Covalent Surface Functionalization of Semiconducting Polymer Dots

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Covalent Surface Functionalization of Semiconducting Polymer Dots with beta-Cyclodextrin for Fluorescent Ratiometric Assay of Cholesterol through Host-guest Inclusion and FRET Junyong Sun, Sufan Wang, and Feng Gao Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03002 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 7, 2016

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Covalent Surface Functionalization of Semiconducting Polymer Dots with beta-Cyclodextrin for Fluorescent Ratiometric Assay of Cholesterol through Host-guest Inclusion and FRET Junyong Sun, Sufan Wang, and Feng Gao*

*Corresponding author. Tel. +86-553-3937137; Fax: +86-553-3869303 E-mail: [email protected].

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ABSTRACT: Special functionalization of semiconducting polymer dots (Pdots) is highly desired to expand

their

applications

in

chemo

/

biosening.

Herein,

carboxyl-functionalized

poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)] dots covalently tagged with aminated beta-cyclodextrin (NH2-CD), have been designed to construct a ratiometric sensor for cholesterol (Cho). Using CD-Pdots as energy donors while rhodamine B (RB) as energy acceptors, a fluorescence resonance energy transfer (FRET) pair has been built because the host-guest interaction between RB and CD attached onto Pdots brings donors and acceptors into close proximity. In the presence of Cho, the acceptors will depart from the donors due to the competitive inclusion interaction between Cho and RB with CD, resulting in the hindering of FRET process between CD-Pdots and RB. Based on the turn-on fluorescence of CD-Pdots and turn-off fluorescence of RB, a sensitive ratiometric method for the determination of Cho in concentration range from 25 to 350 nM with a detection limit of 4.9 nM was achieved. The proposed method was validated to determine free Cho in human serum samples with satisfactory results.

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1. INTRODUCTION Semiconducting polymer dots (Pdots) have recently emerged as soft fluorescent nanomaterials1,2 and displayed more competent applications in fluorescent chemo/biosensing, bioimaging and biomedicine comparing to solid fluorescent nanoparticles owing to their extraordinary fluorescence brightness, amplified energy transfer, fast emission rate, excellent photostability, nonblinking, and nontoxic features.3-6 Currently, much attention has been focused on rationally functionalized modification of Pdots to expand their applications in chemo/biosening and biomedicine.7-9 Although several fluorescent chemo/biosensors based on Pdots have been developed for sensing tyrosinase,10

pH,11

temperature,12

Cu2+,13

F-,14

hypoxia,15

protease

(MMP-2)

16

and

carcinoembryonic antigen (CEA),17 such limited applications do not match with the distinguished properties and intrinsic merits of Pdots. The fabrication of versatile Pdots with special sensing function via universal strategies remains a challenge and is highly desired. Cholesterol (Cho), the most abundant sterol in animal tissues, is an essential constituent of cell membranes for maintaining their function and structure.18 Importantly, Cho is also a precursor for the generation of steroids and bile acids, which is attributed to its solubilization of other lipids and function as signal transducers.19 It has been proved that the abnormal levels of Cho are associated with many diseases including anemia, hyperthyroidism, myxedema, atherosclerosis, diabetes mellitus and jaundice.20 The measurement of Cho in biological fluids and tissues has been considered as an essential routine analysis in diagnosis and treatment of those diseases. Up to now, a variety of methods have been developed for the detection of Cho such as gas chromatography,21 colorimetric methods,22,23 electrochemical methods,24,25 and fluorescence-based methods.26 Among these developed methods, the fluorimetry is a promising method due to its merits including high sensitivity and operation convenience. The majority of the reported fluorescent probes for Cho detection,27-30 which are based on fluorescence enhancement or quenching of probe molecules in the presence of Cho, are generally easily affected by environment change and instrument factors. The 3


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ratiometric fluorescent probes based on the relative intensity variation of two emissions may solve or reduce this interference.31,

