Author's Accepted Manuscript
Comparative syntheses of tetracyclineimprinted polymeric silicate and acrylate on CdTe quantum dots as fluorescent sensors Mu-Rong Chao, Chiung-Wen Hu, Jian-Lian Chen
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Biosensors and Bioelectronics
Received date: 19 February 2014 Revised date: 28 May 2014 Accepted date: 29 May 2014 Cite this article as: Mu-Rong Chao, Chiung-Wen Hu, Jian-Lian Chen, Comparative syntheses of tetracycline-imprinted polymeric silicate and acrylate on CdTe quantum dots as fluorescent sensors, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2014.05.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Comparative syntheses of tetracycline-imprinted polymeric silicate and
2
acrylate on CdTe quantum dots as fluorescent sensors
3 4
Mu-Rong Chaoa, Chiung-Wen Hub, Jian-Lian Chenc,*
5 6 7
a
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402, Taiwan.
9
a
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung
Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung
10
402, Taiwan
11
b
Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan
12
b
Department of Family and Community Medicine, Chung Shan Medical University Hospital,
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Taichung, 402, Taiwan
14
c
15
Taiwan.
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* Corresponding author. Tel.: 886 4 22053366. E-mail address:
[email protected] (J.-L.
17
Chen).
18
Abstract
19
School of Pharmacy, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402,
The amphoteric drug molecule tetracycline, which contains groups with pKa 3.4 to 9.9,
20
was used as a template for conjugating molecularly imprinted polymers (MIPs) and as a
21
quencher for CdTe quantum dot (QD) fluorescence. Two MIP-QD composites were
22
synthesized by a sol-gel method using a silicon-based monomer and a monomer linker
23
between the MIP and QD, i.e., tetraethoxylsilane/3-mercaptopropyltriethoxysilane (MPS) and
24
tetraethoxylsilane/3-aminopropyltriethoxysilane (APS). Another MIP-QD composite was
25
synthesized by the chain-growth polymerization of methacrylic acid (MAA) and an allyl
26
mercaptan linker. The prepared MIP-QDs were characterized by FTIR and SEM and utilized 1
27
at 0.33 mg/mL to determine the tetracycline content in phosphate buffers (pH 7.4, 50 mM)
28
through the Perrin and SternVolmer models of quenching fluorometry. The Perrin model was
29
applied to tetracycline concentrations of 7.4 M to 0.37 mM for MIP-MPS-QD, 7.4 M to
30
0.12 mM for MIP-APS-QD, and 7.4 M to 0.10 mM for MIP-MAA-QD (R2= 0.9988, 0.9978,
31
and 0.9931, respectively). The SternVolmer model was applied to tetracycline concentrations
32
of 0.12 mM to 0.37 mM for MIP-APS-QD (R2 = 0.9983) and 0.10 mM to 0.37 mM for
33
MIP-MAA-QD (R2 = 0.9970). The detection limits were 0.45 M, 0.54 M, and 0.50 M for
34
MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively. Equilibrium times,
35
differences between imprinted and nonimprinted polymers, and MIP-QD quenching
36
mechanisms were discussed. Finally, specificity studies demonstrated that MIP-MAA-QD
37
exhibited optimal recoveries of 96% from bovine serum albumin (n = 5, RSD = 3.6%) and
38
91% from fetal bovine serum (n = 5, RSD = 4.8%).
39
Keywords: molecularly imprinted polymers; polyacrylate; sol-gel; tetracycline; quantum dot
40
1. Introduction
41
Many quantum dots (QDs) and their derivatives have been successfully used as
42
fluorescence probes to detect ions, molecules, proteins, enzymes, and cells (Esteve-Turrillas
43
and Abad-Fuentes, 2013; Kuang et al., 2011; Delehanty et al., 2012; Ruedas-Rama et al.,
44
2012). The effective recognition of a target analyte in a variable biological sample is key for a
45
successful QD probe and is usually achieved using receptor-specific molecules, such as
46
ion-selective ligands (Li et al., 2008), ionophores (Ruedas-Rama and Hall, 2008a),
47
cyclodextrins (Aguilera-Sigalat et al., 2012), dendrimers (Algarra et al., 2012), and
48
biomolecules, including enzymes (Li et al., 2011), antibodies (Esteve-Turrillas and
49
Abad-Fuentes, 2013), and aptamers (Yuan et al., 2012), as the recognition materials coupled
50
to the QD surfaces. Molecularly imprinting polymers (MIPs) create receptor-specific
51
recognition and binding sites that are complementary to a template analyte (Wulff, 2013).
