Author’s Accepted Manuscript A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic Kuncai Chen, Rong He, Xiaoyan Luo, Pengzhe Qin, Lei Tan, Youwen Tang, Zhicong Yang www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(17)30224-5 http://dx.doi.org/10.1016/j.bios.2017.03.059 BIOS9647
To appear in: Biosensors and Bioelectronic Received date: 24 January 2017 Revised date: 21 March 2017 Accepted date: 27 March 2017 Cite this article as: Kuncai Chen, Rong He, Xiaoyan Luo, Pengzhe Qin, Lei Tan, Youwen Tang and Zhicong Yang, A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2017.03.059 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.
A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic Kuncai Chena, Rong Hea, Xiaoyan Luoa, Pengzhe Qina, Lei Tana*, Youwen Tangb, Zhicong Yanga* a
Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China
b
MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou 510006, China
[email protected] [email protected] *Corresponding author: Tel:+86-20-36545121; Fax: +86-20-36545137.
Abstract This paper demonstrates a new strategy for developing a fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic. The technique provides amino modified Mn-doped ZnS QDs as fluorescent supports, 4-vinylphenylbronic acid as a covalent monomer, N-isopropyl acrylamide as a thermo-responsive monomer in combination with acrylamide as a non-covalent monomer, and glycosyl moiety of a glycopeptide antibiotic as a template to produce fluorescent molecularly imprinted polymer (FMIP) in aqueous solution. The FMIP can alter its functional moieties and structure with pH and temperature stimulation. This allows recognition of target molecules through control of pH and temperature. The fluorescence intensity of the FMIP was enhanced gradually as the concentration of telavancin increased, and showed selective recognition toward the target glycopeptide antibiotic preferentially among other antibiotics. Using the FMIP as a sensing material, good linear correlations were obtained over the concentration range of 3.0 to 300.0 μg/L and with a low limit of 1
detection of 1.0 μg/L. The analysis results of telavancin in real samples were consistent with that obtained by liquid chromatography tandem mass spectrometry.
Keywords: molecular imprinting; quantum dots; fluorescence probes; glycopeptide antibiotics.
1. Introduction Antibiotic resistance is the ability of bacteria or other microbes to resist the effects of an antibiotic. Such resistance develops when bacteria change in some way to reduce or eliminate the effectiveness of drugs or other agents designed to cure or prevent infections (Levy and Marshall, 2004). Over the last decades, the number of bacteria resistant to antibiotics has increased significantly, resulting in many infections that are resistant to commonly prescribed antibiotic treatments (Septimus and Kuper, 2009). Glycopeptide antibiotics are used in the treatment of serious gram-positive bacterial infections, including those caused by methicillin-resistant Staphylococcus aureus. Typically, glycopeptide antibiotics are regarded as drugs of last resort for the treatment of otherwise resistant and deadly infections (Kahne et al., 2005). However, unfortunately after more than 50 years of clinical use, the emergence of glycopeptide-resistant Gram-positive pathogens presented a serious threat to public health (James et al., 2012; Pootoolal et al., 2002). Considerable efforts were then made to produce semisynthetic glycopeptide with improved pharmacokinetics and pharmacodynamics properties, but the discovery of new classes of antibiotics remains extremely challenging (Coates et al., 2002). Rational use of antibiotics, avoiding clinical abuse and passive exposure is still the most fundamental way to control the disease. High-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) is widely used for clinical drug testing, but they are currently too expensive and require complex sample pretreatment (Chace, 2001; Badawi et al., 2009). The fluorescence sensing process can directly convert a recognition event into a fluorescence readout signal and attracted considerable attention due to its rapid response, high sensitivity and cost-effective instrumentation (Powe et al., 2004; You 2
et al., 2007). The use of fluorescence methods in bioanalysis, cellular and molecular imaging has become popular largely these days (Vendrell et al., 2012). Compared to the traditional fluorescence dyes, quantum dots (QDs) have plenty of advantages, such as low toxicity, good photo stability, long life time, and little background auto fluorescence, making them appropriate candidates in wide variety of areas (Gill et al., 2008; Resch-Genger et al., 2008). Among them, Mn-doped ZnS QDs have gained increased attention for sensing because of its efficient and characteristic luminescent properties (Chantada-Vazquez et al., 2016; Diaz-Diestra et al., 2017; Ertas and Kara, 2015; Ren and Chen, 2015; Song and Wang, 2014). Antibodies are the most commonly used recognition elements for the sensing systems because of their specificity. However, antibodies are not always desired because of their high cost and low stability, which limit their further application (Haupt, 2010; Whitcombe et al., 2011). Molecular imprinting technology, which involves the formation of artificial recognition cavities in a polymer network with complementary shape and chemical functionality to a target analyte, is an alternative method to overcome the limitations of the antibodies (Chen et al., 2011). Improvements in selectivity of QDs toward a certain analyte can be successfully achieved by embedding the QDs into the matrix of molecularly imprinted polymers (MIPs), resulting in so-called molecularly imprinted sensing materials (Chantada-Vázquez et al., 2016b; Karfa et al., 2016; Li et al., 2016; Lin et al., 2004; Lin et al., 2009; Yang et al., 2016; Zhang and Chen, 2016). The key feature of MIPs is their engineered selectivity of binding to an analyte of interest. Whilst the imprinting of an organic molecule and ion has been well established, the imprinting of a macromolecule has been and continues to be a major challenge (Li et al., 2014). The difficulty is mainly due to structural complexity and large size of macromolecules, which leads to more heterogeneous binding sites and hinders the molecular recognition process (Bossi et al., 2007; Ge and Turner, 2008). To solve this problem, several efforts have been made so far. Epitope imprinting, which uses only a small exposed fragment of a macromolecule as a template, is an effective approach for overcoming the issues which are commonly experienced in the synthesis of MIPs, such as steric hindrance, adsorption 3
thermodynamics and kinetics, and shortage of templates (Rachkov and Minoura, 2000; Nishino et al., 2006; Urraca et al., 2011).
