photograph of fabricated 96-well arrays of Au@PDA NPs hydrogel (100 µL per well). (C) UV-vis absorption spectra of agarose hydrogel (wt 2 %) and Au@PDA NPs hydrogel. (d) ~ (g) SEM images of Au@PDA NPs hydrogel under different magnifications. Au@PDA NPs attached on the framework are marked with red circles. The concentration of agarose is wt 0.2 %. Scale bars: (d) 30 µm, (e) 10 µm, (f) 2 µm and (g) 500 nm. (h) UV-vis absorption spectra and photographs of Au@PDA NPs hydrogel and individual agarose gel response to Ag+ (200 µM).

Figure 2. Au@PDA NPs response to Ag+. (a) Schematic illustration of the response to Ag+ by depositing Ag NPs on surfaces of Au@PDA NPs (Ag_Au@PDA NPs) with core-satellites like structure. (b) UV-vis absorption spectra and optical images (inset) of Au@PDA NPs before and after incubation with 100 µM Ag+. (c) TEM image of Au@PDA NPs (top, scale bar: 100 nm) and the related fast Fourier transform (FFT) pattern of HRTEM image of Au@PDA NPs (down, scale bar: 5 nm). (d) TEM image of Au@PDA NPs after incubation with 30 µM Ag+ (top, scale bar: 100 nm) and the related FFT pattern of HRTEM image (down, scale bar: 5 nm). (e) and (f) TEM images of Au@PDA NPs after incubation with 70 µM Ag+ and 150 µM Ag+. Scale bars: 100 nm. (g). EDX profiles of Au@PDA NPs before and after incubation with Ag+ (30 µM).



Figure 3. Mechanism characterizations. (a) Size of Ag NPs deposited on surfaces of Au@PDA NPs under different concentrations of Ag+. Mean ± s.d. (n ≥ 40). (b) UV-vis absorption spectra of Au@PDA NPs under different concentrations of Ag+. (c) Electric field calculation of Au@PDA NP in the xz plane with λexcitation = 540 nm. For the simplified calculation, PDA of Au@PDA NP is discarded due to it hardly affect the LSPR property of Au NP in real measurements. Color scale bar demonstrates the electric field value of ln(|E|)2 which is normalized by the incident field. (d) Electric field calculations of Ag_Au@PDA NP with different sized Ag NPs. (e) Electric field enhancement curve of Ag_Au@PDA NP with different sized Ag NPs.

Ag+. TEM, HRTEM and the selected area fast Fourier transform (FFT) pattern images (Figure 2c) of Au@PDA NPs indicate that the uniform and clear PDA shell and a typical crystal lattice spacing of ~ 0.236 nm of Au NP core that matched with Au (111). After incubation with Ag+ (30 µM), we observe many small nanoparticles with size ~ 5 nm that positioned at the PDA shell displaying a core-satellites like structure (Figure 1d). The EDX profile (Figure 2g) shows the elements of Ag and Au, revealing that those nanoparticles are Ag NPs. HRTEM and the selected FFT pattern measurements (Figure 1d) also validate the Ag NPs with a crystal lattice spacing of ~ 0.237 nm that matched with Ag (111). Thus, we can presume that the Ag+-induced spectral blue-shift and color change of Au@PDA NPs result from depositing Ag NPs on surfaces of Au@PDA NPs (Ag_Au@PDA NPs) with coresatellites like structure. Furthermore, with the increase of [Ag+], the deposited Ag NPs grow in their size to ~8 nm at [Ag+] = 70 µM and ~11 nm at [Ag+] = 150 µM (Figure 2e and f, Figure 3a). The corresponding absorption peak of Au@PDA NPs gradually blue-shifts to the shorter wavelength as the bigger Ag NPs deposited (Figure 3b). To explore the mechanism of the spectral shift, we study the plasmonic coupling effect of the product Ag_Au@PDA NPs by calculating the local electric fields using the finitedifference time domain (FDTD) method. In the calculations, we simply Au@PDA NP as the 40 nm-sized Au NP model by discarding the PDA shell, since it hardly affects the LSPR property of Au@PDA NP. Calculation of Au NP reveals a strong electric field closer to the surface of Au NP (Figure 3c). As for the Ag_Au@PDA NP, we observe greatly enhanced electric field within the gap regions between Ag NP and Au NP (Figure 3d). The larger Ag NP results in the stronger electric field with a maximum value of 1.2 × 107 for the 11 nm-sized Ag NP (Figure 3e). Such “hot” area indicates the strong plasmonic coupling effect between Ag NP and Au NP.