32

A few ratiometric Cho probes using upconverting inorganic

luminescent nanoparticles have been reported33 and displayed complicated prepared process of probe and high detection limit. Furthermore, the poor biocompatibility hampers these nanoparticles for further bioapplications.1 In this study, we have synthesized novel versatile Pdots (CD-Pdots), carboxyl-functionalized poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)]

dots

(COOH-Pdots)

chemically modified with aminated beta-cyclodextrin (NH2-CD). Using the prepared CD-Pdots as fluorescence signal transducer coupling with the host-guest recognition of beta-cyclodextrin (CD) and fluorescence resonance energy transfer (FRET), a ratiometric sensor for Cho detection has been proposed. As shown in Scheme 1, using CD-Pdots as energy donors while rhodamine B (RB) as energy acceptors, a FRET pair has been built, which turns off the fluorescence of CD-Pdots via FRET because the host-guest interaction between RB and CD attached onto Pdots brings donors-acceptors pair into close proximity. When Cho is introduced into this system, the acceptors RB will depart from the donors CD-Pdots due to the competitive inclusion interaction between Cho and RB with CD, leading to the suppression of FRET process between CD-Pdots and RB, and therefore turning on the fluorescence of CD-Pdots accompanying by the fluorescence quenching of RB. The varies of fluorescence intensities of RB and CD-Pdots are synchronous and provide the precondition for ratiometric sensing. Obviously, the more amount of Cho are introduced, the higher inhibition level of FRET gets, suggesting that the ratiometric signals (i.e., the ratios of fluorescence intensities of CD-Pdots and RB) can be used as the indicators of the amount of added Cho. Based on the restored fluorescence of CD-Pdots and quenched fluorescence of RB, a sensitive and selective fluorescent sensor for the ratiometric determination of Cho has been demonstrated. 4


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Scheme 1. Schematic illustration of functionalized Pdots for ratiometric determination of Cho.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Instrumentals. All chemicals were used as received from the commercial suppliers.

Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1’,3}-thiadiazole)]

(PFBT,

polydispersity 3.4, MW 164000 ) was purchased from ADS Dyes, Inc. (Quebec, Canada). Poly(styrene-co-maleic anhydride) (PSMA), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride

(EDC),

tetrahydrofuran

(THF,

anhydrous,

≥99.9%,

inhibitor-free),

2-(4-(2-hydroxyethyl)-1-piperazinyl) ethanesulfonic acid buffer (HEPES), bovine serum albumin (BSA), polyethylene glycol (PEG, MW 3350), sodium dodecyl sulphate (SDS), dimyristoyl phosphatidyl choline (DMPC), quinidine, pivoxicam, and cortisol were purchased from Sigma-Aldrich. Amino-beta-cyclodextrin (NH2-CD) was procured from Shandong Binzhou Zhiyuan BioTechnology Co., Ltd. (Shandong, China). All other reagents were of analytical reagent grade and obtained from Aladdin (Shanghai, China). Ultrapure water produced by a Milli-Q ultrapure water system was used throughout the experiments.

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UV-vis absorption spectra and fluorescence spectra were acquired on an Lambda 35 spectrophotometer

(PerkinElmer,

USA)

and

an

LS-55

fluorescence

spectrophotometer

(PerkinElmer, USA), respectively. Transmission electron micrograph (TEM) was conducted on a HT 7700 transmission electron microscopy (Hitachi, Japan) at an acceleration voltage of 100 kV. Zeta potential and dynamic light scattering (DLS) were carried out using a ZS90 Zetasizer Nano instrument (Malvern, UK). 1H NMR were recorded on a 500 MHz NMR spectrometer (Burker, Swiss) at room temperature. 2.2. Preparation of COOH-Pdots and NH2-CD Functionalized Pdots. The COOH-Pdots in aqueous solution were prepared according to previously reported procedures. 34, 35 In brief, the polymer PFBT and amphiphilic copolymer PSMA were dissolved in THF to make a stock solution of 1 mg mL-1. Then a 5 mL THF homogeneous solution containing 50 µg mL-1 PFBT and 10 µg mL-1 PSMA was added into 10 mL of ultrapure water quickly with a vigorous bath sonicator. The THF was removed by nitrogen stripping, followed by filtration through a 0.2 µm membrane filter. The as-prepared COOH-Pdots were clear and stable for months without aggregation. Surface conjugation was performed by utilizing the EDC-catalyzed reaction between COOH-Pdots and NH2-CD. 34,36 In a typical conjugation reaction, 100 µL of PEG (5% w/v) and 100 µL of 1.0 M HEPES were added to 5 mL of 50 µg mL-1 COOH-Pdots solution, respectively and then 50 µL of 1.0 mg mL-1NH2-CD was added to the solution and mixed thoroughly on a vortex. After that, 100 µL of 10.0 mg mL-1 freshly-prepared EDC solution was added to the mixed solution, and magnetically stirred for 4 hours at room temperature, and then 100 µL of BSA (10% w/v) solution was added into the reaction solution and incubated with another 20 min at 30 oC. Finally, the resulting CD-Pdots were separated from free molecules through a centrifugal filtration device (Amicon Ultra-4; MWCO: 100 kDa). 2.3. Preparation of RB@CD-Pdots. For the preparation of RB@CD-Pdots, 30 µL of 500 µM RB was added into 3 mL of 50 µg mL-1 CD-Pdots solution and incubated for 3 h at 30oC to 6