However, determining an
appropriate epitope to achieve a better imprinting effect still remains a big question (Yu et al., 2014). Furthermore, macromolecule imprinting conditions need to be as close as possible to their natural environment to maintain their conformational integrity and binding activity (Tan et al., 2015). The use of water as solvent provides a biologically benign environment for recognition of macromolecules in aqueous media. However, water can reduce hydrogen bonding and electrostatic interactions between template molecules and functional monomers (Zhang et al., 2013). Thus, non-covalent molecular imprinting often shows unavoidable difficulties in aqueous solutions. Boronic acids can form cyclic esters, most frequently with 1,2- and 1,3-diols, allowing boronate ester formation with a number of biologically important species, including saccharides, glycopeptides, glycoproteins, and dopamine significantly broadening the application of boronic acids in biology (Brooks and Sumerlin, 2016). The cyclic esters form in alkaline aqueous solution and dissociate at acidic pH. Boronic acid-functionalized molecularly imprinted polymers were first reported by Wulff and Vesper in 1978 as the stationary phase to separate sugar derivatives by chromatography (Wulff and Vesper, 1978). Recently, Liu and coworkers have been exploring the new field of boronic acid-functionalized glycoproteins molecularly imprinted polymers in immunoassay (Bi and Liu, 2014; Li et al., 2013; Ye et al., 2014; Wang et al., 2014). Guo et al., demonstrated a strategy for producing double responsive fluorescent MIP for sensing of glycoprotein using upconversion nanoparticles
as
fluorescence
signal
reporter,
N-isopropylacrylamide
and
4-vinylphenylboronicacid as functional monomers (Guo et al., 2016). Inspired by the fact that most antibodies recognize a conformational epitope of the antigen rather than its three-dimensional structure, we combined glycosyl-based template design and surface molecular imprinting to fabricate a novel sensing material for pH and temperature regulated sensing of telavancin, a semi-synthetic glycopeptide antibiotic. To this end, we used amino modified Mn-doped ZnS QDs as solid supports, 4-vinylphenylbronic acid, acrylamide and N-isopropyl acrylamide as the functional monomers, and the glycosyl moiety of telavancin as a template to 4
produce graft imprinting at the surface of the QDs. The resultant fluorescent molecularly imprinted polymer (FMIP) was used as a sensing material for pH and temperature regulated recognition of telavancin in aqueous solution to eventually realize sensitive and selective detection of target glycopeptide antibiotic in biological samples.
2. Experimental section 2.1. Reagents and chemicals All chemicals used were of analytical grade. ZnSO4·7H2O, Na2S·9H2O, MnCl2·4H2O, were used as received from Alfa Aesar (Tianjin, China). 4-vinylphenylboronic acid (VPBA), N-isopropyl acrylamide
(NIPAAM), acrylamide
(AAM), N, N, N’,
N’,-tetramethylethylethylenediamine (TEMED) and ammonium persulfate (APS), 3-mercaptopropyltriethoxysilane (MPTS), 3-aminopropyltriethoxysilane (APTES, 98%), Mannose and Tryptophan were obtained from Sigma-Aldrich Co. (St. Louis, MO). Telavancin was purchased from Dr. Ehrenstorfer GmbH. Hydrophilic/lipophilic balance cartridge columns were from Waters. Mannose-Tryptophan (MT) was synthesized using a solid phase peptide synthesis technology in our laboratory, as described in the supplementary material. Ultrapure water from a laboratory water purification system (Sartorius, Germany) was used throughout this work.
2.2. Characterizations The morphology and microstructure of the QDs and FMIP were studied using high-resolution transmission electron microscopy (HRTEM) on a JEM-2100HR (JEOL. Japan). UV-visible absorption spectra were obtained from a Shimadzu UV-Vis 1700 Spectrophotometer. Fluorescence spectra were recorded on an F-2500 (Hitachi, Tokyo, Japan). pH of a solution was monitored with a pH211 microprocessor pH meter (Hanna instruments). The chromatographic system consisted of a Waters® ACQUITY UPLC® I-Class System and a 100 mm×2.1 mm i.d., 1.7 μm particle diameter Acquity BEH-C18 analytical column (Waters) was set at a temperature of 40 0C. A mixture of 0.1% formic acid in water and acetonitrile was delivered at a flow rate of 0.3 mL/min. Detection was performed with a Waters® Xevo TQ triple quadrapole 5
mass spectrometer. The experimental conditions of the ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) used have been summarized in the Supplementary Material (SM).
2.3. Synthesis of amino-capped Mn-doped ZnS QDs The procedure for synthetically producing the amino-capped Mn-doped ZnS QDs used in this work was similar to an already published method (Tan et al., 2014). Specifically, 1.8 g of ZnSO4·7H2O, 0.1 g of MnCl2·4H2O, and 20 mL of ultrapure water were added to a 50 mL three-necked flask. After the mixture was stirred under nitrogen at room temperature for 10 min, 5 mL of aqueous solution containing 1.5 g of Na2S·9H2O was added drop wise, and the mixture was kept stirring for 30 min. Then, 5 mL of an ethanol solution containing 0.072 g of MPTS was added, and the mixture was kept stirring for 24 h. The resultant MPTS-capped Mn-doped ZnS QDs were centrifuged and washed with absolute ethanol three times before being dried under vacuum. In the second step, 10 mL of an absolute ethanol solution containing 200 μL of APTES, 0.35 mL of TEOS, and 250 mg of MPTS-capped Mn-doped ZnS QDs were added to a 25-mL flask. After the mixture was stirred for 10 min, a 0.4 mL of concentrated ammonium hydroxide solution and a 2.0 mL of H2O were added, and the mixture was kept stirring for 18 h. The amino modified Mn-doped ZnS QDs were centrifuged, washed with ultrapure water three times and then dried under vacuum at room temperature.