The plasmonic coupling causes the spectral shift of Au NP. In bulk, Ag+-induced deposition of Ag NPs shifts the SPR absorption peak of Au@PDA NPs that couple with color change to yellow. To verify the spectral shift caused by deposition of Ag NPs, we also simulate the related absorption spectra. Figure S6 clearly indicate that introducing Ag NPs results in the blue-shift of absorption spectra of Au NPs, which is greatly aligned with experimental measurement of Ag_Au@PDA NPs.

Figure 4. Stability and anti-interference ability. (a) Normalized UV-vis absorption spectra of Au@PDA NPs hydrogel (solid line) and Au@PDA NPs (dotted line) in the presence of different kinds of cations (20 mM). (b) Optical images of Au@PDA NPs in the presence of different kinds of cations (20 mM). (c) Optical images of Au@PDA NPs hydrogel in response to Ag+ (200 µM) upon different kinds of cations (20 mM).


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Figure 5. Assays of biothiols. (a) Schematic illustration of biothiol sensing using Au@PDA NPs hydrogel. (b) UV-vis absorption spectra and the related optical images of Au@PDA NPs hydrogel respond to Ag+ (200 µM) in the presence and absence of biothiol (200 µM). (c) Concentration-dependent curve of wavelength shift of Au@PDA NPs hydrogel in response to biothiol of GSH (Ag+, 200 µM). (d) Concentration-dependent curve of wavelength shift of Au@PDA NPs hydrogel in response to biothiol of Cys (Ag+, 200 µM). (e) Wavelength shift histogram of Au@PDA NPs hydrogel in response to GSH, Cys and the 1 : 1 mixture of GSH and Cys (Ag+, 200 µM). Mean ± s.d. (n ≥ 3).

Since we confirm the Ag+-responsive SRhG of Au@PDA NPs hydrogel based on the in situ chemical deposition approach, we further test the stability and anti-interference ability of Au@PDA NPs hydrogel. We use commonly encountered metal cations (Na+, K+, Mg2+, Ca2+, Zn2+, Cu2+, Ni2+ and Fe3+) at 100-fold higher concentration to Ag+. Introducing these ions into Au@PDA NPs solution rapidly causes the aggregation of Au@PDA NPs (Figure 4a and b). At the same conditions, Au@PDA NPs hydrogel negatively show any apparent spectral red-shift or increased absorbance at λ > 600 nm that signed to the aggregated Au@PDA NPs. And the color of these Au@PDA NPs hydrogels is red as well (Figure 4c), indicating the great improved stability of Au@PDA NPs hydrogel compared to Au@PDA NPs. Followed by incubation with Ag+, these Au@PDA NPs hydrogels robustly turn the color to yellow. The related absorption peaks of Au@PDA NPs hydrogel undergo the blue-shift similar to that of in the absence of these metal cations (Figure S7). These metal cations hardly generate significant influence on the Ag+responses of Au@PDA NPs hydrogel. Au@PDA NPs hydrogel shows strong anti-interference capacity against other ions for the Ag+-induced spectral shift and color change. In addition, we also test the potential impacts of sunlight and UV-light irradiation. After an exposure of 7 days, it cannot recognize any spectral change or absorbance increase compared to that of freshly prepared Ag+ solution (Figure S8a and b). When incubated with Au@PDA NPs hydrogel, the same blue-shift and color change to yellow are recorded (Figure S8c). These results indicate good stability of the Ag+responsive hydrogel system. Colorimetric sensing of biothiols. With above findings of optical response and proofs, we believe that Ag+-Au@PDA NPs system-derived SRhG of Au@PDA NPs hydrogel can serve as an analytical platform under rational designs for colorimetric sensors or biosensors with certain advantages. Biothiols, vast majority consisting of reduced glutathione