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guarantee the thorough inclusion of CD-Pdots with RB. Thereafter, RB@CD-Pdots were purified by centrifugal filter device (Amicon Ultra-4; MWCO: 100 kDa), and dissolved in 20 mM HEPES buffer (pH 7.4) to get a concentration of ca. 5 µg mL-1 solution. Typically, less than 10 % of the Pdots were lost during the filtration process by comparing the absorbance at 450 nm. 2.4. Procedures for Cho Detection. In a series of 2 mL colorimetric tubes, 100 µL of 5 µg mL-1 RB@CD-Pdots and different amount of Cho were added, and then the mixtures were diluted to 1 mL with HEPES buffer solution (pH 7.4, 20 mM) and incubated at 37 oC for 30 min. The fluorescence spectra were collected at an excitation of 450 nm. The human serum samples were provided and pretreatmented by the hospital of Anhui normal university. Prior to detection, human serum samples were diluted by ethanol with a volume ratio of 1:500. Then, 10µL of human serum samples were collected for assaying free Cho under the conditions described above.

3. RESULTS AND DISCUSSION

3.1. Preparation and Characterization of the RB@CD-Pdots. The prepared Pdots were functionalized with NH2-CD using the typical carboxyl-amine coupling reaction catalyzed by EDC and their morphologies were studied by the TEM and DLS measurements. As shown in Figure 1A and C, the COOH-Pdots display nearly uniform spherical shapes with an average diameter of ca. 23 nm. The sizes of CD-Pdots are slightly larger than those of COOH-Pdots and display an average diameter of ca. 26 nm, as shown in Figure 1B and D, indicating that the covalent immobilization of COOH-Pdots with NH2-CD was achieved successfully. The functionalization processes were also monitored by zeta potential. The surface zeta potentials of COOH-Pdots and CD-Pdots were measured to be -33.3 mV and -30.5 mV, respectively, indicating that the charges of COOH-Pdots

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were reduced after they conjugate with NH2-CD via EDC-catalyzed carboxyl-amine coupling reaction. The coupling reaction between COOH-Pdots and NH2-CD was further confirmed by agarose gel electrophoresis analysis. As shown in Figure 1E, we can see that the signal intensity of the lane for CD-Pdots is lower than that of other lanes, suggesting that COOH-Pdots is successfully conjugated with NH2-CD. The fluorescence quantum yield of the prepared CD-Pdots was determinated to be of 0.26 using a dilute solution of Coumarin 6 in ethanol as a reference substance, and the amounts of CD anchored on the surface of the COOH-Pdots were calculated to be 751 based on the carboxyl group content of COOH-Pdots.34 CD anchored on the surface of nanoparticles can give the capability of further assembly with various functional receptors via host-guest interaction. 37,38 In this study, when RB was mixed with CD-Pdots, the colour of the nanocomposites solution was changed from green yellow to orange under UV light radiation, as shown in Figure 1F, which is attributed to the inclusion of RB with CD anchored on the CD-Pdots surface through host-guest interaction. Taking advantage of the host-guest recognition, the functionalized Pdots, CD-Pdots are envisaged as a selective probe to sense Cho.