2.4. Preparation of fluorescent molecularly imprinted polymer (FMIP) Typically, 100 mg of NH2 modified Mn-doped ZnS QDs were first dispersed in 10.0 mL of carbonate buffer solution (0.01 mol/L, pH 9.5) by ultrasonication. AAM, 4-vinylphenylbronic acid, MT and NIPAAM were dissolved into the above dispersion. The mixture was then purged with nitrogen for 30 min and stirred for 1 h before 10 mg of APS and 5 mg of TEMED was added into it. After addition, the solution was again purged with nitrogen and stirred for 5 h. The fluorescent nonimprinted polymer (FNIP) was synthesized in parallel but without the addition of the template. After the reaction, the FMIP and FNIP composites were centrifuged and washed with 6
ultrapure water to remove all absorbed oligomers and unreacted monomers. Finally, the FMIP composites were washed repeatedly with 0.02 mol/L phosphoric acid solution containing 30% acetonitrile (v/v) and shaken for 2 h to remove the template until no MT in the supernatant was detected in UHPLC-MS/MS.
2.5. Evaluation of the adsorption characteristics of FMIP Ten milligrams of FMIP or FNIP were suspended in 5 mL of aqueous solution of different telavancin concentrations. After incubation at the set temperature for 12 h, the FMIP was removed via centrifugation at 8000 rpm for 6 minutes. The binding amount of telavancin was calculated from the difference in concentration between the initial spike solution and the concentration of the supernatant after equilibration. The experimental data is presented as the adsorption capacity per unit mass (mg) of the nanoparticles, and the adsorption capacity (Q) was calculated using
Q
V (C0 Ce ) , where Q (mg/g) is the mass of the template adsorbed by a unit m
mass of particles, C0 (mg/mL) is the initial template concentration, Ce (mg/mL) is the template concentration of the supernatant, V (mL) is the volume of the initial solution, and m (g) is the mass of the composites.
2.6. Measurement procedure The standard solutions of telavancin for calibration were prepared by dissolving appropriate amount of telavancin in 0.01 mol/L carbonate buff solution (CBS , pH 9.0, 0.01 mol/L) and stored at 4 0C. To a 5 mL calibrated test tube, 0.5 mL of 100 mg/L FMIP or FNIP, 0.5 mL of CBS, and different concentrations of telavancin (1.0 mL) were sequentially added. The mixture was mixed thoroughly followed by incubation at 38 0
C for 15 minutes. In the fluorimeter, the slit widths were kept at 10 nm for both
excitation and emission in the fluorescence mode. The excitation wavelength was set at 340 nm and a PMT voltage of 700 V.
2.7. Serum samples Clinical serum samples were collected from volunteers of Guangzhou 12th people’s hospital and stored at −20 °C until use. Prior to use, the samples were first thawed 7
gently, then treated with 100 μL nitric acid (0.5 mol/L) to precipitate the proteins. Next, the mixture was vortexed for 1 minute and then centrifuged at 10000 rpm for 10 min. An appropriate dilution of serum (20-fold, carbonate buffer solution, 0.01 mol/L, pH 9.0) was adopted before detection. In a typical test, to a 5-mL calibrated test tube, were added 0.5 mL of FMIP composites (100 mg/L), 0.5 mL of carbonate buffer solution (CBS) (0.01 mol/L, pH 9.0) and 1.0 mL of the diluted serum sample in sequence. The mixture was mixed thoroughly followed by incubation at 38 0C for 15 minutes.
2.8. Solid phase extraction procedure The solid phase extraction procedure is based on the previous report with slight modification (Wang et al., 2014). Hydrophilic/lipophilic balance cartridges were conditioned and equilibrated by 3 mL of methanol and 3 mL of ultrapure water before 1 mL serum sample loading. After sample loading, the cartridges were rinsed with 3 mL of ultrapure water and vacuum-dried for 5 min. The target compounds were eluted with 3 mL of methanol. After evaporation to dryness under a gentle stream of N2 at 40 0C, the residue was re-dissolved in 1.0 mL of 0.1% formic acid solution for UHPLC-MS/MS analysis.