(GSH), cysteine (Cys) and homocysteine (Hcy), play crucial roles in maintaining functionality of biological systems. Lacking or overdosing of these biothiols causes/indicates disorder of physiological functions and diseases like slowgrowing syndrome, Alzheimer's disease, angiocardiopathy and cancer.56 Thiol-containing molecules (RSH) can coordinate with Ag+ and form metal polymers through the metal-metal bonding among coordinated silver ions.57, 58 Those polymers are stable enough with aggregation induced-emission (AIE) or quenching properties according to their chromophore ligands.59-61 In our designs, biothiols like GSH/Cys or their mixtures produce the coordinated Ag-Ag polymer that prevent Ag+ breaking through the interface between Au@PDA NPs hydrogel and solution (Figure 5a). The polymerized Ag+ is not available to the in situ chemical deposition of Ag_Au@PDA NPs. As a result, Ag+-induced color transformation and spectral shift of Au@PDA NPs hydrogel are inhibited by the biothiols. Absorption spectra of Au@PDA NPs hydrogel incubated with different components (Figure 5b) show that biothiols like GSH and Cys recover the absorption peak blueshift caused by Ag+. And the color change to yellow of Au@PDA NPs hydrogel return to red. Before colorimetric sensing of biothiols, we optimize the experimental conditions of [Ag+] and incubation time using the Au@PDA NPs hydrogel 96-arrays (Figure S9). 200 µM Ag+ and 40 min incubation time are selected for the rest experiments. UV-vis absorption spectra of mixtures of GSH/Cys and Ag+ depict two absorption peaks near λ = 355 nm and λ = 276 nm (Figure S10). By measuring the timedependent spectra, we observe the coordination between GSH/Cys and Ag+ is a very fast process. Within two minutes, the related peak absorbance reaches a maximum without increasing to longer time (Figure S11). To fully ensure the reaction, we pick a mixing time of 5 min. We incubate the Au@PDA NPs hydrogel with mixtures of 200 µM Ag+ and various amounts of biothiols. As the [GSH] or [Cys]



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Figure 6. Assays of AChE activity and inhibitor test. (a) Schematic illustration of AChE activity sensing using Au@PDA NPs hydrogel. (b) UV-vis absorption spectral and optical images of Au@PDA NPs hydrogel after incubation with Ag+ (600 µM), ATCh (300 µM) and different amounts of AChE. (c) The concentration-dependent wavelength change curve of Au@PDA NPs hydrogel after incubation with Ag+ (600 µM), ATCh (300 µM) and different amounts of AChE. AChE concentrations: 1.25, 2.5, 7.5, 12.5, 20, 25, 50, 75, 125 and 250 mU/mL. The inset: the linear fitting range of 2.5 ~ 25 mU/mL. (d) The selectivity of Au@PDA NPs hydrogel upon 8 kinds of proteins including bovine serum albumin (BSA, wt 0.1 %) and alkaline phosphatase (ALP), glucoamylase, glucose oxidase (GOD), cholesterol esterase, invertase, catalase and lipase at a concentration 100-times higher than AChE. Mean ± s.d. (n ≥ 3). (e) The inhibition efficiency of huperzine A tested using the Au@PDA NPs hydrogel. The inset: the chemical structure of huperzine A.

