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Comparative syntheses of tetracycline-imprinted polymeric silicate and
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acrylate on CdTe quantum dots as fluorescent sensors
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Mu-Rong Chaoa, Chiung-Wen Hub, Jian-Lian Chenc,*
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a
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402, Taiwan.
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a
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung
Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung
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402, Taiwan
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b
Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan
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b
Department of Family and Community Medicine, Chung Shan Medical University Hospital,
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Taichung, 402, Taiwan
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c
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Taiwan.
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* Corresponding author. Tel.: 886 4 22053366. E-mail address: cjl@mail.cmu.edu.tw (J.-L.
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Chen).
School of Pharmacy, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402,
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Abstract
The amphoteric drug molecule tetracycline, which contains groups with pKa 3.4 to 9.9,
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was used as a template for conjugating molecularly imprinted polymers (MIPs) and as a
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quencher for CdTe quantum dot (QD) fluorescence. Two MIP-QD composites were
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synthesized by a sol-gel method using a silicon-based monomer and a monomer linker
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between the MIP and QD, i.e., tetraethoxylsilane/3-mercaptopropyltriethoxysilane (MPS) and
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tetraethoxylsilane/3-aminopropyltriethoxysilane (APS). Another MIP-QD composite was
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synthesized by the chain-growth polymerization of methacrylic acid (MAA) and an allyl
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mercaptan linker. The prepared MIP-QDs were characterized by FTIR and SEM and utilized
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at 0.33 mg/mL to determine the tetracycline content in phosphate buffers (pH 7.4, 50 mM)
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through the Perrin and Stern−Volmer models of quenching fluorometry. The Perrin model was
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applied to tetracycline concentrations of 7.4 μM to 0.37 mM for MIP-MPS-QD, 7.4 μM to
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0.12 mM for MIP-APS-QD, and 7.4 μM to 0.10 mM for MIP-MAA-QD (R2= 0.9988, 0.9978,
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and 0.9931, respectively). The Stern−Volmer model was applied to tetracycline concentrations
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of 0.12 mM to 0.37 mM for MIP-APS-QD (R2 = 0.9983) and 0.10 mM to 0.37 mM for
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MIP-MAA-QD (R2 = 0.9970). The detection limits were 0.45 μM, 0.54 μM, and 0.50 μM for
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MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively. Equilibrium times,
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differences between imprinted and nonimprinted polymers, and MIP-QD quenching
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mechanisms were discussed. Finally, specificity studies demonstrated that MIP-MAA-QD
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exhibited optimal recoveries of 96% from bovine serum albumin (n = 5, RSD = 3.6%) and
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91% from fetal bovine serum (n = 5, RSD = 4.8%).
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Keywords: molecularly imprinted polymers; polyacrylate; sol-gel; tetracycline; quantum dot
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1. Introduction
Many quantum dots (QDs) and their derivatives have been successfully used as
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fluorescence probes to detect ions, molecules, proteins, enzymes, and cells (Esteve-Turrillas
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and Abad-Fuentes, 2013; Kuang et al., 2011; Delehanty et al., 2012; Ruedas-Rama et al.,
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2012). The effective recognition of a target analyte in a variable biological sample is key for a
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successful QD probe and is usually achieved using receptor-specific molecules, such as
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ion-selective ligands (Li et al., 2008), ionophores (Ruedas-Rama and Hall, 2008a),
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cyclodextrins (Aguilera-Sigalat et al., 2012), dendrimers (Algarra et al., 2012), and
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biomolecules, including enzymes (Li et al., 2011), antibodies (Esteve-Turrillas and
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Abad-Fuentes, 2013), and aptamers (Yuan et al., 2012), as the recognition materials coupled
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to the QD surfaces. Molecularly imprinting polymers (MIPs) create receptor-specific
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recognition and binding sites that are complementary to a template analyte (Wulff, 2013).
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Coating MIPs on nanoparticles increases their surface area-to-volume ratios and improves
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their template-binding kinetics (Poma et al., 2010).