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Figure 1. (A, B) TEM measurements of COOH-Pdots and CD-Pdots. (C, D) Dynamic light scattering of COOH-Pdots and CD-Pdots. (E) Agarose gel electrophoresis assays of different Pdots composites. (F) Photographs of CD-Pdots before and after including with RB under UV light radiation.

3.2. Studies on Fluorescence Spectra of RB@CD-Pdots. To demonstrate the feasibility of the host-guest inclusion complex39 of CD-Pdots and RB, RB@CD-Pdots for Cho detection, the fluorescence spectra of RB@CD-Pdots in the absence and presence of Cho were investigated. As shown in Figure 2A, CD-Pdots exhibit a strong fluorescence emission at 544 nm (curve 1 in Figure 2A) with an excitation of 450 nm. When RB was introduced into the CD-Pdots solution, inclusion complexes, RB@CD-Pdots were formed and displayed two typical fluorescence emission bands at 544 nm and 582 nm, respectively (curve 3 in Figure 2A). The fluorescence emission at 544 nm is ascribed to CD-Pdots while the fluorescence emission at 582 nm is ascribed to RB. Furthermore, by comparing curve 1 with curve 3 in Figure 2A, we can see that the fluorescence emission band at 544 nm was dramatically quenched and we contributed this quenching to the FRET from CD-Pdots to RB, which can be confirmed by the large spectral overlay between fluorescence emission spectrum of CD-Pdots and absorption spectrum of RB (Figure 2B). In the presence of the Cho, it can be clearly observed that the two emission bands of RB@CD-Pdots display different change tendencies, and the fluorescence of CD-Pdots emission band (544 nm) was restored while the RB emission band (582nm) was decreased, as shown in curve 4 in Figure 2A. This result suggests that the competitive inclusion interaction between Cho and RB with CD occurs and the acceptors RB molecules depart from the donors CD-Pdots, leading to the suppression of FRET, and therefore the fluorescence restoring of the CD-Pdots and decreasing of RB. 9


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Furthermore, the more Cho were added into this system, the more fluorescence recovery at 544 nm and quenching at 582 nm were observed (curve 5 in Figure 2A). The FRET process allowed us to develop ratiometric fluorescence sensing for Cho based on the ratio of fluorescence variation of CD-Pdots/RB FRET pair.

Figure 2. (A) Fluorescence emission spectra of CD-Pdots (curve 1), CD-Pdots + 300nM Cho (curve 2), RB@CD-Pdots (curve 3), RB@CD-Pdots + 75 nM Cho (curve 4) and RB@CD-Pdots + 300 nM Cho (curve 5) in HEPES buffer (20.00 mM, pH 7.4). The concentrations of CD-Pdots and RB@CD-Pdots are both 0.5 µg mL-1. (B) Emission spectrum of CD-Pdots and the absorption spectrum of RB.

To unveil the competitive host-guest inclusion mechanism of RB and Cho with CD anchored on Pdots, the geometric configurations of the inclusion complexes including RB with CD (RB-CD) and Cho with CD (Cho-CD) were studied using the density functional theory (DFT) method with the Becke three-parameter Lee-Yang-Parr (B3LYP) level, respectively. Figure 3 shows that RB and Cho with CD can form different types of host-guest inclusion complexes, and one is exo-inclusion complex formed by RB and CD and the other is endo-inclusion complex formed by Cho and CD. The bonding energy of the inclusion between Cho and CD is calculated to be 11.64 kcal mol -1, which is higher than that of the inclusion between RB and CD (8.04 kcal mol -1). This result suggests that a more stable inclusion complex can be formed between Cho and CD, and Cho can 10


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dissociate the inclusion complex of RB-CD and therefore release RB to suppress the FRET, providing a precondition for sensing Cho with RB@CD-Pdots as fluorescent probes.

Figure 3. Energy-minimized structures of RB-CD (A) and Cho-CD (B) complexes in the ground state using balls for representing atoms.

