Amphiphilic Polymer-Mediated Aggregation Induced Emission

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Biological and Medical Applications of Materials and Interfaces

Amphiphilic Polymer-Mediated Aggregation Induced Emission Nanoparticles for Highly Sensitive Organophosphorus Pesticide Biosensing Jianling Chen, Xiaojie Chen, Qiuyi Huang, Wenlang Li, Qiaoxi Yu, Longji Zhu, Tianwen Zhu, Siwei Liu, and Zhenguo Chi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10237 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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ACS Applied Materials & Interfaces

Amphiphilic Polymer-Mediated Aggregation Induced Emission Nanoparticles for Highly Sensitive Organophosphorus Pesticide Biosensing

Jianling Chen, Xiaojie Chen, Qiuyi Huang, Wenlang Li, Qiaoxi Yu, Longji Zhu, Tianwen Zhu, Siwei Liu*, and Zhenguo Chi*

PCFM Lab, GD HPPC Lab, Guangdong Engineering Technology Research Center for High Performance Organic and Polymer Photoelectric Functional Films, State Key Laboratory of Optoelectronic Material and Technologies, School of Chemistry, Sun Yat-sen University, Guangzhou, 510275, China

* Corresponding author. E-mail: [email protected]; [email protected]

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ABSTRACT Biosensing applications require signal reporters to be sufficiently stable and biosafe, as well as highly efficient. Aggregation induced emission (AIE) nanoparticles have proven to be capable of cell imaging and cancer therapy, however, realizing biomolecules sensitive detection remains great challenges because of their instability, biotoxicity and lack of modifiable functional groups. Herein, we report a self-assembling strategy to fabricate AIE nanoparticles (PTDNPs) through dispersion of amphiphilic polymers (PTD) in phosphate buffer saline (PBS). The PTD were prepared through radical co-polymerization of N-(1,2,2-triphenylvinyl)-4-acetylaniline (TPE-N-A) and dimethyl diallyl ammonium chloride (DMDAAC). We found that the particle size, morphology, functional groups and fluorescence property of PTDNPs can be fine-tuned. Further, PTDNPs-0.10 were chosen as signal reporters to detect organophosphorus pesticides (OPs) with the aid of gold nanoparticles (AuNPs). Their sensing performance on OPs is superior to that using C-dot/quantum dot/ rhodamine B as signal reporter. This study not only provides new possibilities to fabricate novel AIE nanoparticles with exceptional properties, but also facilitates the AIE nanoparticle’s application for target analytes biosensing. .

KEYWORDS:

Amphiphilic

polymer;

aggregation

induced

organophosphorus pesticide; gold nanoparticles; biosensing


emission

nanoparticle;

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INTRODUCTION Organophosphorus pesticides (OPs) play a key determinant in agricultural products growth and storage because of high efficacy and low toxicity.1,2 Meanwhile, it has been well accepted that OPs can depress the cholinesterase activity at low concentration,3,4 and thus are closely relevant to many diseases.5 In this context, it is highly urgent to develop sensitive OPs detection methods. Although many techniques, such as colorimetric,6,7 chemiluminescent8 and electrochemical assays9,10 have been designed to probe OPs, the disadvantages including low sensitivity, complex operation procedure and obligatory involvements of nucleic acid,11 antibody12 and multiple enzymes13 still exist, and significantly hinder their advanced utilizations. Fluorescent biosensor has been widely concerned in simple, fast and sensitive aspects,14,15 and has become a powerful tool for OPs biosensing. Further, it is accepted that analytical performance of a fluorescent biosensor depends mainly on characteristics of signal reporters. However, most of them applied in fluorescent analysis of OPs suffer from high toxicity,16 low luminescence efficiency17 and weak stability,18 making difficult to apply for practical application in biological samples. So, to realize OPs practical detection, the key challenge is to design and fabricate novel fluorescence materials that can address these above problems. Aggregation induced emission (AIE) nanoparticles possess advantages of strong brightness, low photobleaching, and high stability, and thus provide an alternative way for addressing the issues above mentioned.19-24 Up to now, various AIE nanoparticles have been fabricated and intensively used in biomedical field.25 However, it should be noted that these AIE nanoparticles have some intractable drawbacks. (1) Most of AIE nanoparticles suffered from