increasing, the Ag+-induced blue-shift of absorption peak gradually recovers to the pristine wavelength of λ = 526 nm of Au@PDA NPs hydrogel (Figure S12). The concentrationdependent wavelength change (∆λ = λ - λ0, λ0 means absorption peak in the absence of biothiol; λ means absorption peak in the presence of biothiol) depicts the nonlinear curve for GSH as well as Cys (Figure 5c and d). While for the mixtures of GSH and Cys with molar ratio of 1:1, we observe a weakened wavelength change at low concentrations (50 µM and 90 µM) compared to a single component of GSH or Cys (Figure 5e). The analysis of biothiols depends on the coordination of thiols and Ag+, and the hydrodynamic diameter of product polymer (e.g. molecular weight) plays an important role. We use dynamic light scattering (DLS) to measure the sizes of product in solutions containing 200 µM Ag+ and different mixtures of GSH and Cys (Figure S13). The hydrodynamic diameter related to low concentration mixtures is apparently below 200 nm. For high concentration mixture (150 µM) the corresponding hydrodynamic diameter is greater than 500 nm. The low concentration mixtures produce low molecular weight polymers which can partially diffuse into Au@PDA NPs hydrogel and be available to form Ag_Au@PDA NPs. The high molecular weight polymers generated from 150 µM biothiol mixture can hardly diffuse into Au@PDA NPs hydrogel. As a result, Ag+-induced wavelength shift of Au@PDA NPs hydrogel fully recover as well as individual GSH or Cys. We test the selectivity of Au@PDA NPs hydrogel by using 19 kinds of common amino acids containing thiol-free groups (Table S1). At the same amount, most of amino acids do not

produce any effect on the Ag+-Au@PDA NPs system without any wavelength change of Au@PDA NPs hydrogel. Except for histidine and the acidic amino acid of aspartic acid and glutamic acid, they generate a weak wavelength change. As for a 10-folds concentration, these three molecules result in the significant wavelength change recovery of Au@PDA NPs hydrogel. This is due to the chelating interaction between carboxyl or imidazole group and Ag+. Colorimetric sensing of AChE activity and inhibitor effect test. The successful analysis of biothiols using Au@PDA NPs hydrogel inspires that the SRhG platform can be implemented to follow activation of biocatalytic cascades that involve thiol-generating esterase. We demonstrate it by following acetylcholinesterase/acetylthiocholine (AChE/ATCh) enzymatic cascade and detection of AChE activity and AChE inhibitor as a model system for sensing chemical warfare agents.62, 63 Acetylcholine (ACh) is a central neurotransmitter and involved in neurological disorders such as Alzheimer's disease. AChE-catalyzed hydrolysis of acetylcholine is the main regulating process of the neural response.64 The inhibition of AChE, for example, by nerve gases, leads to perturbations of nerval conduction process and rapid paralysis of vital living functions.65 Figure 6a shows the colorimetric approach to follow the AChE/ATCh cascade using Au@PDA NPs hydrogel. AChE catalyzes hydrolysis of ATCh into thiocholine. The resulting thiocholines coordinate with Ag+ producing macromolecular polymer, which prevent from forming Ag_Au@PDA NPs in hydrogel and recover the absorption peak of Au@PDA NPs hydrogel and the red color. We optimize the concentration of ATCh and Ag+ to ensure the stable and sensitive detection of AChE activity using


