Water-compatible ‘aspartame’-imprinted polymer grafted on silica surface for selective recognition in aqueous solution Meenakshi Singh, Abhishek Kumar & Nazia Tarannum
Analytical and Bioanalytical Chemistry ISSN 1618-2642 Anal Bioanal Chem DOI 10.1007/s00216-013-6812-6
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Author's personal copy Anal Bioanal Chem DOI 10.1007/s00216-013-6812-6
RESEARCH PAPER
Water-compatible ‘aspartame’-imprinted polymer grafted on silica surface for selective recognition in aqueous solution Meenakshi Singh & Abhishek Kumar & Nazia Tarannum
Received: 6 December 2012 / Revised: 1 February 2013 / Accepted: 1 February 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Molecularly imprinted polymers selective for aspartame have been prepared using N-[2-ammonium-ethylpiperazinium) maleimidopropane sulfonate copolymer bearing zwitterionic centres along the backbone via a surfaceconfined grafting procedure. Aspartame, a dipeptide, is commonly used as an artificial sweetener. Polymerisation on the surface was propagated by means of Michael addition reaction on amino-grafted silica surface. Electrostatic interactions along with complementary H-bonding and other hydrophobic interactions inducing additional synergetic effect between the template (aspartame) and the imprinted surface led to the formation of imprinted sites. The MIP was able to selectively and specifically take up aspartame from aqueous solution and certain pharmaceutical samples quantitatively. Hence, a facile, specific and selective technique using surface-grafted specific molecular contours developed for specific and selective uptake of aspartame in the presence of various interferrants, in different kinds of matrices is presented. Keywords Aspartame . Dipeptide . Molecularly imprinted polymer . Sulfobetaine polymer
Introduction In 1996, Lemieux published an excellent account on ‘role of water in Molecular Recognition’ and concluded that ‘water plays a central role in all biological associations because its interactions with molecular surfaces lead to perturbation by either ordering (nonpolar surface) or strain (polyamphiphilic surface). This bottled energy is always on tap to help drive M. Singh (*) : A. Kumar : N. Tarannum Department of Chemistry, MMV, Banaras Hindu University, Varanasi 221005, India e-mail:
[email protected]
specific molecular events. Like a chaperon, water accompanies the reactants in their search for each other’ [1]. Molecular imprinting is one of few general, nonbiological methods for creating molecular receptors. The approach of molecular imprinting creates populations of specific recognition sites in synthetic network polymers [2]. Polymerisation of monomers in the presence of target molecule to imprint the structural information is known as molecular imprinting of polymers [3]. This is a scientific field which is rapidly gaining significance for a wide range of applications in chemistry, biotechnology and pharmaceutical research. Due to their analytically useful properties, such as selectivity, shelf stability, robustness and reusability, molecular imprinting of polymers offers potential for the synthesis of artificial recognition material, and they are being proposed for the development of novel biorecognition elements for drugs, environmental pollutants and other toxic substances [4–7]. Water incompatibility is the primary concern of imprinting fraternity [8]. This severely hampers applications in aqueous media and poses problem in real analysis. Efforts are being made to improve water compatibility of MIPs. Dirion et al. imprinted bupivacaine in aqueous medium with good selectivity [9, 10], and Pan et al. were able to synthesise water-compatible MIP microspheres with hydrophilic macromolecular chain transfer agentmediated RAFT precipitation polymerisation [11]. Ciprofloxacin was also imprinted in water-compatible medium [12], and sildenafil and its metabolite were imprinted in watercompatible medium [13]. Au-MIP nanocomposites were prepared as highly selective and sensitive functional materials using bis-aniline-crosslinked Au nanoparticles for plasmonic sensing of certain antibiotic substrates (neomycin, kanamycin, streptomycin) via boronic acid ligands; for chiroselctive sensing of certain amino acids (L-glutamic acid, L-aspartic acid, Lhistidine, L-phenylalanine) and the explosive trinitrotoluene sensor [14–16]. Our group has shown water-compatible imprinting with novel polymer format in which the monomers