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A sol-gel technique was utilized in the fabrication of MIP-QD composites for the
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optosensing of phenols (Wang et al., 2009), pyrethoids (Ge et al., 2011), ractopamine (Liu et
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al., 2013), and proteins (Zhang et al., 2012). In those studies, the silicon-based MIP-QDs were
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formed by the step-growth polymerization of tetraethoxysilane with the functional monomer,
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3-mercapto- or 3-aminopropyltriethoxysilane, which could simultaneously interact with the
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templates to form the imprinted cavities and anchor the QDs to the silica monoliths by
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electron pair donation from the sulfur or nitrogen atoms to the QD metals. In contrast, organic
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polymer-based MIPs conjugated with QDs were developed for optosensing of small
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molecules (Lin et al., 2004; Stringer et al., 2012; Liu et al., 2012; Zhao et al., 2012) and DNA
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(Diltemiz et al., 2008) by a fluorescence quenching mechanism. However, these
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polymer-based MIPs were synthesized primarily via chain-growth polymerization of ethylene
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glycol dimethacrylate crosslinker and methacrylic acid (MAA) functional monomer with
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radial initiator. Unlike the functional monomers in sol-gel processes, MAAs lacking a strong
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donor cannot conjugate with QDs. To address this problem, QDs have been functionalized
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with an anchor, such as 4-vinylpyridine (Lin et al., 2004), 1-vinyl-3-octylimidazolium (Liu et
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al., 2012), methacryloylamidocysteine (Diltemiz et al., 2008), or an amino compound
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(Stringer et al., 2012), before polymerization with the acrylate monomers.
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In this study, two silicon-based MIP-QDs formed from 3-mercapto- and
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3-aminopropyltriethoxysilane and one methacrylate-based MIP-QD formed from allyl
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mercaptan anchor were synthesized and characterized by FTIR, SEM, and fluorometry. The
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polyketide antibiotic tetracycline, which possesses a four-ring skeleton with amphoteric
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functional groups, was used for the first time as an MIP template and QD quencher to study
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MIP polymer matrix effects on equilibrium times, differences between imprinted and
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nonimprinted polymers (NIPs), and fluorescence quenching mechanisms. Tetracycline
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recoveries from bovine serum albumin and fetal bovine serum were determined for the
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MIP-QDs.
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2. Experimental
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2.1. Materials and chemicals
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CdCl2 · 2.5H2O, tellurium powder, NaBH4, thioglycolic acid (TGA), ethanol, ammonia, and
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phosphate salts were purchased from Acros (Thermo Fisher Scientific, Geel, Belgium).
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3-Mercaptopropyltriethoxysilane (MPS), 3-aminopropyltriethoxysilane (APS), allyl
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mercaptan (AM) and methacrylic acid (MAA) were purchased from Alfa Aesar (Ward Hill,
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MA, USA). Tetracycline (Tc), oxytetracycline, doxycycline, hydocorticone, cholic acid,
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β-estradiol, tetraethoxysilane (TEOS), ethylene glycol dimethacrylate (EGDMA),
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α,α’-azobisisobutyronitrile (AIBN), 1-propanol, and 1,4-butanediol were purchased from
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Aldrich (Milwaukee, WI, USA). Bovine serum albumin (BSA, lyophilized powder, mol. wt.
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~66 kDa) and fetal bovine serum (FBS, hemoglobin ≤ 25 mg/dL) were purchased from Sigma
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(Milwaukee, WI, USA). Purified water (18 MΩ-cm) from a Milli-Q® water purification
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system (Millipore, Bedford, MA, USA) was used to prepare the standard salt and buffer
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solutions. All standard solutions were protected from light and stored in a refrigerator at 4 °C.
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2.2. Synthesis and characterization of Tc-templated MIP-QD and NIP-QD composites
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TGA-capped CdTe was synthesized according to a previously published method (Chao et
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al., 2013). In brief, NaHTe was prepared by reducing tellurium powder with excess NaBH4 in
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water while stirring under N2. This NaHTe solution was injected into aqueous TGA-CdCl2.