To further investigated the CD-based host-guest inclusion interaction, 1H NMR spectra was performed and displayed in Figure 4. As shown in Figure 4, the chemical shifts of aromatic protons of RB shift slightly to upfield by ca. 0.03 ppm (Figure 4B and 4C), revealing the formation of RB-CD inclusion complexes. In the presence of Cho, the aromatic protons of RB shift back to downfield by ca. 0.03 ppm (Figure 4C and 4D), which indicates the release of RB after adding Cho. The slight proton chemical shifts of RB upon inclusion with CD is probably attributed to the exo-inclusion complex formed by RB and CD. Moreover, the 1H chemical shift of Cho shift slighly to upfield (ca. 0.02 ppm), suggesting that CD can bind Cho more affinity to release RB by comparing Figure 4D to Figure 4E. The protons of Cho underwent slightly upfield shifting because Cho molecules enter into the inside of CD with alkyl chain.

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Figure 4. 1H NMR spectra (DMSO, 500 MHz, 298K) of (A) CD (10 mM each), (B) RB (10 mM), (C) CD and RB (10 mM each), (D) CD, RB and Cho(10 mM each), and (E) Cho (10 mM).

3.3. Analytical Applications of the Biosensor for Cho Measurements. We have optimized the experimental conditions including incubation time and pH for sensing Cho using RB@CD-Pots system (Figure S1, supporting information). Under the optimal conditions, the effects of various concentrations of Cho on the fluorescence spectra of RB@CD-Pots system have been investigated in HEPES buffer (20 mM, pH 7.4) with 30 min incubation. As shown Figure 5A, the intensity ratio of CD-Pots over RB (I544 / I582) is highly sensitive to Cho and increases gradually with increasing the amount of Cho, accompanying a distinct colour change from orange to green emission under a 365 nm UV lamp (inset in Figure 5A). The calibration curve clearly indicates that Cho can be satisfactorily detected in the concentration range from 25 nM to 350 nM with a sensitivity of 0.332% per nM Cho, a correlation coefficient (R) of 0.993 (inset in Figure 5B) and a detection limit of 4.9 nM. To our knowledge, the present analytical performances including the linear range and the detection limit are comparable or better than those of previous fluorescent nanosensing systems with inorganic nanoparticles (Table S1). The distinguished analytical performances may attributed 12


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to the inherent properties of the prepared Pdots such as extraordinary fluorescence brightness and amplified energy transfer derived from exciton diffusion.4

Figure 5. (A) Fluorescence spectra of the sensing system at various Cho concentrations and the corresponding visual photographs under UV light (365 nm) (inset). (B) The plots of intensity ratios (I544/I582) for against the Cho concentrations and the linear regression curve (inset). RB@CD-Pots: 0.5 µg mL-1; HEPES buffer (20 mM, pH 7.4). (C) The plots of intensity ratios (I544/I582) towards various interference species. RB@CD-Pots: 0.5 µg mL-1; Cho: 300 nM; Interfering substances: 15 µM; HEPES buffer (20.00 mM, pH 7.4). (D) The plots of the intensity ratios (I544/I582) of Pdots with and without NH2-CD modification versus different concentrations of Cho. RB@CD-Pots: 0.5 µg mL-1; RB@Pots: 0.5 µg mL-1; HEPES buffer (20.00 mM, pH 7.4).

For the practical application of the RB@CD-Pots system in biological sample analysis, we investigated the influences of various interfering substances such as salts, amino acids, surfactant, 13


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lecithin, DMPC, quinidine, pivoxicam, and cortisol on the detection of Cho. The intensity ratio with and without different interfering substance in the RB@CD-Pots system was measured, respectively, at identical conditions. As shown in Figure 5C, the result indicates that these interfering substances do not show obvious effect on the fluorescence response in the Cho assay, suggesting that the proposed method has a good selectivity for Cho detection. In this study, in order to avoid the nonspecific adsorption of RB onto the Pdots surface, the Pdots were passivated with BSA, which can maintain long-term colloidal stability, block hydrophobic surfaces, and reduce nonspecific binding in subsequent experiment.40 A control experiment was carried out to investigate the effect of the nonspecific adsorption of RB on the assay of Cho. The COOH-Pdots prepassivated with BSA and CD-Pots were mixed with same amount of RB, and then purified with centrifugal filter, respectively. The obtained RB@Pots and RB@CD-Pots were used as fluorescent probes for Cho detection under same conditions, respectively. As show in Figure 5D, the intensity ratios of the system using RB@Pots as fluorescent probes change very slightly with different concentrations of Cho compared with those of RB@CD-Pots, indicating the present nanosenor using RB@CD-Pots as fluorescent probes shows much lower nonspecific adsorption after passivated with BSA. The developed sensor was further validated to detect the free Cho level in serum samples from healthy people and patients using the standard addition method. As shown in Table 1, the Cho levels of the serum samples determinated by the proposed method are in good agreement with the clinical data with the enzymatic analysis method. The recoveries in the range from 97.41% to 103.52% were obtained, indicating the practicability of the proposed sensor for clinical analysis of free Cho.