3. Results and discussion 3.1. Preparation and characterization of FMIP As shown in Fig, S1, telavancin, a typical glycopeptide antibiotic has a homologous heptapeptide scaffold and both covalently attached glycosyl and long chain acyl groups. Note that the cis-diols of telavancin can react with boronic acids monomers to form cyclic esters in an alkaline aqueous solution, and the N-and C-terminals can form hydrogen bonds with non-covalent functional monomers. We selected the glycosyl moiety of telavancin to design and synthesize a new compound, (Fig. S2) which can function as a template to prepare molecularly imprinted polymer receptors for telavancin. . Fig. 1. Schematic illustration of the synthesis of FMIP composites. 8
Traditional MIPs generally show good selectivity but do not possess the ability for intrinsic signal transduction required for sensing applications. To solve this difficulty, incorporation of a fluorescent signal unit within the imprinted cavity of molecularly imprinted polymers was proposed. Among the various luminophores, quantum dots (QDs) have been widely employed due to their high luminescence efficiency, photo-stability and easy preparation. More interestingly, intelligent imprinted polymers have been reported recently, in which the recognition sites can be switched on and off by triggers (Zhang et al., 2014; Ge et al., 2013). As shown in Fig. 1, VPBA was chosen as one of the functional monomers based on the consideration that the boronic acid group could covalently react with glycopeptides or glycoproteins to form cyclic esters in an alkaline aqueous solution. NIPAAM was chosen as a thermoresponsive monomer which would undergo swelling and shrinking with change in temperature (Yoshimatsu et al., 2012). AAM was chosen as a non-covalent functional monomer based on the consideration that effective electrostatic interaction can occur between the template and AAM. After removing the template, recognition cavities complementary to the template molecule in shape, size, and chemical functionality were formed in the cross-linked polymer matrix. Note that the recognition sites were related to the molar ratio of the template and the functional monomers. We designed and prepared several different FMIP to find the optimal monomer composition and the optimal ratio of template to the functional monomer. Telavancin was chosen as the target analyte for evaluation of the synthesized FMIP to find the optimal condition. As shown in Table S1, the adsorption capacity for telavancin was 6.26 mg/g, which yielded a maximum imprinting factor (IF) value of 3.0, obtained using an FMIP comprised of 20% AAM and 20% VPBA at a molar ratio of the template to VPBA equal to 1:1. As shown in Table S1, FMIP possesses a high adsorption capacity and selectivity at a molar ratio of 1:1, while the other molar ratios showed a low adsorption capacity or selectivity. This is possibly because low VPBA to AAM ratios induced fewer non-specific binding sites in the polymer due to formation of fewer complexes between the functional monomer and the template. On the other hand, if VPBA was used alone, the 9
resultant polymer may contain some residual boronic acid moieties so that non-specific binding sites are present outside the imprinted cavities. Considering these two aspects, the molar ratio of 1:1 of VPBA to AAM was chosen to develop the FMIP composites. High-resolution transmission electron microscopy (HRTEM) images revealed that the QDs were spherical in shape and almost uniform in size, with a diameter of approximately 3 nm (Fig. S5 (A)). Fig. S5 (B) shows that the QDs aggregated into larger particles after surface imprinting on the QDs. The thickness of the molecularly imprinted shell was difficult to characterize by HRTEM due to aggregation of the FMIP.
3.2. Effect of pH on fluorescence detection of the target glycopeptide antibiotic . Fig. 2. Influence of pH on fluorescence quenching efficiency of FMIP (green) and FNIP (red) after adding telavancin (20.0 μg/L) using the following buffer solutions and concentrations: pH of 4 Potassium phthalate monobasic (0.05 mol/L); 5-6 CH3COOH-CH3COONa (0.01 mol/L); 7-8 NaH2PO4-Na2HPO4 (0.01 mol/L); 9-11 Na2CO3-NaHCO3 (0.01 mol/L). The enhanced fluorescence intensity (△F) normalized to the initial fluorescence intensity (F0) was used to evaluate the enhanced efficiency of telavancin in increasing the fluorescence intensity of the FMIP. As shown in Fig. 2, the interaction between the FMIP and telavancin was actually pH-dependent. Both the FMIP and FNIP composites exhibited a higher fluorescence enhancement in alkaline environment than in an acidic one. It indicated that the alkaline system indeed influenced the uptake capacity due to lower dissociation extent of the boronic acid groups in the FMIP. The maximum enhancement percentage of fluorescence intensity occurs at a pH of 9.0, so we used a 1.0 mL carbonate buffer solution (CBS 0.01 mol L-1 pH 9.0) to obtain stable fluorescence intensity and high sensitivity in further experiments. Note that this buffer solution is also a typical condition for boronate affinity interaction. 10
The phenomenon can be explained by the fact that boronic acids formed reversible covalent bonds with cis-diols in alkaline pH, whereas the reversible covalent bond dissociated when the medium turned acidic. In acidic pH, boronic acid groups in the cavities were protonated and positively charged; when the pH was increased to neutral or basic levels, the boronic acid groups were deprotonated and became negatively charged. Thus, a change in pH did influence the reversible covalent binding between boronic acids and the target glycopeptide antibiotics, and thereby, influenced the interactions between the template and the cavities of FMIP (Brooks and Sumerlin, 2016). This behavior is an apparent advantage to avoid non-specific adsorption.
3.3. Effect of temperature on fluorescence detection of the target glycopeptide antibiotics
Fig. 3. (A) Effect of temperature on the fluorescence intensity of FNIP and FMIP composites by 10.0 μg/L telavancin, (B) temperature-responsive fluorescence quenching efficiency of FMIP (20.0 mg/L) by telavancin. Prior using the FMIP composites for fluorescence detection of the target glycopeptide antibiotic, we investigated the effect of temperature on the response of the sensing material. As shown in Fig. 3A, both FMIP and FNIP showed relatively low fluorescence intensities at temperature below the lower critical solution temperature of poly (NIPAAM). However, a relatively stable fluorescence enhancement percentage could be observed in the range 35 0C – 50 0C. A similar trend was also exhibited by FNIP with smaller fluorescent response ability due to absence of specific cavities inside the polymer matrix. FMIP exhibited a temperature-responsive behavior. It became swollen at low temperatures, and the imprinted cavities in the FMIP were no longer complementary to the template, thus weakening their interactions. When the temperature was alternated between 10 oC and 38 oC, fluorescence response of the FMIP in the presence of the target glycopeptide antibiotic was first inhibited at 10 oC then restored at 38 oC and this 11
went repeatedly with temperature variation (Fig. 3B). Therefore, to obtain a stable fluorescence intensity and high sensitivity, further experiments were conducted at 38 oC.
3.4. Evaluation of analytical performance of FMIP Selective binding of target molecules to the artificial receptor is the most important objective of developing a sensing material. Therefore, we evaluated the selectivity of our FMIP composites to telavancin by comparing the composite’s fluorescence enhancement response in presence of telavancin and other common antibiotics. As shown in Fig. S6, FMIP showed very sensitive responses toward the template and telavancin, but was almost insensitive to other antibiotics. The most relevant ions in biological fluids had no effect on the detection of telavancin (Table S2). With a boronic acid−functionalized molecularly imprinted sensing material, interference from other cis-diol compounds is always a major concern. The concentration of other monosaccharaides in blood is very low (
PII: DOI: Reference:
S0956-5663(17)30224-5 http://dx.doi.org/10.1016/j.bios.2017.03.059 BIOS9647
To appear in: Biosensors and Bioelectronic Received date: 24 January 2017 Revised date: 21 March 2017 Accepted date: 27 March 2017 Cite this article as: Kuncai Chen, Rong He, Xiaoyan Luo, Pengzhe Qin, Lei Tan, Youwen Tang and Zhicong Yang, A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2017.03.059 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.