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After sparging with N2 for 20 min, the 1.0:0.5:2.4 molar ratio Cd2+/HTe−/TGA solution was
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heated at 100 °C for 8 h, affording hydrophilic TGA-CdTe QDs that were used to create
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MIP-QDs. The MIP-QD synthesis is illustrated in Fig. 1.
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In the sol-gel preparation of MIP-QDs, a mixture of 0.5 mL QDs, 4.0 mmol functional
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monomer (MPS or APS), 0.5 mmol TEOS, 2.0 mL 1/1 (v/v) 1-propanol/1,4-butanediol, 5.0
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mg tetracycline, and 0.5 mL 20% (w/v) ammonia was stirred at room temperature for 16 h.
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The tetracycline-incorporated MIP-QDs (i.e., Tc-MIP-MPS-QD and Tc-MIP-APS-QD) were
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isolated through centrifugation. To obtain the tetracycline-free MIP-MPS-QDs and
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MIP-APS-QDs, the Tc-MIP-QD products were washed with 5.0 mL ethanol, sonicated for 15
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min, and then centrifuged at 4000 r.p.m. for 30 min. Washing was repeated until tetracycline
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was not detected in the supernatant by a UV-VIS spectrophotometer (UV-1800, Shimadzu).
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To prepare the polyacrylate-based MIP-QD, 5.0 mg tetracycline, 3.0 mmol AM, 1.0 mmol
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MAA, 1.0 mmol EGDMA, 2.0 mL 1/1 (v/v) 1-propanol/1,4-butanediol, and 0.5 mL 1/1/1
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(v/v/v) 1-propanol/1,4-butanediol/H2O saturated with AIBN were charged sequentially to 0.5
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mL of the bare QD solution. The mixture was then heated at 70 °C for 3.5 h to complete the
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radical-initiated polymerization. To remove adsorbed tetracycline, the polymerization
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products were isolated by filtration and successively washed with 80 mL increments of 3/1
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(v/v) ethanol/H2O, affording the tetracycline-free MIP-MAA-QD.
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For comparison with MIP-QDs, five additional materials were synthesized. First,
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NIP-QDs (i.e., NIP-MPS-QD and NIP-APS-QD) were obtained through the MIP-QD
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procedure, except tetracycline was not added. Second, MPS-TEOS, APS-TEOS, and
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AM-MAA-EGDMA were prepared using their respective monomer mixtures without the
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addition of tetracycline and QDs.
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After washing thoroughly and drying in an oven at 40 °C for 30 min, the MIP-QDs and
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their comparative samples were characterized by FTIR (Prestige-21, Shimadzu) and scanning
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electron microscopy (SEM; JSM-6700F, JEOL) at an accelerating voltage of 3.0 kV. The
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MIP-QD solutions (1.0 mg/3 mL phosphate buffer (pH 7.5, 50 mM)) were characterized by a
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transmission electron microscope (TEM; JEM-1400, JEOL) and a Zetasizer (Zetasizer Nano
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ZS90, Malvern) for the measurements of particle size using dynamic light scattering (DLS)
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and zeta potential using laser Doppler microelectrophoresis.
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2.3. Fluorescence measurement of Tetracycle by MIP-QDs
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Fluorescence spectra were collected at an excitation of 315 nm using a
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spectrofluorophotometer (LS55, Perkin Elmer). Samples were prepared by adding 1.0 mg of
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MIP-QDs or NIP-QDs to 3.0 mL of phosphate buffers (pH 7.5, 50 mM) containing different
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tetracycline or interferent concentrations. Fluorescence intensities were recorded at 540 nm
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for MIP-MPS-QD and MIP-APS-QD and at 580 nm for MIP-MAA-QD. BSA (1.2 mg; 6 μM)
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or FBS (0.15 mL; 5% (v/v)) were added to the phosphate buffers to account for matrix effects.