poisonous organic solvents/surfactants.26,27 (2) Usually, these AIE nanoparticles are unstable in biological solution and their diameters are larger than 10 nm, making them detrimental to practical applications.28,29 (3) Most of them lack modifiable functional groups, which are not of benefit for them used in chemo/biosensing field.30,31 (4) Moreover, most of them are mainly used in cell imaging and cancer treatment, and there is little information on the biological sensing of target metabolites.32,33 (5) In addition, previously reported AIE nanoparticles were independent and unrelated, leading to a lack of systematic investigation.34,35 Self assembly of amphiphilic polymer is an effective strategy for fabricating well-defined nanoparticles, which proves that the method has better uniform dispersion, biosafety and surface function than precipitation or emulsion polymerization methods.36,37 If AIE moiety and amphiphilic polymer are combined, AIE amphiphilic polymer could be prepared, and in turn can regulate the performance of AIE nanoparticles by adjusting the composition. Herein, a novel kind of AIE amphiphilic polymers, PTD, has been successfully prepared through radical co-polymerization of N-(1,2,2-triphenylvinyl)-4-acetylaniline (TPE-N-A) and dimethyl diallyl ammonium chloride (DMDAAC). The self-assembling behavior of the PTD was comprehensively investigated via changing the mass ratio between TPE-N-A and DMDAAC. The results demonstrated AIE assemblies (PTDNPs) with controllable size, fluorescence brightness and surface charge density could be readily prepared through self-assembling of PTD in buffer solution. In view of the unique features of PTDNPs, we apply them as signal reporters in development of biosensor for OPs sensitive detection with the aid of AuNPs. Compared with other OPs biosensors,6-8 the detection limit is relatively low, and the linear range is wide. We believe this strategy will present a new path to prepare AIE


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nanoparticles and broaden their application in the field of biosensing. EXPERIMENTAL SECTION Synthesis of TPE-N-A. Acrylic acid (0.58 g, 0.008 mol) was dissolved in 50 mL CH2Cl2, and then EDC (3.54g, 0.008 mol) and NHS (0.18g, 0.0016 mol) were added. The reaction solution was stirred for 30 min at room temperature. Subsequently, TPE-NH2 (1.39 g, 0.004 mol) was added and the mixture was allowed to react for 6.0 h. The crude product was purified through silica gel column chromatography (n-hexane:CH2Cl2, 2:1) to obtain the pure product (1.14 g, yield 71%). 1H NMR (300 MHz, CDCl3) δ: 5.72-5.76 (d, 1H), 6.16-6.23 (dd, 1H), 6.36-6.42 (d, 1H), 6.95-7.20 (m, 18H), 7.3-7.35 (d, 2H); FT-IR (KBr) υ/cm-1: 571, 612, 669, 698, 746, 759, 796, 815, 849, 956, 974, 1034, 1189, 1244, 1320, 1406, 1443, 1493, 1514, 1664 (C=O), 3020 (C-H, Ar-H), 3047 (C-H, Ar-H), 3331 (N-H). HRMS, m/z: 402.2 ([M]+, calcd for C29H23NO, 402). Synthesis of amphiphilic polymer PTD. DMDAAC (0.37 g) was dissolved in 5 mL H2O, then 25 mL dioxane solution containing 0.10 g TPE-N-A was added. After stirring for 10 min, AIBN was added and the mixture was stirred at 70 oC for 24 h. The precipitate was sequentially washed with CH2Cl2 and ethanol to obtain white powder, denoted as PTD-0.10. FT-IR (KBr) υ/cm-1: 618, 671, 720, 845, 950, 983, 1078, 1196, 1320, 1340, 1418, 1452, 1479, 1635 (C=O), 2942 (C-H, Ar-H), 3014 (C-H, Ar-H), 3445 (N-H). As control samples, PTD-0.05, PTD-0.15, and PTD-0.20 were fabricated through the same procedure. Preparation of PTDNPs through self assembling process. 2.4 mg PTD-0.10 was dispersed in 15 mL of PBS buffer (10 mM, pH 8.5), then the solution was heated to 80 oC for