A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic Kuncai Chena, Rong Hea, Xiaoyan Luoa, Pengzhe Qina, Lei Tana*, Youwen Tangb, Zhicong Yanga* a
Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China
b
MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou 510006, China
[email protected] [email protected] *Corresponding author: Tel:+86-20-36545121; Fax: +86-20-36545137.
Abstract This paper demonstrates a new strategy for developing a fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic. The technique provides amino modified Mn-doped ZnS QDs as fluorescent supports, 4-vinylphenylbronic acid as a covalent monomer, N-isopropyl acrylamide as a thermo-responsive monomer in combination with acrylamide as a non-covalent monomer, and glycosyl moiety of a glycopeptide antibiotic as a template to produce fluorescent molecularly imprinted polymer (FMIP) in aqueous solution. The FMIP can alter its functional moieties and structure with pH and temperature stimulation. This allows recognition of target molecules through control of pH and temperature. The fluorescence intensity of the FMIP was enhanced gradually as the concentration of telavancin increased, and showed selective recognition toward the target glycopeptide antibiotic preferentially among other antibiotics. Using the FMIP as a sensing material, good linear correlations were obtained over the concentration range of 3.0 to 300.0 μg/L and with a low limit of 1
detection of 1.0 μg/L. The analysis results of telavancin in real samples were consistent with that obtained by liquid chromatography tandem mass spectrometry.
Keywords: molecular imprinting; quantum dots; fluorescence probes; glycopeptide antibiotics.
1. Introduction Antibiotic resistance is the ability of bacteria or other microbes to resist the effects of an antibiotic. Such resistance develops when bacteria change in some way to reduce or eliminate the effectiveness of drugs or other agents designed to cure or prevent infections (Levy and Marshall, 2004). Over the last decades, the number of bacteria resistant to antibiotics has increased significantly, resulting in many infections that are resistant to commonly prescribed antibiotic treatments (Septimus and Kuper, 2009). Glycopeptide antibiotics are used in the treatment of serious gram-positive bacterial infections, including those caused by methicillin-resistant Staphylococcus aureus. Typically, glycopeptide antibiotics are regarded as drugs of last resort for the treatment of otherwise resistant and deadly infections (Kahne et al., 2005). However, unfortunately after more than 50 years of clinical use, the emergence of glycopeptide-resistant Gram-positive pathogens presented a serious threat to public health (James et al., 2012; Pootoolal et al., 2002). Considerable efforts were then made to produce semisynthetic glycopeptide with improved pharmacokinetics and pharmacodynamics properties, but the discovery of new classes of antibiotics remains extremely challenging (Coates et al., 2002). Rational use of antibiotics, avoiding clinical abuse and passive exposure is still the most fundamental way to control the disease. High-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) is widely used for clinical drug testing, but they are currently too expensive and require complex sample pretreatment (Chace, 2001; Badawi et al., 2009). The fluorescence sensing process can directly convert a recognition event into a fluorescence readout signal and attracted considerable attention due to its rapid response, high sensitivity and cost-effective instrumentation (Powe et al., 2004; You 2
et al., 2007). The use of fluorescence methods in bioanalysis, cellular and molecular imaging has become popular largely these days (Vendrell et al., 2012). Compared to the traditional fluorescence dyes, quantum dots (QDs) have plenty of advantages, such as low toxicity, good photo stability, long life time, and little background auto fluorescence, making them appropriate candidates in wide variety of areas (Gill et al., 2008; Resch-Genger et al., 2008). Among them, Mn-doped ZnS QDs have gained increased attention for sensing because of its efficient and characteristic luminescent properties (Chantada-Vazquez et al., 2016; Diaz-Diestra et al., 2017; Ertas and Kara, 2015; Ren and Chen, 2015; Song and Wang, 2014). Antibodies are the most commonly used recognition elements for the sensing systems because of their specificity. However, antibodies are not always desired because of their high cost and low stability, which limit their further application (Haupt, 2010; Whitcombe et al., 2011). Molecular imprinting technology, which involves the formation of artificial recognition cavities in a polymer network with complementary shape and chemical functionality to a target analyte, is an alternative method to overcome the limitations of the antibodies (Chen et al., 2011). Improvements in selectivity of QDs toward a certain analyte can be successfully achieved by embedding the QDs into the matrix of molecularly imprinted polymers (MIPs), resulting in so-called molecularly imprinted sensing materials (Chantada-Vázquez et al., 2016b; Karfa et al., 2016; Li et al., 2016; Lin et al., 2004; Lin et al., 2009; Yang et al., 2016; Zhang and Chen, 2016). The key feature of MIPs is their engineered selectivity of binding to an analyte of interest. Whilst the imprinting of an organic molecule and ion has been well established, the imprinting of a macromolecule has been and continues to be a major challenge (Li et al., 2014). The difficulty is mainly due to structural complexity and large size of macromolecules, which leads to more heterogeneous binding sites and hinders the molecular recognition process (Bossi et al., 2007; Ge and Turner, 2008). To solve this problem, several efforts have been made so far. Epitope imprinting, which uses only a small exposed fragment of a macromolecule as a template, is an effective approach for overcoming the issues which are commonly experienced in the synthesis of MIPs, such as steric hindrance, adsorption 3
thermodynamics and kinetics, and shortage of templates (Rachkov and Minoura, 2000; Nishino et al., 2006; Urraca et al., 2011).