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3. Results and Discussion
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3.1. Characterization of Tc-templated MIP-QDs
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The Tc-templated MIP composites with QDs (TGA-CdTe) – MIP-MPS-QD,
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MIP-APS-QD, and MIP-MAA-QD – were synthesized according to the scheme in Fig. 1 and
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were characterized by FTIR, SEM, and spectrofluorometry. As elaborated upon in the
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Electronic Supplementary Material (ESM), the FTIR spectra (ESM S-1~S-3) possessed peaks
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characteristic of MIP-QDs and therefore confirmed successful syntheses. However, the FTIR
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spectra of the MIP-QDs and their respective NIP-QDs were barely distinguishable.
Differences in the morphologies of the NIP-QDs and MIP-QDs were noted using SEM
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(Fig. 2). The images of NIP-MPS-QD (Fig. 2(a)), NIP-APS-QD (Fig. 2(c)), and
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NIP-MAA-QD (Fig. 2(e)) revealed that most of the NIP-QD particles were embedded in the
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silica and polyacrylate coating materials. In addition, more MIP-QD particles appeared in the
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MIP-MPS-QD (Fig. 2(b)), MIP-APS-QD (Fig. 2(d)), and MIP-MAA-QD images (Fig. 2(f))
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than did the NIP-QD particles in the NIP-QD images. The greater number of observable
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particles resulted from Tc template molecules competing with the electron-donating
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monomers, such as MPS in MPS-QD, APS in APS-QD, and AM in MAA-QD, for adsorption
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onto the CdTe particle surfaces. This competition impeded propagation of the respective
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polymer chains. Without Tc molecules, the NIP-QD particles became wrapped in polymer
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networks and therefore were not observable by SEM. Moreover, the polymerization method
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affected the SEM morphology. In the sol-gel synthesis, which is a step-growth polymerization,
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the silane monomers, including MPS, APS, and TEOS, were linked stepwise, forming the
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silica gel bulk. The QDs were likely excluded from the silane linkages and separated from the
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blocky silica matrices, which are shown in Fig. 2(a)-2(d). By contrast, in the chain-growth
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polymerization with AIBN, the MAA- and EGDMA-monomer radicals tended to
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cross-propagate with AM-modified QDs rather than homopropagate. Consequently, the QDs
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were blended well into the polyacrylate matrices, as seen in Fig. 2(e) and 2(f). Notably, the
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type of electron-donating monomer also affected the SEM morphology. It demonstrated that
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the MIP-MPS-QD (Fig. 2 (b)) and MIP-MAA-QD (Fig. 2 (f)) granules produced with the
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sulfur donating monomers, MPS and AM, were smaller than the MIP-APS-QD (Fig. 2 (d))
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granules produced with the nitrogen donating monomer, APS. The diameter size of the
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MIP-QDs dissolved in pH 7.4 phosphate buffer was measured by TEM and was close to the
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size measured by SEM. Their average sizes, which were near to 150 nm, 950 nm, and 135 nm
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for MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively, and their size
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distributions are shown in ESM S-4. The TEM images present the MIP-QD particles were
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well dispersive without aggregation. Moreover, their averaged DLS size and zeta potential
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values, which were measured every 30 min by Zetasizer, were 146.4±1.6 nm/-17.2±0.1 mV,
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945.6±2.5 nm/-20.1±0.1 mV, and 130.6±1.3 nm/-31.1±0.2 mV (mean±σ, n=20) for
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MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively. These values were not
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significantly varied with the time of 10-hour monitoring, and no aggregation was also
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revealed.