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Table 1. The results of Cho assay in human serum matrix by the standard addition method. clinical results

Content

Added

Found

Recovery

samples / nM

/ nM

/ nM

/ nM

/%

1

35.00

36.22±0.02

100

139.74±0.03

103.52±2.1

2

97.40

98.61±0.01

100

196.02±0.02

97.41±1.5

3

64.20

64.94±0.03

100

163.89±0.03

98.95±0.6

4. CONCLUSIONS

In summary, we have constructed an ultrasensitve ratiometric sensor for Cho detection based on the host-guest supramolecular recognition interaction and the amplified FRET technique of the Pdots resulted from exciton diffusion. The proposed ratiometric sensor exhibits excellent sensing performances in both aqueous solutions and serum samples. As fluorescent organic nanoparticles, Pdots-based fluorescent probes exhibit relatively low cytotoxicity compared with hard nanoparticles due to the biocompatible nature of most organic materials, displaying great potential for biological applications.

ACKNOWLEDGEMENTS This work was financially supported by the Natural Science Foundation of China (no. 21575004, 21605001), Program for New Century Excellent Talents in University (NCET-12-0599), the project sponsored by SRF for ROCS, SEM, and the Foundation for Innovation Team of Bioanalytical Chemistry of Anhui Province.

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Supporting Information The information including the influences of incubation time and pH value on the assay of cholesterol in this system, and the comparison of the analytical performances of different cholesterol sensing systems is available free of charge via the Internet at http://pubs.acs.org/. REFERENCES

(1) Peng, H. S.; Chiu, D. T. Soft fluorescent nanomaterials for biological and biomedical imaging. Chem. Soc. Rev. 2015, 44, 4699-4722. (2) Maskey, S.; Osti, N. C.; Grest, G. S.; Perahia, D. Dynamics of Polydots: Soft Luminescent Polymeric Nanoparticles.Macromolecules 2016, 49, 2399-2407. (3) Liu, Z.; Sun, Z.; Di, W.; Qin, W.; Yuan, Z.; Wu, C. Brightness calibrates particle size in single particle fluorescence imaging. Opt. Lett. 2015, 40, 1242-1245. (4)Tian, Z.; Yu, J.; Wu, C.; Szymanski, C.; McNeill, J., Amplified energy transfer in conjugated polymer nanoparticle tags and sensors. Nanoscale 2010, 2, 1999-2011. (5) Wu, C.; Chiu, D. T. Highly fluorescent semiconducting polymer dots for biology and medicine. Angew. Chem. Int. Ed. 2013, 52, 3086-3109. (6) Chen, H.; Chang, K.; Men, X.; Sun, K.; Fang, X.; Ma, C.; Zhao, Y.; Yin, S.; Qin, W.; Wu, C. Covalent Patterning and rapid visualization of latent fingerprints with photo-cross-linkable semiconductor polymer dots. ACS Appl. Mater. Interfaces 2015, 7, 14477-14484. (7) Kuo, S. Y.; Li, H. H.; Wu, P. J.; Chen, C. P.; Huang, Y. C.; Chan, Y. H. Dual Colorimetric and Fluorescent Sensor Based On Semiconducting Polymer Dots for Ratiometric Detection of Lead Ions in Living Cells. Anal. Chem. 2015, 87, 4765-4771.

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Covalent Surface Functionalization of Semiconducting Polymer Dots

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