However, determining an
appropriate epitope to achieve a better imprinting effect still remains a big question (Yu et al., 2014). Furthermore, macromolecule imprinting conditions need to be as close as possible to their natural environment to maintain their conformational integrity and binding activity (Tan et al., 2015). The use of water as solvent provides a biologically benign environment for recognition of macromolecules in aqueous media. However, water can reduce hydrogen bonding and electrostatic interactions between template molecules and functional monomers (Zhang et al., 2013). Thus, non-covalent molecular imprinting often shows unavoidable difficulties in aqueous solutions. Boronic acids can form cyclic esters, most frequently with 1,2- and 1,3-diols, allowing boronate ester formation with a number of biologically important species, including saccharides, glycopeptides, glycoproteins, and dopamine significantly broadening the application of boronic acids in biology (Brooks and Sumerlin, 2016). The cyclic esters form in alkaline aqueous solution and dissociate at acidic pH. Boronic acid-functionalized molecularly imprinted polymers were first reported by Wulff and Vesper in 1978 as the stationary phase to separate sugar derivatives by chromatography (Wulff and Vesper, 1978). Recently, Liu and coworkers have been exploring the new field of boronic acid-functionalized glycoproteins molecularly imprinted polymers in immunoassay (Bi and Liu, 2014; Li et al., 2013; Ye et al., 2014; Wang et al., 2014). Guo et al., demonstrated a strategy for producing double responsive fluorescent MIP for sensing of glycoprotein using upconversion nanoparticles
as
fluorescence
signal
reporter,
N-isopropylacrylamide
and
4-vinylphenylboronicacid as functional monomers (Guo et al., 2016). Inspired by the fact that most antibodies recognize a conformational epitope of the antigen rather than its three-dimensional structure, we combined glycosyl-based template design and surface molecular imprinting to fabricate a novel sensing material for pH and temperature regulated sensing of telavancin, a semi-synthetic glycopeptide antibiotic. To this end, we used amino modified Mn-doped ZnS QDs as solid supports, 4-vinylphenylbronic acid, acrylamide and N-isopropyl acrylamide as the functional monomers, and the glycosyl moiety of telavancin as a template to 4
produce graft imprinting at the surface of the QDs. The resultant fluorescent molecularly imprinted polymer (FMIP) was used as a sensing material for pH and temperature regulated recognition of telavancin in aqueous solution to eventually realize sensitive and selective detection of target glycopeptide antibiotic in biological samples.
2. Experimental section 2.1. Reagents and chemicals All chemicals used were of analytical grade. ZnSO4·7H2O, Na2S·9H2O, MnCl2·4H2O, were used as received from Alfa Aesar (Tianjin, China). 4-vinylphenylboronic acid (VPBA), N-isopropyl acrylamide
(NIPAAM), acrylamide
(AAM), N, N, N’,
N’,-tetramethylethylethylenediamine (TEMED) and ammonium persulfate (APS), 3-mercaptopropyltriethoxysilane (MPTS), 3-aminopropyltriethoxysilane (APTES, 98%), Mannose and Tryptophan were obtained from Sigma-Aldrich Co. (St. Louis, MO). Telavancin was purchased from Dr. Ehrenstorfer GmbH. Hydrophilic/lipophilic balance cartridge columns were from Waters. Mannose-Tryptophan (MT) was synthesized using a solid phase peptide synthesis technology in our laboratory, as described in the supplementary material. Ultrapure water from a laboratory water purification system (Sartorius, Germany) was used throughout this work.
2.2. Characterizations The morphology and microstructure of the QDs and FMIP were studied using high-resolution transmission electron microscopy (HRTEM) on a JEM-2100HR (JEOL. Japan). UV-visible absorption spectra were obtained from a Shimadzu UV-Vis 1700 Spectrophotometer. Fluorescence spectra were recorded on an F-2500 (Hitachi, Tokyo, Japan). pH of a solution was monitored with a pH211 microprocessor pH meter (Hanna instruments). The chromatographic system consisted of a Waters® ACQUITY UPLC® I-Class System and a 100 mm×2.1 mm i.d., 1.7 μm particle diameter Acquity BEH-C18 analytical column (Waters) was set at a temperature of 40 0C. A mixture of 0.1% formic acid in water and acetonitrile was delivered at a flow rate of 0.3 mL/min. Detection was performed with a Waters® Xevo TQ triple quadrapole 5
mass spectrometer. The experimental conditions of the ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) used have been summarized in the Supplementary Material (SM).
2.3. Synthesis of amino-capped Mn-doped ZnS QDs The procedure for synthetically producing the amino-capped Mn-doped ZnS QDs used in this work was similar to an already published method (Tan et al., 2014). Specifically, 1.8 g of ZnSO4·7H2O, 0.1 g of MnCl2·4H2O, and 20 mL of ultrapure water were added to a 50 mL three-necked flask. After the mixture was stirred under nitrogen at room temperature for 10 min, 5 mL of aqueous solution containing 1.5 g of Na2S·9H2O was added drop wise, and the mixture was kept stirring for 30 min. Then, 5 mL of an ethanol solution containing 0.072 g of MPTS was added, and the mixture was kept stirring for 24 h. The resultant MPTS-capped Mn-doped ZnS QDs were centrifuged and washed with absolute ethanol three times before being dried under vacuum. In the second step, 10 mL of an absolute ethanol solution containing 200 μL of APTES, 0.35 mL of TEOS, and 250 mg of MPTS-capped Mn-doped ZnS QDs were added to a 25-mL flask. After the mixture was stirred for 10 min, a 0.4 mL of concentrated ammonium hydroxide solution and a 2.0 mL of H2O were added, and the mixture was kept stirring for 18 h. The amino modified Mn-doped ZnS QDs were centrifuged, washed with ultrapure water three times and then dried under vacuum at room temperature.