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The fluorescence spectra of QDs coated with the MPS-TEOS, APS-TEOS, and
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AM-MAA-EGDMA copolymers are provided in Fig. 3(a), (b), and (c), respectively. Curves
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(1), (2), and (3) represent the QD, NIP-QD, and Tc-MIP-QD nanoparticles before Tc removal,
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respectively, whereas curve (4) represents the MIP-QD after Tc removal. The emission
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wavelength of curve (2) in Fig. 3(a) and Fig. 3(b) appear near 540 nm; however, the emission
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wavelength of the curve (2) in Fig. 3(c) is red-shifted to approximately 580 nm because of
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AM sulfur atom donation to the QD. Red-shifts are usually attributed to the recombination of
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trapped charge carriers and require dangling bonds at the particle surface. The S-atoms can
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function as hole traps, occupying the places where Te atoms on the tetrahedron faces do not
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fully coordinate to Cd vacancies (Rockenberger et al., 1998). Here, the AM molecules acted
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like 4-mercaptophenol and β-mercaptoethanol (Wuister et al., 2004; Nadeau et al., 2012),
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which possessed higher redox energy levels than the top valence band of the QDs, trapped the
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photogenerated hole, and resulted in the recombination redshift. Without the hole stabilization
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by the AM allyl group, the MPS nanoparticle derivatives did not exhibit the redshift. In
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contrast to the NIP-QDs in curves (2) of Fig. 3, the Tc-MIP-QD emission intensities in curves
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(3) of Fig. 3 decreased because of the apparent nonradiative annihilation of charged CdTe
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from surface traps to tetracycline template. In addition to the positively charged ammonium
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group of tetracycline being attracted by the negatively charged TGA capping reagent (Gao et
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al., 2013), electrostatic attraction between the charged tetracycline and different ligands in the
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imprinted cavities could have occurred and is illustrated in Fig. 1. Stripping tetracycline from
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Tc-MIP-QDs therefore restored the PL intensity to NIP-QD levels. The slightly higher
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intensities observed in curves (4) of Fig. 3(b) and Fig. 3(c) for MIP-APS-QD and
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MIP-MAA-QD, respectively, resulted primarily from the thinner MIP layers coatings on the
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MIP-QDs than on the corresponding NIP-QDs, as observable in the SEM images of Fig.
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2(c)−(f). Therefore, the CdTe particles per unit weight of the MIP-QDs were greater than the
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CdTe particles per unit weight for the corresponding NIP-QDs.
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3.2. Fluorescent sensing of tetracycline by MIP-QDs
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The Tc-templated MIP-QDs (i.e., MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD)
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were used as homogeneous fluorescence-sensing platforms. Response time, dosage response,
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and specificity for tetracycline were studied in sequence.
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3.2.1. Time response
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After exposing the QD probes (0.33 mg/mL) to the tetracycline analyte (0.1 mM), the
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ratios of the observed PL intensities (I) to initial PL intensities (I0) were plotted against
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response time (Fig. 4). This figure revealed that the tetracycline coordination with
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MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD reached equilibrium at approximately 10,
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15, and 25 min, respectively, after tetracycline addition to the pH 7.4 phosphate buffer. The
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most effective PL quenching caused by the tetracycline coordination occurred with
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MIP-MAA-QD, although its equilibration time was longest. As evident in Fig. 2’s SEM
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images, the heavy polyacrylate coating of the NIP-QDs decreased the fluorescent core
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numbers per unit weight and blocked most tetracycline diffusive paths to exterior binding
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sites and to interior binding sites through cavity channels. Accordingly, for the NIP-QDs, the
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increase in PL intensity was observed later than for the corresponding MIP-QDs. However,
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the I/Io standard deviation (1.5%, n=5) for each NIP-QD was smaller than the I/Io standard
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deviation (3.0%, n=5) for each MIP-QDs because of the extra synthetic steps required to form
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the MIP-QDs imprinted cavities.
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After equilibrium, an imprinted factor (IF), defined as ΔFMIP/ΔFNIP, in which ΔF is the
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change in PL intensity after template binding (Liu et al., 2011), was obtained from Fig. 4 for
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each MIP-QD. MIP-MAA-QD, with the most effective PL quenching, had a higher IF (3.0)
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than MIP-MPS-QD (1.5) and MIP-APS-QD (2.0). Strong interactions between template and
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the imprinted cavities in MIP-MAA-QD created higher recognition ability and therefore a
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higher IF value. As illustrated in Fig. 1(b), the tricarbonyl (pKa ≈ 3.4) and
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dimethylammonium (pKa ≈ 9.9) groups of tetracycline are capable of electrostatic attractions
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to the AM thiol (pKa ≈ 10) and the MAA carboxylate (pKa ≈ 4.7) in MIP-MAA-QD,
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respectively, in pH 7.4 phosphate buffer (Şanli et al., 2009). However, tetracycline is
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electrostatically attracted to only one group in MIP-MPS-QD (the MPS thiol, pKa ≈ 10) and
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one group in MIP-APS-QD (the APS amine, pKa ≈ 10) (Fig. 1(a)).