52
Coating MIPs on nanoparticles increases their surface area-to-volume ratios and improves 2
53 54
their template-binding kinetics (Poma et al., 2010). A sol-gel technique was utilized in the fabrication of MIP-QD composites for the
55
optosensing of phenols (Wang et al., 2009), pyrethoids (Ge et al., 2011), ractopamine (Liu et
56
al., 2013), and proteins (Zhang et al., 2012). In those studies, the silicon-based MIP-QDs were
57
formed by the step-growth polymerization of tetraethoxysilane with the functional monomer,
58
3-mercapto- or 3-aminopropyltriethoxysilane, which could simultaneously interact with the
59
templates to form the imprinted cavities and anchor the QDs to the silica monoliths by
60
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
62
molecules (Lin et al., 2004; Stringer et al., 2012; Liu et al., 2012; Zhao et al., 2012) and DNA
63
(Diltemiz et al., 2008) by a fluorescence quenchingmechanism. However, these
64
polymer-based MIPs were synthesized primarily via chain-growth polymerization of ethylene
65
glycol dimethacrylate crosslinker and methacrylic acid (MAA) functional monomer with
66
radial initiator. Unlike the functional monomers in sol-gel processes, MAAs lacking a strong
67
donor cannot conjugate with QDs. To address this problem, QDs have been functionalized
68
with an anchor, such as 4-vinylpyridine (Lin et al., 2004), 1-vinyl-3-octylimidazolium (Liu et
69
al., 2012), methacryloylamidocysteine (Diltemiz et al., 2008), or an amino compound
70
(Stringer et al., 2012), before polymerization with the acrylate monomers.
71
In this study, two silicon-based MIP-QDs formed from 3-mercapto- and
72
3-aminopropyltriethoxysilane and one methacrylate-based MIP-QD formed from allyl
73
mercaptan anchor were synthesized and characterized by FTIR, SEM, and fluorometry. The
74
polyketide antibiotic tetracycline, which possesses a four-ring skeleton with amphoteric
75
functional groups, was used for the first time as an MIP template and QD quencher to study
76
MIP polymer matrix effects on equilibrium times, differences between imprinted and
77
nonimprinted polymers (NIPs), and fluorescence quenching mechanisms. Tetracycline
78
recoveries from bovine serum albumin and fetal bovine serum were determined for the 3
79
MIP-QDs.
80 81
2. Experimental
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2.1. Materials and chemicals
83
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
86
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,
88
-estradiol, tetraethoxysilane (TEOS), ethylene glycol dimethacrylate (EGDMA),
89
,’-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
92
(Milwaukee, WI, USA). Purified water (18 M-cm) from a Milli-Q® water purification
93
system (Millipore, Bedford, MA, USA) was used to prepare the standard salt and buffer
94
solutions. All standard solutions were protected from light and stored in a refrigerator at 4 °C.
95
2.2. Synthesis and characterization of Tc-templated MIP-QD and NIP-QD composites
96
TGA-capped CdTe was synthesized according to a previously published method (Chao et
97
al., 2013). In brief, NaHTe was prepared by reducing tellurium powder with excess NaBH4 in
98
water while stirring under N2. This NaHTe solution was injected into aqueous TGACdCl2.
99
After sparging with N2 for 20 min, the 1.0:0.5:2.4 molar ratio Cd2+/HTe/TGA solution was
100
heated at 100 °C for 8 h, affording hydrophilic TGA-CdTe QDs that were used to create
101
MIP-QDs. The MIP-QD synthesis is illustrated in Fig. 1.
102 103
In the sol-gel preparation of MIP-QDs, a mixture of 0.5 mL QDs, 4.0 mmol functional monomer (MPS or APS), 0.5 mmol TEOS, 2.0 mL 1/1 (v/v) 1-propanol/1,4-butanediol, 5.0
4
104
mg tetracycline, and 0.5 mL 20% (w/v) ammonia was stirred at room temperature for 16 h.