30 min to make PTD fully dispersed. After that, the mixture was cooled down to 37 oC and sonicated for 2.0 h to provide a homogeneous dispersion. Subsequently, PTD nanoparticles, denoted as PTDNPs-0.10, generated. For PTD-0.05, PTD-0.15, and PTD-0.20, the assemblies were fabricated using the same procedure with PTDNPs-0.10, and denoted as PTDNPs-0.05, PTDNPs-0.15, and PTDNPs-0.20, respectively. Preparation of AuNPs. Negative AuNPs were synthesized according to previously reported method. Briefly, 1 mL chloroauric acid was dissolved in 24 mL H2O, then the mixture was stirred at 100 oC for 10 min. Subsequently, 9.7 mM trisodium citrate solution (2.5 mL) was added, and the reaction solution was further stirred for 10 min to prepare AuNPs. Fluorescence detection of AChE based on PTDNPs-0.10 and AuNPs. First, 50 µL of AuNPs solution (16 nM) and 50 µL of PTDNPs-0.10 solution (640 µg/mL) were mixed at 37 oC.

Subsequently, 50 µL of ATCh solution (12 µM) and 50 µL of AChE solution with

different concentrations were successively added into the reaction solution. The resulting solution was permitted to react for 25 min at 37 oC before fluorescence measurement. Fluorescence detection of OPs based on PTDNPs-0.10 and AuNPs. Paraoxon dissolves in a PBS buffer containing 5% ethanol to form a different concentration of paraoxon solution. Prior to be analyzed, the paraoxon solution was heated to 90 oC for 10 min. After cooling down to 37 oC, 25 µL of paraoxon solution with different concentrations and 25 µL of AChE solution (72 mU/mL) were mixed and incubated for 20 min. Subsequently, 50 µL of ATCh solution (12 µM) and 100 µL mixed solution comprising of AuNPs and PTDNPs-0.10


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solution were added. The resulting solution was permitted to react for 25 min at 37 oC before fluorescence measurement.

Figure 1. Synthetic route to polymer PTD. RESULTS AND DISCUSSION Characterization of PTDNPs. The synthetic route to PTD is depicted in Figure 1. Monomer TPE-N-A was prepared in a 71% yield via Suzuki and dehydration condensation reaction with AIE characteristic in THF-H2O mixture (Figure S1). PTD was obtained through radical co-polymerization of DMDAAC and TPE-N-A. For PTD-0.10, 1H NMR result demonstrated that the real mass ratio of TPE-N-A and DMDAAC was 0.29 (Figure S2), which is close to feed ratio (0.10/0.37). In addition, viscosity average molecular weight was detected to be 99800 by using Ubbelohde Viscometer technique.38 Further, fluorescence technique was applied to investigate the optical property of PTD with different compositions (PTD-0.05, PTD-0.10, PTD-0.15 and PTD-0.20). As shown in Figure 2A-D, FL intensity gradually increased with elevating PTD concentration. Based on the luminescence mechanism of AIE dyes, the PTD aggregates (PTDNPs) generated at higher concentration due to their 7 ACS Paragon Plus Environment


enhanced light emission, and the corresponding schematic diagram was demonstrated in Figure 2E. When they were added into the buffer solution, the hydrophobic segments (TPE-N-A) are prone to aggregate into nuclear while the hydrophilic segments (DMDAAC) tend to dissolve in solution, subsequently contributing to the formation of assemblies. Meanwhile, fluorescence tests demonstrated that the FL intensity increased with the increase in feeding ratio of TPE-N-A and DMDAAC (PTD concentration: 80 μg/mL; Figure S3). Evidently, the weak signal of 3.56 a.u. was obtained for PTD-0.05, as well as relatively high FL intensity of 117.8 a.u. was observed for PTD-0.10. When the mass ratio of TPE-N-A/DMDAAC was further increased to 0.15/0.37 and 0.20/0.37, FL intensity was increased to 903 a.u. and 2105 a.u, respectively. It could be ascribed to different self assembling behavior of PTD with different components. With the amount of DMDAAC increasing, the prepared PTD holds fewer fluorescence units as well as more quaternary ammonium groups, which contributed to their excellent solubility, subsequently resulting in weak fluorescence. Therefore, FL intensity can be improved by increasing the amount of TPE-N-A. However, the prepared PTD would lack of polar groups, leading to poor water dispersibility (Figure S4) and irregular morphology.