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Analytical Chemistry Au@PDA NPs hydrogel 96-arrays. Their concentrations are 300 µM and 600 µM for the next experiments (Figure S14). Also, we confirm that only in the presence of both AChE and ATCh the absorption peak of Au@PDA NPs hydrogel mostly recovers to the pristine state (Figure S15). Notably, the reaction time of enzyme-catalyzed hydrolysis directly affects the yield of thiocholine. The absorption spectra of Au@PDA NPs hydrogel after incubation with hydrolysis mixture solutions under different reaction time reveal that a reaction time of 60 minutes that can generate enough thiocholine recovering the absorption peak of hydrogel (Figure S16). For quantification, we introduce various amounts of AChE to the above described sensing system. With increasing [AChE], we observe that the absorption peak of Au@PDA NPs hydrogel (Figure 6b and c) gradually recover to longer wavelength with a maximum value of λ = 523 nm that near the pristine value of Au@PDA NPs hydrogel (λ=526 nm). The color of hydrogel also returns from yellow to red. The wavelength change of Au@PDA NPs hydrogel show a well linear fitting range of 2.5 ~ 25 mU/mL with R2 = 0.999. The limit of detection (LOD) value is 0.9 mU/mL (3.3σ/S), which is lower than many reported methods (Table S2). We test the selectivity of detecting AChE activity using several kinds of protein with a 100-fold higher concentration compared to AChE. Figure 6d depicts the wavelength change of Au@PDA NPs hydrogel upon different proteins (AChE, cholesterol esterase, alkaline phosphatase, glucoamylase, glucose oxidase, BSA, invertase, catalase and lipase). Even though some proteins especially BSA can bind with Ag+, the weak bindings do not involve chemical changes on Ag+ valence. Ag+ is still able to diffuse into hydrogel and available for the in situ chemistry-guided reduction and deposition of Ag NPs on surfaces of Au@PDA NPs. Among the tested proteins only AChE results in significant wavelength change, indicating the colorimetric SRhG platform of Au@PDA NPs hydrogel is highly selective for AChE activity assays. From a practical point of view, it is important to follow the inhibition assays of AChE. Toward this goal, we measure the inhibition of AChE by 5,9-methanocycloocta(b)pyridin-2(1h)one,5-amino-11-ethylidene-5,6,9,10-tetrah (huperzine-A) that mimics the function of nerve gases. In the presence of inhibited AChE/ATCh cascade system, the formation of thiolcholine is blocked, thus permitting the effective formation of Ag_Au@PDA NPs in hydrogel by Ag+-Au@PDA NPs reporter system. Accordingly, the AChE/ATCh enzymatic system is subjected to variable concentrations of huperzine-A at a fixed incubation time of 30 min. The wavelength change of incubated Au@PDA NPs hydrogel is recorded to probe the inhibition efficiency of huperzine-A (Figure 6e). Evidently, as the concentration of inhibitor increases, the formation of Ag_Au@PDA NPs successfully proceeds, that consistent with blue-shifting absorption peak of Au@PDA NPs hydrogel. The IC50 value of huperzine-A is 53.8 nM using Au@PDA NPs hydrogel. This value is similar to that of tested by other methods,66, 67 revealing the colorimetric SRhG of Au@PDA NPs hydrogel is reliable.

CONCLUSIONS In conclusion, we present here a new developed plasmonic SRhG of Au@PDA NPs hydrogel by combining the core-shell structured Au@PDA NPs with agarose hydrogel that can respond to Ag+ showing spectral shift and color change. By using TEM and FDTD simulations, we explore the mechanism

of producing Ag_Au@PDA NPs with core-satellites like structure in hydrogel that based on the in situ chemical deposition of Ag NPs on surfaces of Au@PDA NPs. The strongly coupling effect between Ag NPs and Au NP results SPR absorption peak blue-shift and color turn from red to yellow. Compared to the SRhG-based sensors that undergo structural changes, this rationally designed colorimetric SRhG of Au@PDA NPs hydrogel is more simple and convenient and portable. The solid Au@PDA NPs hydrogel shows greatly improved stability and anti-interference ability compared to the Au@PDA NPs solution, and thus exhibits advantage in analytical science. To high throughput analysis, the Au@PDA NPs hydrogel can be easily fabricated to 96-arrays using commercial 96-well plates. With rational designs, we use Au@PDA NPs hydrogel for colorimetric sensing of biothiols. And further supplementing with enzymatic-cascade system, Au@PDA NPs hydrogel can successfully apply for AChE activity assay and the related inhibitor effect tests with good sensitivity and selectivity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The detailed experimental protocols and extended characterizations of TEM, DLS, spectral simulation and UV-vis spectra.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Fax: +86-10-8254 5620 Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the Ministry of Science and Technology of China (2013YQ190467), Chinese Academy of Sciences (XDA09030305) and the National Science Foundation of China (81361140345, 21535001 and 81730051) for financial support.

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Hydrogels Incorporating Au@polydopamine Nanoparticles: Robust

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