105
The tetracycline-incorporated MIP-QDs (i.e., Tc-MIP-MPS-QD and Tc-MIP-APS-QD) were
106
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
108
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
114
radical-initiated polymerization. To remove adsorbed tetracycline, the polymerization
115
products were isolated by filtration and successively washed with 80 mL increments of 3/1
116
(v/v) ethanol/H2O, affording the tetracycline-free MIP-MAA-QD.
117
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
119
procedure, except tetracycline was not added. Second, MPS-TEOS, APS-TEOS, and
120
AM-MAA-EGDMA were prepared using their respective monomer mixtures without the
121
addition of tetracycline and QDs.
122
After washing thoroughly and drying in an oven at 40 °C for 30 min, the MIP-QDs and
123
their comparative samples were characterized by FTIR (Prestige-21, Shimadzu) and scanning
124
electron microscopy (SEM; JSM-6700F, JEOL) at an accelerating voltage of 3.0 kV. The
125
MIP-QD solutions (1.0 mg/3 mL phosphate buffer (pH 7.5, 50 mM)) were characterized by a
126
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)
128
and zeta potential using laser Doppler microelectrophoresis.
129
2.3. Fluorescence measurement of Tetracycle by MIP-QDs 5
130
Fluorescence spectra were collected at an excitation of 315 nm using a
131
spectrofluorophotometer (LS55, Perkin Elmer). Samples were prepared by adding 1.0 mg of
132
MIP-QDs or NIP-QDs to 3.0 mL of phosphate buffers (pH 7.5, 50 mM) containing different
133
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 M)
135
or FBS (0.15 mL; 5% (v/v)) were added to the phosphate buffers to account for matrix effects.
136 137
3. Results and Discussion
138
3.1. Characterization of Tc-templated MIP-QDs
139
The Tc-templated MIP composites with QDs (TGA-CdTe) – MIP-MPS-QD,
140
MIP-APS-QD, and MIP-MAA-QD – were synthesized according to the scheme in Fig. 1 and
141
were characterized by FTIR, SEM, and spectrofluorometry. As elaborated upon in the
142
Electronic Supplementary Material (ESM), the FTIR spectra (ESM S-1~S-3) possessed peaks
143
characteristic of MIP-QDs and therefore confirmed successful syntheses. However, the FTIR
144
spectra of the MIP-QDs and their respective NIP-QDs were barely distinguishable.
145
Differences in the morphologies of the NIP-QDs and MIP-QDs were noted using SEM
146
(Fig. 2). The images of NIP-MPS-QD (Fig. 2(a)), NIP-APS-QD (Fig. 2(c)), and
147
NIP-MAA-QD (Fig. 2(e)) revealed that most of the NIP-QD particles were embedded in the
148
silica and polyacrylate coating materials. In addition, more MIP-QD particles appeared in the
149
MIP-MPS-QD (Fig. 2(b)), MIP-APS-QD (Fig. 2(d)), and MIP-MAA-QD images (Fig. 2(f))
150
than did the NIP-QD particles in the NIP-QD images. The greater number of observable
151
particles resulted from Tc template molecules competing with the electron-donating
152
monomers, such as MPS in MPS-QD, APS in APS-QD, and AM in MAA-QD, for adsorption
153
onto the CdTe particle surfaces. This competition impeded propagation of the respective
154
polymer chains. Without Tc molecules, the NIP-QD particles became wrapped in polymer
155
networks and therefore were not observable by SEM. Moreover, the polymerization method 6
156
affected the SEM morphology. In the sol-gel synthesis, which is a step-growth polymerization,
157
the silane monomers, including MPS, APS, and TEOS, were linked stepwise, forming the
158
silica gel bulk. The QDs were likely excluded from the silane linkages and separated from the
159
blocky silica matrices, which are shown in Fig. 2(a)-2(d). By contrast, in the chain-growth
160
polymerization with AIBN, the MAA- and EGDMA-monomer radicals tended to
161
cross-propagate with AM-modified QDs rather than homopropagate. Consequently, the QDs
162
were blended well into the polyacrylate matrices, as seen in Fig. 2(e) and 2(f). Notably, the
163
type of electron-donating monomer also affected the SEM morphology. It demonstrated that
164
the MIP-MPS-QD (Fig. 2 (b)) and MIP-MAA-QD (Fig. 2 (f)) granules produced with the
165
sulfur donating monomers, MPS and AM, were smaller than the MIP-APS-QD (Fig. 2 (d))
166
granules produced with the nitrogen donating monomer, APS. The diameter size of the
167
MIP-QDs dissolved in pH 7.4 phosphate buffer was measured by TEM and was close to the
168
size measured by SEM. Their average sizes, which were near to 150 nm, 950 nm, and 135 nm
169
for MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively, and their size
170
distributions are shown in ESM S-4. The TEM images present the MIP-QD particles were
171
well dispersive without aggregation. Moreover, their averaged DLS size and zeta potential
172
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±, n=20) for
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MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD, respectively. These values were not
175
significantly varied with the time of 10-hour monitoring, and no aggregation was also
176
revealed.