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With the best recognition ability among the MIP-QDs, MIP-MAA-QD was used to test
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other tetracycline analogues (0.1 mM), such as oxytetracycline and doxycycline. However,
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the specificity to tetracycline over its analogues was obscure as their IF values were also
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nearly close to 3.0. By contrast, the non-specificity to the steroids (0.1 mM), including
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hydocorticone, cholic acid, and β-estradiol, that are like tetracycline having four fused rings
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but without tricarbonyl and dimethylammonium functionalities, was obvious as their ΔFMIP
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and ΔFNIP values were nearly unchanged within standard derivations.
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3.2.2. Dose response
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As discussed for Fig. 3 and Fig. 4, the addition of tetracycline template to the MIP-QDs
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solutions was expected to quench their PL intensities. The PL intensities of three prepared
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MIP-QDs were measured after equilibrium with tetracycline in pH 7.4 phosphate buffer and
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plotted against the tetracycline concentrations ranging from 7.4 μM to 0.37 mM (Fig. 5). The
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Stern−Volmer equation is frequently applied to describe quenching kinetics:
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I0/I = 1 + KSV · [Q]
in which I and I0 are the PL intensities of the MIP-QDs at a given quencher concentration, [Q],
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and in a quencher free solution, respectively. KSV is the slope of the linear equation. Figure 5
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shows the Stern−Volmer quenching curves describing I0/I of the MIP-QDs as a function of
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tetracycline concentration. However, the Stern−Volmer linearity was not observed in
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MIP-MPS-QD. Moreover, linearity was observed in MIP-APS-QD (R2 = 0.9983) only for
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0.12 mM to 0.37 mM tetracycline concentrations and in MIP-MAA-QD (R2 = 0.9970) only
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for 0.10 mM to 0.37 mM tetracycline concentrations. Derivations from Stern−Volmer
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linearity are frequently attributed to a combination of static and dynamic quenching. While
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typical Stern−Volmer quenching behavior is driven by dynamic collisions between quencher
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and fluorescent molecules, static quenching may arise from charge transfer, electron tunneling,
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or overlap of molecular orbitals. The non-linear quenching observed in other semiconductor
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nanoparticles (Laferrière et al., 2006) and modified QDs (Chen et al., 2006; Ruedas-Rama
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and Hall, 2008b) manifested itself in curvatures identical to the non-linear regions seen in Fig.
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5. In these instances, a simple Perrin model, in which quenching of the excited states occurs
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only within a sphere of action for immobile quencher, has been proposed:
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I0/I = eα[Q]
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in which α =NAV, in which NA is Avogadro’s number and V the quenching or ‘action’ volume.
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From this relationship, a plot of ln(I0/I) against [Q] reveals V, and hence the radius (r) of the
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action sphere can be calculated. In this instance (Fig. 5), the Perrin linearities for
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MIP-MPS-QD from 7.4 μM to 0.37 mM tetracycline, MIP-APS-QD from 7.4 μM to 0.12 mM
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tetracycline, and MIP-MAA-QD from 7.4 μM to 0.10 mM tetracycline were obtained (R2=
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0.9988, 0.9978, and 0.9931, respectively). The radii (r) of the action sphere calculated from
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the slope of the linear relationship were 17 nm, 21 nm, and 20 nm, respectively. The action
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radius defines how far in from the MIP-QD exterior surface a 50% quenching efficiency
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occurs. In this study, the thickness of the MIPs, whose imprinted cavities electrostatically
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attracted templates and annihilated the charged QDs’ surface traps, are approximately 20 nm
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in the buffer solution.