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Figure 2. Fluorescence spectra of PTD with different concentrations in PBS buffer: (A) PTD-0.05: 5, 10, 20, 40, 80, 160, 320, 480, 960, and 1000 µg/mL; (B) PTD-0.10: 5, 10, 20, 40, 80, 160, 320, 480, and 500 µg/mL; (C) PTD-0.15: 1, 2, 3, 4, 5, 10, 20, 40, 80, 160, and 320 µg/mL; (D) PTD-0.20: 0.1, 0.5, 1, 5, 10, 20, 40, 80, and 160 µg/mL; (E) Schematic representation for PTDNPs fabrication through self-assembling strategy. TEM technique was further employed to characterize the size and morphology of PTDNPs (Figure 3). Spherical nanoparticles with size of 4.3 nm were seen for PTD-0.10, while larger nanoparticles of 120 nm in diameter were obtained for PTD-0.15. With the amount of TPE-N-A further increasing, for PTD-0.20, the morphology presented an irregular structure (Figure 3D). However, for PTD-0.05, there were no aggregates observed. It could be ascribed



to different feeding ratio of TPE-N-A and DMDAAC. With the amount of TPE-N-A increasing, PTD possesses more and more hydrophobic units, which make the size growing until irregular assemblies appeared. On the contrary, with the amount of DMDAAC increasing, the as-synthesized PTD has more hydrophilic units, causing it to dissolve completely in solution, thus preventing the formation of aggregates. On this basis, we could adjust the morphology and particle size of PTDNPs by changing the mass ratio between TPE-N-A and DMDAAC. Furthermore, zeta potential measurements showed that PTDNPs-0.10 and PTDNPs-0.15 possessed positive charge with +23.56 mV and +17.83 mV, respectively, indicating good stability and positive charge on the surface. The reason for this is due to quaternary ammonium groups in polymer skeleton. In addition, it is noted that the zeta value decreased with increasing the amount of TPE-N-A, which is consistent with the fact that only DMDAAC contributes to positive charge.

Figure 3. TEM images of PTDNPs-0.05 (A), PTDNPs-0.1 (B), PTDNPs-0.15 (C), and PTDNPs-0.20 (D) in PBS buffer. 10 ACS Paragon Plus Environment

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Figure 4. (A) FL intensity of PTDNPs-0.10 vs different pH values. (B) FL intensity of PTDNPs-0.10 in different solution: (a) blank, (b) NaCl, (c) KCl, (d) MgCl2, (e) CaCl2, (f) Na2SO4, (g) Na2CO3, (h) H2O2, and (i) thiophenol. The concentration of all substances was 10 mM. As is well known, strong fluorescence, functional groups, as well as small size are beneficial for AIE nanoparticles to develop biosensors with high sensitivity and good selectivity. Taking account of these factors, PTDNPs-0.10 were chosen as the ideal fluorescence nanoparticles for developing bioassay. For advanced utilization of PTDNPs-0.10 in sensing field, both stability and anti-interference ability are studied (Figure 4). The FL intensity of PTDNPs-0.10 changed negligibly with pH value elevating from 6.0 to 10.5. Moreover, various metal ions, anions, thiol compound, and reactive oxygen species (ROS) made little influence too. In addition, PTDNPs-0.10 made little influence on AChE activity and enjoyed excellent biocompatibility (Figure S5). This information means that the prepared PTDNPs-0.10 has significant stability and good biocompatibility, and makes it a promising candidate for biosensor development. Design of PTDNPs-AuNPs platform. Based on the above analysis, PTDNPs-0.10 was chosen as signal reporters to construct platform for OPs assay with the aid of AuNPs. AuNPs 11 ACS Paragon Plus Environment