177
The fluorescence spectra of QDs coated with the MPS-TEOS, APS-TEOS, and
178
AM-MAA-EGDMA copolymers are provided in Fig. 3(a), (b), and (c), respectively. Curves
179
(1), (2), and (3) represent the QD, NIP-QD, and Tc-MIP-QD nanoparticles before Tc removal,
180
respectively, whereas curve (4) represents the MIP-QD after Tc removal. The emission
181
wavelength of curve (2) in Fig. 3(a) and Fig. 3(b) appear near 540 nm; however, the emission 7
182
wavelength of the curve (2) in Fig. 3(c) is red-shifted to approximately 580 nm because of
183
AM sulfur atom donation to the QD. Red-shifts are usually attributed to the recombination of
184
trapped charge carriers and require dangling bonds at the particle surface. The S-atoms can
185
function as hole traps, occupying the places where Te atoms on the tetrahedron faces do not
186
fully coordinate to Cd vacancies (Rockenberger et al., 1998). Here, the AM molecules acted
187
like 4-mercaptophenol and -mercaptoethanol (Wuister et al., 2004; Nadeau et al., 2012),
188
which possessed higher redox energy levels than the top valence band of the QDs, trapped the
189
photogenerated hole, and resulted in the recombination redshift. Without the hole stabilization
190
by the AM allyl group, the MPS nanoparticle derivatives did not exhibit the redshift. In
191
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
193
from surface traps to tetracycline template. In addition to the positively charged ammonium
194
group of tetracycline being attracted by the negatively charged TGA capping reagent (Gao et
195
al., 2013), electrostatic attraction between the charged tetracycline and different ligands in the
196
imprinted cavities could have occurred and is illustrated in Fig. 1. Stripping tetracycline from
197
Tc-MIP-QDs therefore restored the PL intensity to NIP-QD levels. The slightly higher
198
intensities observed in curves (4) of Fig. 3(b) and Fig. 3(c) for MIP-APS-QD and
199
MIP-MAA-QD, respectively, resulted primarily from the thinner MIP layers coatings on the
200
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
202
CdTe particles per unit weight for the corresponding NIP-QDs.
203
3.2. Fluorescent sensing of tetracycline by MIP-QDs
204
The Tc-templated MIP-QDs (i.e., MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD)
205
were used as homogeneous fluorescence-sensing platforms. Response time, dosage response,
206
and specificity for tetracycline were studied in sequence.
207
3.2.1. Time response 8
208
After exposing the QD probes (0.33 mg/mL) to the tetracycline analyte (0.1 mM), the
209
ratios of the observed PL intensities (I) to initial PL intensities (I0) were plotted against
210
response time (Fig. 4). This figure revealed that the tetracycline coordination with
211
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
213
most effective PL quenching caused by the tetracycline coordination occurred with
214
MIP-MAA-QD, although its equilibration time was longest. As evident in Fig. 2’s SEM
215
images, the heavy polyacrylate coating of the NIP-QDs decreased the fluorescent core
216
numbers per unit weight and blocked most tetracycline diffusive paths to exterior binding
217
sites and to interior binding sites through cavity channels. Accordingly, for the NIP-QDs, the
218
increase in PL intensity was observed later than for the corresponding MIP-QDs. However,
219
the I/Io standard deviation (1.5%, n=5) for each NIP-QD was smaller than the I/Io standard
220
deviation (3.0%, n=5) for each MIP-QDs because of the extra synthetic steps required to form
221
the MIP-QDs imprinted cavities.