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Logically, if the quenching conjugation is strong enough, static quenching will first occur
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for the template quencher at low concentration, and then dynamic quenching will possibly
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follow. Therefore, the Perrin model (which accounts for both static and dynamic concerns)
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was fitted to MIP-APS-QD and MIP-MAA-QD in the lower concentration range, whereas the
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Stern−Volmer model (which accounts for dynamics only) was fitted to MIP-APS-QD and
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MIP-MAA-QD in the higher tetracycline concentration range. Compared with MIP-APS-QD
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and MIP-MAA-QD PL intensities at 0 M tetracycline in Fig. 5, the high MIP-MPS-QD PL
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intensity indicated that more QDs were embedded in MIP-MPS-QD and more binding sites in
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imprinted cavities were available for tetracycline conjugation. Therefore, MIP-MPS-QD fitted
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to the Perrin model over the entire tetracycline concentration range.
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Using Perrin linearity, the tetracycline detection limits (0.45 μM, 0.54 μM, and 0.50 μM
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(3σ, n = 10)) were determined for the MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD
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solutions (0.33 mg/mL), respectively, without tetracycline added. The RSD values for
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identical solutions with 7.4 μM tetracycline were 3.2%, 3.4%, and 3.0% (n = 10), respectively,
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for other higher concentrations were between 3.0% and 3.5% (n = 5).
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3.2.3. Specificity study
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As discussed in Fig. 3, MIP-MAA-QDs had a higher tetracycline specificity in the simple
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phosphate buffer because that material possessed stronger interactions between the template
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and its imprinted cavities than the other MIP-QDs. Protein matrices (BSA (6 μM) or FBS (5%
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(v/v)) were added to the tetracycline solutions (7.4 μM) to approximate the complexity of real
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world samples and to further test MIP function. The percentage recovery, which was defined
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as the ratio of PL intensity measured without protein matrix over the PL intensity measured
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with protein matrix in the identical phosphate buffer, was calculated.
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For bare QDs, the tetracycline recoveries from spiked BSA and FBS were approximately
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135% (n = 5, RSD = 13.8%) and 122% (n = 5, RSD = 12.6%), respectively, after a 60 min
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equilibrium period. According to Podery’s report (Poderys et al., 2011), a high recovery from
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bovine serum albumin results from the formation of a bovine serum albumin coating on bare
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QD surface that prevents QD aggregation and rapid washout of the TGA passivant. In this
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study, the formation of a FBS coating and its preventative effects were less pronounced;
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therefore, tetracycline recovery was lower for FBS than BSA.
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For the MIP-MAA-QDs, which afforded optimal recoveries of 96% from bovine serum
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albumin (n = 5, RSD = 3.6%) and 91% from fetal bovine serum (n = 5, RSD = 4.8%), the
308
outer MIP shell largely reduced protein wrapping, QD aggregation, and matrix inclusion.
309
Deviation from the ideal 100% may have arisen from the interaction of the protein matrices
310
with tetracycline analytes, which would have interrupted tetracycline entering the binding
311
sites. Regarding their selectivity and changeable target template, MIP-MAA-QDs were
312
comparable to the other QD materials used for the fluorimetric tetracycline determination (see
313
ESM S-5), except MIP-MAA-QD preparation required additional steps that reduced the
314
sensitivity.
315
The biocompatible and hydrophilic acrylate-based MIP resists nonspecific protein
316
adsorption better than the hydrophobic silicon-based MIP. For the silicon-based MIP,
317
high-molecular-weight protein wrapping may have interfered with analyte binding, and
318
low-molecular-weight protein matrix may have blocked the active sites, therefore affecting
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QD quenching. For MIP-MPS-QD, the BSA recoveries were 82% (n = 5, RSD = 3.6%), and
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the FBS recoveries were 75% (n = 5, RSD = 4.3%). For MIP-APS-QD, the BSA and FBS
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recoveries were 86% (n = 5, RSD = 3.3%) and 80% (n = 5, RSD = 4.2%), respectively.