have been widely applied in analysis of biomolecules through mixing with polymers.39-41 In the present study, AuNPs were fabricated via a redox reaction between sodium citrate and HAuCl4, and characterized by TEM, UV-vis and zeta potential techniques (Figure S6). The results illustrated that the AuNPs were spherical with the diameter of 13.5 nm and negatively charged with zeta value of -25.64 mV. In addition, AuNPs have a broad UV-Vis absorption spectrum (400-700 nm) and a high molar absorptivity [3.578×108L/(mol·cm)] at 520 nM, which decide in favor of PTDNPs’ fluorescence quenching.42,43 The interaction between AuNPs and PTDNPs-0.10 are depicted in Figure 5A. Without conjugation to AuNPs, PTDNPs-0.10 emits strong blue fluorescence. However, the absorption spectrum of AuNPs exhibits complete overlap with the emission band of PTDNPs-0.10 (Figure 5B), which provided an opportunity for fluorescence resonance energy transfer (FRET) between PTDNPs-0.10 and AuNPs. As expected, the fluorescence of PTDNPs-0.10 can be quenched by AuNPs, and the FL intensity is negatively related to AuNPs amount (Figure 5C and Figure S7). For example, with AuNPs concentration increasing from 0 to 4.0 nM, the FL intensity reduced from 231.8 to 17.6 a.u.. With AuNPs concentration further increasing, the FL intensity decreased slightly, attributable to saturated electrostatic interaction between them. In view of the cost and sensing ability, 160 µg/mL PTDNPs-0.10 and 4.0 nM AuNPs were selected for the subsequent biosensor development. To investigate the interaction mechanism, UV-vis and TEM characterizations were conducted and the results were demonstrated in Figure 5D and E. Evidently, the absorption peak of AuNPs changed from 520 to 639 nm in conjugation with PTDNPs-0.10, strongly arguing the aggregation occurrence induced by PTDNPs. TEM image in Figure 5E can


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directly verify the formation of AuNPs aggregates. This may be because PTDNPs-0.10 possessed abundant reaction sites (positive charge), which reacted with negative charge on AuNPs

surface,

subsequently

resulting

in

the

aggregation.

Negatively

charged

deoxyribonucleic acid (DNA) was used to further justify the electrostatic interaction between PTDNPs-0.10 and AuNPs (Figure 5F and G). PTDNPs-0.10 was first incubated with DNA for 30 min to generate DNA-coated PTDNPs-0.10. The DNA-coated PTDNPs-0.10 had the zeta value of -16.57 mV, which was different from that of PTDNPs-0.10 alone. It demonstrated the positively charged PTDNPs-0.10 were covered with negative charges after DNA treatment. As expected, the fluorescence of PTDNPs-0.10 could not be quenched by AuNPs due to electrostatic repulsion. Moreover, metal ions, anions, and ROS have little interference with the interaction between PTDNPs and AuNPs. Thiol compound exerts a significant influence due to strong interaction of –SH and Au. Fortunately, this can be readily addressed through heating treatment (Figure S8). In short, we get the exciting information that PTDNPs-0.10 can effectively bind to AuNPs, and the fluorescence can be quenched through the FRET effect.



Figure 5. (A) Schematic representation of interaction between PTDNPs and AuNPs. (B) Normalized fluorescence spectrum of PTDNPs-0.10 (a) and absorption spectrum of AuNPs (b). (C) Fluorescence response of PTDNPs-0.10 to different AuNPs concentrations: 0, 1, 2, 3, 4, 5, 6, and 7 nM. (D) Normalized absorption spectra of PTDNPs-0.10 (a), AuNPs (b), and PTDNPs-AuNPs (c). (E) TEM image of PTDNPs-AuNPs. (F) Scheme of influence of DNA on interaction of PTDNPs-AuNPs. (G) FL intensity vs different samples: (a) PTDNPs-0.10, (b) PTDNPs-AuNPs, (c) PTDNPs-DNA, and (d) PTDNPs-DNA-AuNPs.


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To prove the advantages of PTDNPs-0.10 in development of biosensor, we chose PTDNPs-0.15, PTDNPs-0.20, TPE-N-A and methylene blue (MB) as contrast substances to evaluate the quenching behavior under the same conditions (Figure 6A). Among them, PTDNPs-0.10 participated biosensor shows the best quenching efficiency of 92.41%, which is superior to that of contrast substances. It can be attributed to its ultra-small size and high positive surface charge, allowing infinite contact and sufficient interaction with AuNPs. Further, the response of PTDNPs-AuNPs to acetylcholinesterase (AChE) and OPs was investigated (Figure 6B). It is worth noting that AChE-catalyzed hydrolysis product (thiocholine) competitively interacts with AuNPs and keeps AuNPs away from PTDNPs, subsequently blocking FRET and enhancing the fluorescence. Other quaternary ammonium, such as lecithin or glycine betaine, can not increase the fluorescence signal because of no hydrosulfide group (Figure S9). However, when paraoxon (selected as model OP) was pre-incubated with AChE, the activity of AChE was depressed effectively,