222
After equilibrium, an imprinted factor (IF), defined as ǻFMIP/ǻFNIP, in which ǻF is the
223
change in PL intensity after template binding (Liu et al., 2011), was obtained from Fig. 4 for
224
each MIP-QD. MIP-MAA-QD, with the most effective PL quenching, had a higher IF (3.0)
225
than MIP-MPS-QD (1.5) and MIP-APS-QD (2.0). Strong interactions between template and
226
the imprinted cavities in MIP-MAA-QD created higher recognition ability and therefore a
227
higher IF value. As illustrated in Fig. 1(b), the tricarbonyl (pKa 3.4) and
228
dimethylammonium (pKa 9.9) groups of tetracycline are capable of electrostatic attractions
229
to the AM thiol (pKa 10) and the MAA carboxylate (pKa 4.7) in MIP-MAA-QD,
230
respectively, in pH 7.4 phosphate buffer ( anli et al., 2009). However, tetracycline is
231
electrostatically attracted to only one group in MIP-MPS-QD (the MPS thiol, pKa 10) and
232
one group in MIP-APS-QD (the APS amine, pKa 10) (Fig. 1(a)).
233
With the best recognition ability among the MIP-QDs, MIP-MAA-QD was used to test 9
234
other tetracycline analogues (0.1 mM), such as oxytetracycline and doxycycline. However,
235
the specificity to tetracycline over its analogues was obscure as their IF values were also
236
nearly close to 3.0. By contrast, the non-specificity to the steroids (0.1 mM), including
237
hydocorticone, cholic acid, and -estradiol, that are like tetracycline having four fused rings
238
but without tricarbonyl and dimethylammonium functionalities, was obvious as their ǻFMIP
239
and ǻFNIP values were nearly unchanged within standard derivations.
240
3.2.2. Dose response
241
As discussed for Fig. 3 and Fig. 4, the addition of tetracycline template to the MIP-QDs
242
solutions was expected to quench their PL intensities. The PL intensities of three prepared
243
MIP-QDs were measured after equilibrium with tetracycline in pH 7.4 phosphate buffer and
244
plotted against the tetracycline concentrations ranging from 7.4 M to 0.37 mM (Fig. 5). The
245
SternVolmer equation is frequently applied to describe quenching kinetics:
246
I0/I = 1 + KSV · [Q]
247
in which I and I0 are the PL intensities of the MIP-QDs at a given quencher concentration, [Q],
248
and in a quencher free solution, respectively. KSV is the slope of the linear equation. Figure 5
249
shows the SternVolmer quenching curves describing I0/I of the MIP-QDs as a function of
250
tetracycline concentration. However, the SternVolmer linearity was not observed in
251
MIP-MPS-QD. Moreover, linearity was observed in MIP-APS-QD (R2 = 0.9983) only for
252
0.12 mM to 0.37 mM tetracycline concentrations and in MIP-MAA-QD (R2 = 0.9970) only
253
for 0.10 mM to 0.37 mM tetracycline concentrations. Derivations from SternVolmer
254
linearity are frequently attributed to a combination of static and dynamic quenching. While
255
typical SternVolmer quenching behavior is driven by dynamic collisions between quencher
256
and fluorescent molecules, static quenching may arise from charge transfer, electron tunneling,
257
or overlap of molecular orbitals. The non-linear quenching observed in other semiconductor
258
nanoparticles (Laferrière et al., 2006) and modified QDs (Chen et al., 2006; Ruedas-Rama
259
and Hall, 2008b) manifested itself in curvatures identical to the non-linear regions seen in Fig. 10
260
5. In these instances, a simple Perrin model, in which quenching of the excited states occurs
261
only within a sphere of action for immobile quencher, has been proposed:
262
I0/I = e[Q]
263
in which =NAV, in which NA is Avogadro’s number and V the quenching or ‘action’ volume.
264
From this relationship, a plot of ln(I0/I) against [Q] reveals V, and hence the radius (r) of the
265
action sphere can be calculated. In this instance (Fig. 5), the Perrin linearities for
266
MIP-MPS-QD from 7.4 M to 0.37 mM tetracycline, MIP-APS-QD from 7.4 M to 0.12 mM
267
tetracycline, and MIP-MAA-QD from 7.4 M to 0.10 mM tetracycline were obtained (R2=
268
0.9988, 0.9978, and 0.9931, respectively). The radii (r) of the action sphere calculated from
269
the slope of the linear relationship were 17 nm, 21 nm, and 20 nm, respectively. The action
270
radius defines how far in from the MIP-QD exterior surface a 50% quenching efficiency
271
occurs. In this study, the thickness of the MIPs, whose imprinted cavities electrostatically
272
attracted templates and annihilated the charged QDs’ surface traps, are approximately 20 nm
273
in the buffer solution.