2.4. Preparation of fluorescent molecularly imprinted polymer (FMIP) Typically, 100 mg of NH2 modified Mn-doped ZnS QDs were first dispersed in 10.0 mL of carbonate buffer solution (0.01 mol/L, pH 9.5) by ultrasonication. AAM, 4-vinylphenylbronic acid, MT and NIPAAM were dissolved into the above dispersion. The mixture was then purged with nitrogen for 30 min and stirred for 1 h before 10 mg of APS and 5 mg of TEMED was added into it. After addition, the solution was again purged with nitrogen and stirred for 5 h. The fluorescent nonimprinted polymer (FNIP) was synthesized in parallel but without the addition of the template. After the reaction, the FMIP and FNIP composites were centrifuged and washed with 6
ultrapure water to remove all absorbed oligomers and unreacted monomers. Finally, the FMIP composites were washed repeatedly with 0.02 mol/L phosphoric acid solution containing 30% acetonitrile (v/v) and shaken for 2 h to remove the template until no MT in the supernatant was detected in UHPLC-MS/MS.
2.5. Evaluation of the adsorption characteristics of FMIP Ten milligrams of FMIP or FNIP were suspended in 5 mL of aqueous solution of different telavancin concentrations. After incubation at the set temperature for 12 h, the FMIP was removed via centrifugation at 8000 rpm for 6 minutes. The binding amount of telavancin was calculated from the difference in concentration between the initial spike solution and the concentration of the supernatant after equilibration. The experimental data is presented as the adsorption capacity per unit mass (mg) of the nanoparticles, and the adsorption capacity (Q) was calculated using
Q
V (C0 Ce ) , where Q (mg/g) is the mass of the template adsorbed by a unit m
mass of particles, C0 (mg/mL) is the initial template concentration, Ce (mg/mL) is the template concentration of the supernatant, V (mL) is the volume of the initial solution, and m (g) is the mass of the composites.
2.6. Measurement procedure The standard solutions of telavancin for calibration were prepared by dissolving appropriate amount of telavancin in 0.01 mol/L carbonate buff solution (CBS , pH 9.0, 0.01 mol/L) and stored at 4 0C. To a 5 mL calibrated test tube, 0.5 mL of 100 mg/L FMIP or FNIP, 0.5 mL of CBS, and different concentrations of telavancin (1.0 mL) were sequentially added. The mixture was mixed thoroughly followed by incubation at 38 0
C for 15 minutes. In the fluorimeter, the slit widths were kept at 10 nm for both
excitation and emission in the fluorescence mode. The excitation wavelength was set at 340 nm and a PMT voltage of 700 V.
2.7. Serum samples Clinical serum samples were collected from volunteers of Guangzhou 12th people’s hospital and stored at −20 °C until use. Prior to use, the samples were first thawed 7
gently, then treated with 100 μL nitric acid (0.5 mol/L) to precipitate the proteins. Next, the mixture was vortexed for 1 minute and then centrifuged at 10000 rpm for 10 min. An appropriate dilution of serum (20-fold, carbonate buffer solution, 0.01 mol/L, pH 9.0) was adopted before detection. In a typical test, to a 5-mL calibrated test tube, were added 0.5 mL of FMIP composites (100 mg/L), 0.5 mL of carbonate buffer solution (CBS) (0.01 mol/L, pH 9.0) and 1.0 mL of the diluted serum sample in sequence. The mixture was mixed thoroughly followed by incubation at 38 0C for 15 minutes.
2.8. Solid phase extraction procedure The solid phase extraction procedure is based on the previous report with slight modification (Wang et al., 2014). Hydrophilic/lipophilic balance cartridges were conditioned and equilibrated by 3 mL of methanol and 3 mL of ultrapure water before 1 mL serum sample loading. After sample loading, the cartridges were rinsed with 3 mL of ultrapure water and vacuum-dried for 5 min. The target compounds were eluted with 3 mL of methanol. After evaporation to dryness under a gentle stream of N2 at 40 0C, the residue was re-dissolved in 1.0 mL of 0.1% formic acid solution for UHPLC-MS/MS analysis.