6-10

indicating

there was no thiocholine generated. Thus, the depression of AChE activity caused FRET effect to be blocked, and no enhanced fluorescence was detected. To confirm the working principle, fluorescent experiments were carried out under different conditions (Figure 6C). When PTDNPs-0.10, AuNPs, AChE, and ATCh coexist in solution, FL intensity is significantly enhanced compared with individual PTDNPs-AuNPs. However, when AChE or ATCh is absent from the system, the FL intensity is barely increased. This is in good agreement with the sensing principle that only thiocholine originating from AChE-catalyzed hydrolysis on ATCh can result in separation of PTDNPs-0.10 and AuNPs, subsequently enhancing the fluorescence. When paraoxon is added, AChE activity is inhibited,



making PTDNPs and AuNPs unable to stay away from each other, so FL intensity cannot be enhanced. The results suggested that the proposed electrostatic adsorption/repulsion strategy was feasible for developing PTDNPs-AuNPs biosensor for AChE and OPs assay.

Figure 6. (A) Quenching percentages of PTDNPs-0.10 and compared materials by AuNPs. (B) Fluorescence curves of PTDNPs-AuNPs under different conditions: (a) PTDNPs-AuNPs, (b) PTDNPs-AuNPs + AChE, (c) PTDNPs-AuNPs + ATCh, (d) PTDNPs-AuNPs + paraoxon, (e) PTDNPs-AuNPs + ATCh + AChE, and (f) PTDNPs-AuNPs + ATCh + AChE + paraoxon. (C) Schematic representation of PTDNPs-0.10 participated biosensor for highly sensitive detection of AChE and OPs. AChE-initiated fluorescence recovery. Before using our platform for OPs assay, we investigated the response of PTDNPs-AuNPs to AChE and carefully optimized the working conditions that might affect sensing performance (Figure S10). Firstly, the ATCh concentration was studied. When the concentration of ATCh gradually increased to 3.0 µM, FL intensity was positively correlated with its content and reached a plateau, indicating that 16 ACS Paragon Plus Environment

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the optimum concentration was 3.0 µM. Secondly, the incubation time of AChE, ATCh and PTDNPs-AuNPs was another key factor affecting fluorescence signals. To achieve the highest sensitivity, sufficient time is required to keep PTDNPs-0.10 away from AuNPs and break FRET. However, too long time would not make fluorescence stronger. Thus, 25 min was chosen as the optimal time for whole experiments (Figure S10B). Further, the responsive ability of PTDNPs-AuNPs to different AChE amounts was evaluated, and the results were manifested in Figure 7. It can be seen that the FL intensity increases gradually with the increase of AChE concentration, and shows a positive linear relationship between 0.10 ~ 9.0 mU/mL. The linear equation was calculated to be F = 18.7CAChE + 24.83, with R2 = 0.9930. These results have been successful in proving that PTDNPs-AuNPs has excellent AChE sensing performance.

Figure 7. (A) Fluorescence curves of PTDNPs-AuNPs to different AChE concentrations: 0.1, 0.5, 1, 3, 5, 7, 9, and 12 mU/mL. (B) Working curve for AChE detection. Sensitive detection of paraoxon. Based on the unique response of PTDNPs-AuNPs toward AChE and effect inhibition of OPs on AChE, we proposed a fluorescent OPs sensing strategy. Under the optimized conditions (Figure S11), paraoxon was applied as model OPs to verify the sensitivity and selectivity. It was noted that FL intensity decreased gradually 17 ACS Paragon Plus Environment


with paraoxon concentration increasing (Figure 8), presenting a clear indication of effective inhibition of paraoxon on AChE and negative correlation between paraoxon and FL intensity. In order to quantitatively detect paraoxon, standard curve was fitted between FL intensity and paraoxon concentration in the range of 0.8-60 ng/mL. The linear regression equation is F = -2.63Cparaoxon + 186.59 (R2=0.9910). The detection limit (LOD) is calculated to be 0.38 ng/mL, which satisfies the test requirements of paraoxon in real samples (10 ng/mL), and is comparable or better than other methods (Table S1). Furthermore, it should be noted that the linear range is wider and LOD is lower than that of previously developed sensors, which used C-dot/ quantum dot/ rhodamine B44-46 as signal reporters and AuNPs as nanoquencher.