274
Logically, if the quenching conjugation is strong enough, static quenching will first occur
275
for the template quencher at low concentration, and then dynamic quenching will possibly
276
follow. Therefore, the Perrin model (which accounts for both static and dynamic concerns)
277
was fitted to MIP-APS-QD and MIP-MAA-QD in the lower concentration range, whereas the
278
SternVolmer model (which accounts for dynamics only) was fitted to MIP-APS-QD and
279
MIP-MAA-QD in the higher tetracycline concentration range. Compared with MIP-APS-QD
280
and MIP-MAA-QD PL intensities at 0 M tetracycline in Fig. 5, the high MIP-MPS-QD PL
281
intensity indicated that more QDs were embedded in MIP-MPS-QD and more binding sites in
282
imprinted cavities were available for tetracycline conjugation. Therefore, MIP-MPS-QD fitted
283
to the Perrin model over the entire tetracycline concentration range.
284 285
Using Perrin linearity, the tetracycline detection limits (0.45 M, 0.54 M, and 0.50 M (3, n = 10)) were determined for the MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD 11
286
solutions (0.33 mg/mL), respectively, without tetracycline added. The RSD values for
287
identical solutions with 7.4 M tetracycline were 3.2%, 3.4%, and 3.0% (n = 10), respectively,
288
for other higher concentrations were between 3.0% and 3.5% (n = 5).
289
3.2.3. Specificity study
290
As discussed in Fig. 3, MIP-MAA-QDs had a higher tetracycline specificity in the simple
291
phosphate buffer because that material possessed stronger interactions between the template
292
and its imprinted cavities than the other MIP-QDs. Protein matrices (BSA (6 M) or FBS (5%
293
(v/v)) were added to the tetracycline solutions (7.4 M) to approximate the complexity of real
294
world samples and to further test MIP function. The percentage recovery, which was defined
295
as the ratio of PL intensity measured without protein matrix over the PL intensity measured
296
with protein matrix in the identical phosphate buffer, was calculated.
297
For bare QDs, the tetracycline recoveries from spiked BSA and FBS were approximately
298
135% (n = 5, RSD = 13.8%) and 122% (n = 5, RSD = 12.6%), respectively, after a 60 min
299
equilibrium period. According to Podery’s report (Poderys et al., 2011), a high recovery from
300
bovine serum albumin results from the formation of a bovine serum albumin coating on bare
301
QD surface that prevents QD aggregation and rapid washout of the TGA passivant. In this
302
study, the formation of a FBS coating and its preventative effects were less pronounced;
303
therefore, tetracycline recovery was lower for FBS than BSA.
304
For the MIP-MAA-QDs, which afforded optimal recoveries of 96% from bovine serum
305
albumin (n = 5, RSD = 3.6%) and 91% from fetal bovine serum (n = 5, RSD = 4.8%), the
306
outer MIP shell largely reduced protein wrapping, QD aggregation, and matrix inclusion.
307
Deviation from the ideal 100% may have arisen from the interaction of the protein matrices
308
with tetracycline analytes, which would have interrupted tetracycline entering the binding
309
sites. Regarding their selectivity and changeable target template, MIP-MAA-QDs were
310
comparable to the other QD materials used for the fluorimetric tetracycline determination (see
311
ESM S-5), except MIP-MAA-QD preparation required additional steps that reduced the 12
312
sensitivity.
313
The biocompatible and hydrophilic acrylate-based MIP resists nonspecific protein
314
adsorption better than the hydrophobic silicon-based MIP. For the silicon-based MIP,
315
high-molecular-weight protein wrapping may have interfered with analyte binding, and
316
low-molecular-weight protein matrix may have blocked the active sites, therefore affecting
317
QD quenching. For MIP-MPS-QD, the BSA recoveries were 82% (n = 5, RSD = 3.6%), and
318
the FBS recoveries were 75% (n = 5, RSD = 4.3%). For MIP-APS-QD, the BSA and FBS
319
recoveries were 86% (n = 5, RSD = 3.3%) and 80% (n = 5, RSD = 4.2%), respectively.