3. Results and discussion 3.1. Preparation and characterization of FMIP As shown in Fig, S1, telavancin, a typical glycopeptide antibiotic has a homologous heptapeptide scaffold and both covalently attached glycosyl and long chain acyl groups. Note that the cis-diols of telavancin can react with boronic acids monomers to form cyclic esters in an alkaline aqueous solution, and the N-and C-terminals can form hydrogen bonds with non-covalent functional monomers. We selected the glycosyl moiety of telavancin to design and synthesize a new compound, (Fig. S2) which can function as a template to prepare molecularly imprinted polymer receptors for telavancin. . Fig. 1. Schematic illustration of the synthesis of FMIP composites. 8
Traditional MIPs generally show good selectivity but do not possess the ability for intrinsic signal transduction required for sensing applications. To solve this difficulty, incorporation of a fluorescent signal unit within the imprinted cavity of molecularly imprinted polymers was proposed. Among the various luminophores, quantum dots (QDs) have been widely employed due to their high luminescence efficiency, photo-stability and easy preparation. More interestingly, intelligent imprinted polymers have been reported recently, in which the recognition sites can be switched on and off by triggers (Zhang et al., 2014; Ge et al., 2013). As shown in Fig. 1, VPBA was chosen as one of the functional monomers based on the consideration that the boronic acid group could covalently react with glycopeptides or glycoproteins to form cyclic esters in an alkaline aqueous solution. NIPAAM was chosen as a thermoresponsive monomer which would undergo swelling and shrinking with change in temperature (Yoshimatsu et al., 2012). AAM was chosen as a non-covalent functional monomer based on the consideration that effective electrostatic interaction can occur between the template and AAM. After removing the template, recognition cavities complementary to the template molecule in shape, size, and chemical functionality were formed in the cross-linked polymer matrix. Note that the recognition sites were related to the molar ratio of the template and the functional monomers. We designed and prepared several different FMIP to find the optimal monomer composition and the optimal ratio of template to the functional monomer. Telavancin was chosen as the target analyte for evaluation of the synthesized FMIP to find the optimal condition. As shown in Table S1, the adsorption capacity for telavancin was 6.26 mg/g, which yielded a maximum imprinting factor (IF) value of 3.0, obtained using an FMIP comprised of 20% AAM and 20% VPBA at a molar ratio of the template to VPBA equal to 1:1. As shown in Table S1, FMIP possesses a high adsorption capacity and selectivity at a molar ratio of 1:1, while the other molar ratios showed a low adsorption capacity or selectivity. This is possibly because low VPBA to AAM ratios induced fewer non-specific binding sites in the polymer due to formation of fewer complexes between the functional monomer and the template. On the other hand, if VPBA was used alone, the 9
resultant polymer may contain some residual boronic acid moieties so that non-specific binding sites are present outside the imprinted cavities. Considering these two aspects, the molar ratio of 1:1 of VPBA to AAM was chosen to develop the FMIP composites. High-resolution transmission electron microscopy (HRTEM) images revealed that the QDs were spherical in shape and almost uniform in size, with a diameter of approximately 3 nm (Fig. S5 (A)). Fig. S5 (B) shows that the QDs aggregated into larger particles after surface imprinting on the QDs. The thickness of the molecularly imprinted shell was difficult to characterize by HRTEM due to aggregation of the FMIP.
3.2. Effect of pH on fluorescence detection of the target glycopeptide antibiotic . Fig. 2. Influence of pH on fluorescence quenching efficiency of FMIP (green) and FNIP (red) after adding telavancin (20.0 μg/L) using the following buffer solutions and concentrations: pH of 4 Potassium phthalate monobasic (0.05 mol/L); 5-6 CH3COOH-CH3COONa (0.01 mol/L); 7-8 NaH2PO4-Na2HPO4 (0.01 mol/L); 9-11 Na2CO3-NaHCO3 (0.01 mol/L). The enhanced fluorescence intensity (△F) normalized to the initial fluorescence intensity (F0) was used to evaluate the enhanced efficiency of telavancin in increasing the fluorescence intensity of the FMIP. As shown in Fig. 2, the interaction between the FMIP and telavancin was actually pH-dependent. Both the FMIP and FNIP composites exhibited a higher fluorescence enhancement in alkaline environment than in an acidic one. It indicated that the alkaline system indeed influenced the uptake capacity due to lower dissociation extent of the boronic acid groups in the FMIP. The maximum enhancement percentage of fluorescence intensity occurs at a pH of 9.0, so we used a 1.0 mL carbonate buffer solution (CBS 0.01 mol L-1 pH 9.0) to obtain stable fluorescence intensity and high sensitivity in further experiments. Note that this buffer solution is also a typical condition for boronate affinity interaction. 10
The phenomenon can be explained by the fact that boronic acids formed reversible covalent bonds with cis-diols in alkaline pH, whereas the reversible covalent bond dissociated when the medium turned acidic. In acidic pH, boronic acid groups in the cavities were protonated and positively charged; when the pH was increased to neutral or basic levels, the boronic acid groups were deprotonated and became negatively charged. Thus, a change in pH did influence the reversible covalent binding between boronic acids and the target glycopeptide antibiotics, and thereby, influenced the interactions between the template and the cavities of FMIP (Brooks and Sumerlin, 2016). This behavior is an apparent advantage to avoid non-specific adsorption.
3.3. Effect of temperature on fluorescence detection of the target glycopeptide antibiotics
Fig. 3. (A) Effect of temperature on the fluorescence intensity of FNIP and FMIP composites by 10.0 μg/L telavancin, (B) temperature-responsive fluorescence quenching efficiency of FMIP (20.0 mg/L) by telavancin. Prior using the FMIP composites for fluorescence detection of the target glycopeptide antibiotic, we investigated the effect of temperature on the response of the sensing material. As shown in Fig. 3A, both FMIP and FNIP showed relatively low fluorescence intensities at temperature below the lower critical solution temperature of poly (NIPAAM). However, a relatively stable fluorescence enhancement percentage could be observed in the range 35 0C – 50 0C. A similar trend was also exhibited by FNIP with smaller fluorescent response ability due to absence of specific cavities inside the polymer matrix. FMIP exhibited a temperature-responsive behavior. It became swollen at low temperatures, and the imprinted cavities in the FMIP were no longer complementary to the template, thus weakening their interactions. When the temperature was alternated between 10 oC and 38 oC, fluorescence response of the FMIP in the presence of the target glycopeptide antibiotic was first inhibited at 10 oC then restored at 38 oC and this 11
went repeatedly with temperature variation (Fig. 3B). Therefore, to obtain a stable fluorescence intensity and high sensitivity, further experiments were conducted at 38 oC.
3.4. Evaluation of analytical performance of FMIP Selective binding of target molecules to the artificial receptor is the most important objective of developing a sensing material. Therefore, we evaluated the selectivity of our FMIP composites to telavancin by comparing the composite’s fluorescence enhancement response in presence of telavancin and other common antibiotics. As shown in Fig. S6, FMIP showed very sensitive responses toward the template and telavancin, but was almost insensitive to other antibiotics. The most relevant ions in biological fluids had no effect on the detection of telavancin (Table S2). With a boronic acid−functionalized molecularly imprinted sensing material, interference from other cis-diol compounds is always a major concern. The concentration of other monosaccharaides in blood is very low (