Figure 8. Fluorescence spectra of PTDNPs-AuNPs in response to different paraoxon amount: 0.8, 3, 5, 10, 20, 40, 60, 80, and 100 ng/mL. (B) Linear plot of fluorescence change at 478 nm vs paraoxon concentration. In view of the sensing results and working principle, we expect that PTDNPs-AuNPs would be also suitable for other OPs sensing. To illustrate the universality, we selected four other OPs (chlorpyrifos, malathion, diazinon and parathion) for comparative experiments (Figure S12). It has been reported that chlorpyrifos/malathion/diazinon/parathion can be used like paraoxon to inhibit AChE activity.11-13 Thus, in the presence of one of these four OPs, the 18 ACS Paragon Plus Environment

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reaction solution was expected to emit weak fluorescence. FL intensity at 478 nm was determined to be only 36/34/38/34.6 a.u. for chlorpyrifos/malathion/diazinon/parathion, respectively, suggesting the good suitability and versatility of the established platform for OPs biosensing. Meanwhile, three other substances including nitrobenzene, 4-nitrophenol, and diethyl(4-methoxybenzyl)phosphonate that having the similar structure with paraoxon were applied to confirm the selectivity of the proposed biosensor for OPs biosensing (Figure S12). Strong fluorescence was observed due to the weak inhibition on AChE activity, indicating the good specificity. Table 1. Detection of paraoxon spiked in East Lake water (A) and cabbage extract (B) samples (n = 3)

Sample No.

A

B aRecovery

Added (ng/mL)

Mean measured (ng/mL) This method

HPLC

Mean recovery a (%)

RSD (%)

1

5

5.38

5.17

107.20

3.16

2

20

18.33

20.13

96.65

2.67

3

40

37.20

38.56

93

3.01

1

5

4.74

4.92

94.48

5.71

2

20

21.08

22.63

105.40

4.29

3

40

42.36

43.08

105.90

4.58

(%) = 100  (cmean measured / cadded)

The practical application of PTDNPs-AuNPs in real sample was studied by probing paraoxon in East Lake water and cabbage extract through the standard addition method, respectively. Prior to recovery study, the amounts of paraoxon in East Lake water and cabbage extract were measured by our sensing strategy and high-performance liquid chromatography (HPLC), respectively. The experimental results show that the test samples contained no or trace amount of paraoxon, which could not be detected by HPLC or our 19 ACS Paragon Plus Environment


established platform. As shown in Table 1, for 5, 20 and 40 ng/mL paraoxon detection, the recoveries of 107.2%, 96.5%, 93%, 94.48%, 105.40%, and 105.90% were gotten with low relative standard deviations (RSDs). In addition, the sensing results were consistent with that of HPLC, suggesting the success of OPs detection in real samples. CONCLUSIONS In summary, we have successfully fabricated a novel kind of amphiphilic polymers containing AIE-active fluorophores and quaternary ammonium groups through radical polymerization. The polymers could self-assemble into nanoparticles (PTDNPs) with the features of controllable size, morphology, functional groups and fluorescence property. Meanwhile, PTDNPs have significant stability and strong anti-interference ability. Further, with the aid of AuNPs, a high-performance platform was constructed from PTDNPs-0.10, and it was proved to have exceptional sensing performance on AChE and OPs, such as the enhanced sensitivity and wider linear range. We expect this work will not only paves a new way to development of high-performance AIE nanoparticles, but also enriches the AIE nanoparticle functionalization with other materials, such as enzyme, protein and metal oxide, which are of benefit to broaden the application scope of AIE nanoparticles. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (NSFC: 51733010, 21672267), Science and Technology Planning Project of Guangdong (2015B090913003), and the Fundamental Research Funds for the Central Universities.


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ASSOCIATED CONTENT Supporting Information Available: Real sample analysis; fluorescence information of PTDNPs and PTDNPs-AuNPs under different conditions; images of PTDNPs; TEM image of AuNPs; UV-vis spectrum of AuNPs. These materials are available free of charge via the Internet at http://pubs.acs.org.



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SYNOPSIS TOC


Amphiphilic Polymer-Mediated Aggregation Induced Emission

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