320
Obviously, these recoveries were affected by the protein matrices, particularly fetal bovine
321
serum, which contains many low-molecular-weight molecules.
322 323 324
4. Conclusion Two silicon-based MIPs, prepared from the sol-gel monomers of TEOS/MPS and
325
TEOS/APS, and one acrylate-based MIP, prepared from the polymerization of MAA and AM,
326
were incorporated in situ with CdTe QDs and successfully characterized by FTIR and SEM.
327
The prepared MIP-QD composites were utilized to detect the drug molecule, tetracycline,
328
acting both as an MIP template and a QD quencher. The equilibration times between the
329
template and the MIP-QDs were approximately 10, 15, and 25 min, respectively, for
330
MIP-MPS-QD, MIP-APS-QD, and MIP-MAA-QD in phosphate buffer. For MIP-APS-QD
331
and MIP-MAA-QD, the Perrin model was applied at low tetracycline concentrations to
332
simultaneously account for static and dynamic quenching, whereas the SternVolmer model
333
described the dynamic quenching behavior well for high tetracycline concentrations.
334
MIP-MAA-QD’s stronger electrostatic interactions with the template resulted in better
335
recognition ability (IF = 3.0) than MIP-MPS-QD (IF = 1.5) and MIP-APS-QD (IF = 2.0). In
336
addition, more favorable recoveries of tetracycline from BSA (96%, n = 5, RSD = 4.8%) and
337
FBS (91%, n = 5, RSD = 4.8%) were found in MIP-MAA-QD. 13
338 339
Acknowledgements
340
We gratefully acknowledge the support for this work provided by the National Science
341
Council of Taiwan under Grant no. NSC–101–2113–M–039–001–MY3 and by the China
342
Medical University under Grant no. CMU101–S–11.
343 344
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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)
392 393
MAA-QD. Fig. 2. Scanning electron microscope images of NIP-QDs and MIP-QDs. (a) NIP-MPS-QD,
394
(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 (ex = 315 nm) of (1) bare QD (1 x10-7 mol), (2) NIP-QD (1.0
397
mg), (3) Tc-MIP-QD (1.0 mg), and (4) MIP-QD (1.0 mg) nanoparticles in 3 mL
398
phosphate buffer (pH 7.6, 50 mM). The polymer bases used in the NIP and MIP were
399
the (a) MPS-TEOS, (b) APS-TEOS, and (c) AM-MAA-EGDMA copolymers.
400
Fig. 4. Kinetic binding of tetracycline (0.1 mM) onto the different nanoparticles (0.33 mg/mL),
401
( ) NIP-MPS-QD, ( ) MIP-MPS-QD, () NIP-APS-QD, (i) MIP-APS-QD, ()
402
NIP-MAA-QD, and () MIP-MAA-QD, with PL detection (ex = 315 nm) in
403
phosphate buffer (pH 7.6, 50 mM). ( ), (), ( ), and (i) were monitored at em = 540
404
nm; () and () were monitored at em = 580 nm.
405
Fig. 5. Fluorescence spectra and PL intensity plots (ex = 315 nm) of the different
406
nanoparticles (0.33 mg/mL), (a) MIP-MPS-QD ( ; em = 540 nm), (b) MIP-APS-QD
407
(i; em = 540 nm), and (c) MIP-MAA-QD (; em = 580 nm), against tetracycline
408
concentration in phosphate buffer (pH 7.6, 50 mM). I /I0 and ln(I /I0), in which I and I0
409
are the PL intensities of the MIP-QDs at a given concentration of tetracycline and in a
410
tetracycline free solution, are fitted to SternVolmer and Perrin linearity, respectively.
411
Highlights:
412 413
1. Tc behaved both as an MIP template and a quantum dot quencher. 2. MIP-QDs determined Tc through the Perrin and SternVolmer quench models.
414 415 416
3. Quenching sphere radius was approximately 20 nm for all of the synthetic MIP-QDs. 4. Acrylate MIP’s stronger affinity for Tc showed higher imprinted factors than silica MIPs. 5. Acrylate MIP afforded higher Tc recoveries from spiked BSA and FBS than silica MIPs.
16
Figure-1(a)
Figure-1(b)
Figure-2
Figure-3
Figure-4
Figure-5