322
Obviously, these recoveries were affected by the protein matrices, particularly fetal bovine
323
serum, which contains many low-molecular-weight molecules.
324
325
326
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4. Conclusion
Two silicon-based MIPs, prepared from the sol-gel monomers of TEOS/MPS and
TEOS/APS, and one acrylate-based MIP, prepared from the polymerization of MAA and AM,
13
328
were incorporated in situ with CdTe QDs and successfully characterized by FTIR and SEM.
329
The prepared MIP-QD composites were utilized to detect the drug molecule, tetracycline,
330
acting both as an MIP template and a QD quencher. The equilibration times between the
331
template and the MIP-QDs were approximately 10, 15, and 25 min, respectively, for
332
MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD in phosphate buffer. For MIP-APS-QD
333
and MIP-MAA-QD, the Perrin model was applied at low tetracycline concentrations to
334
simultaneously account for static and dynamic quenching, whereas the Stern−Volmer model
335
described the dynamic quenching behavior well for high tetracycline concentrations.
336
MIP-MAA-QD’s stronger electrostatic interactions with the template resulted in better
337
recognition ability (IF = 3.0) than MIP-MPS-QD (IF = 1.5) and MIP-APS-QD (IF = 2.0). In
338
addition, more favorable recoveries of tetracycline from BSA (96%, n = 5, RSD = 4.8%) and
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FBS (91%, n = 5, RSD = 4.8%) were found in MIP-MAA-QD.
340
341
Acknowledgements
342
We gratefully acknowledge the support for this work provided by the National Science
343
Council of Taiwan under Grant no. NSC–101–2113–M–039–001–MY3 and by the China
344
Medical University under Grant no. CMU101–S–11.
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Figure captions and legends
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Fig. 1. Synthetic scheme for MIP-coated CdTe QDs. (a) MPS-QD and APS-QD; (b)
394
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MAA-QD.
Fig. 2. Scanning electron microscope images of NIP-QDs and MIP-QDs. (a) NIP-MPS-QD,
396
(b) MIP-MPS-QD, (c) NIP-APS-QD, (d) MIP-APS-QD, (e) NIP-MAA-QD, (f)
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MIP-MAA-QD.
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Fig. 3. Fluorescence spectra (λex = 315 nm) of (1) bare QD (1 x10-7 mol), (2) NIP-QD (1.0
399
mg), (3) Tc-MIP-QD (1.0 mg), and (4) MIP-QD (1.0 mg) nanoparticles in 3 mL
400
phosphate buffer (pH 7.6, 50 mM). The polymer bases used in the NIP and MIP were
401
the (a) MPS-TEOS, (b) APS-TEOS, and (c) AM-MAA-EGDMA copolymers.
402
Fig. 4. Kinetic binding of tetracycline (0.1 mM) onto the different nanoparticles (0.33 mg/mL),
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(□) NIP-MPS-QD, (■) MIP-MPS-QD, (◊) NIP-APS-QD, () MIP-APS-QD, (Δ)
404
NIP-MAA-QD, and (▲) MIP-MAA-QD, with PL detection (λex = 315 nm) in
405
phosphate buffer (pH 7.6, 50 mM). (□), (◊), (■), and () were monitored at λem = 540
406
nm; (Δ) and (▲) were monitored at λem = 580 nm.
407
Fig. 5. Fluorescence spectra and PL intensity plots (λex = 315 nm) of the different
408
nanoparticles (0.33 mg/mL), (a) MIP-MPS-QD (■; λem = 540 nm), (b) MIP-APS-QD
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(; λem = 540 nm), and (c) MIP-MAA-QD (▲; λem = 580 nm), against tetracycline
410
concentration in phosphate buffer (pH 7.6, 50 mM). I /I0 and ln(I /I0), in which I and I0
411
are the PL intensities of the MIP-QDs at a given concentration of tetracycline and in a
412
tetracycline free solution, are fitted to Stern−Volmer and Perrin linearity, respectively.
17