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were water soluble and entire imprinting protocol is followed in water only for selective and sensitive detection of ‘baclofen’ by surface imprinting on silica particles [17]. An added restraint is high crosslinking nature of MIPs which limits the extraction of the original templates located at interior area of the bulk materials which results in incomplete template removal, small binding capacity and slow mass transfer [18–20]. Surface imprinting largely circumvents these limitations, as the imprinted templates are situated at the surface or in the proximity of the material’s surface. Several strategies for surface imprinting have been attempted such as use of immobilised template [21], initiator on supporting matrix [22] and combined surface imprinting with controlled living radical polymerisation. Many particles have been used as supports in the surface imprinting process, such as activated silica gel [23, 24], Fe3O4 magnetic nanoparticles [25], chitosan [26], activated polystyrene beads [22], quantum dots [27, 28] and alumina membranes [29, 30]. Among all these support particles, activated silica gel has shown better prospects. Hence, surface-imprinting of the polymers in a two-dimensional domain can be an attractive alternative [31–34]. Dipeptide, aspartame (L-aspartyl-L-phenylalanylmethyl ester) consists of a hydrophilic and a hydrophobic amino acid providing some exclusive characteristics to it. It is a high potency non-carbohydrate sweetener (approximately 100– 150 times sweeter than sucrose) universally used in soft drinks, tabletop sweeteners, chewing gums, etc. [35]. Aspartame, on ingestion, is immediately absorbed from the intestinal lumen and metabolised to phenylalanine, aspartic acid and methanol [36]. Aspartic acid is transformed into alanine and oxaloacetate while phenylalanine is transformed into tyrosine, and, to a lesser extent, phenylethylalanine and methanol are converted into formaldehyde which oxidises to formic acid [37]. Phenylalanine is known to be neurotoxic and affects synthesis of inhibitory monoamine neurotransmitters [38]. Genotoxic effects of aspartame are also reported as it induces chromosome aberration, micronuclei formation, but it does not induce mutagenesis [39]. Clinical studies showed that usage of the excess aspartame leads to various health problems such as headaches, migraines and memory loss [40]. Since aspartame is widely used in foods, soft drinks and dietary products, and due to its increased application in food industry, fast and efficient methods are needed for its determination. Various methods including spectrophotometry, potentiometry and chromatography have been proposed for the determination of aspartame [41–55]. Various biosensing systems based on enzymes and microbial organisms have also been developed as alternatives methods for aspartame analysis [56–58]. In lieu of these, developing a cost-effective aspartame imprinted polymer under water-compatible medium will prove to be a more realistic option for separation and evaluation of aspartame as well as for its specific and selective measurement in its formulated products. Here, we report on
the sensitive and selective detection of aspartame by an imprinted zwitterionic polymeric format grafted on silica surface. The development of electrostatic interactions along with other non-covalent interactions facilitated the creation of specific and selective imprints of aspartame.
Experimental Reagents Maleimide, N(2-aminoethyl) piperazine, toluene and methanol were purchased from Merck. 1,3 Propane sultone 98 % purity were procured from Fluka (Steinheim, Germany). (3Aminopropyl)triethoxy silane (97 %) (APES) was purchased from Lancaster. Silica gel (pore size 60 Å, 230–400 mesh size) was procured from Sigma Aldrich (Steinheim, Germany). The interferrants used such as L-aspartic acid, acetanilide, Daspartic acid, L-glutamic acid and L-phenylalanine were purchased from Spectrochem Pvt Ltd., Mumbai, India. Toluene and hydrochloric acid, sodium hydroxide and dimethylsulfoxide were procured from Fischer Scientific (Mumbai, India). All the chemicals and solvents were of AR grade and used as received. The pharmaceutical samples analyzed were sugarfree gold sachet (artificial sweetener) from Acme Diet Care Pvt. Ltd. (Chhatral, India). Apparatus A Cary 50 Bio (Varian instruments Inc, Melbourne Australia) was used for all spectrophotometric measurements. Standard solutions of aspartame and other crossselective analytes have been prepared, and calibration plots were constructed between the concentration and absorbance at 208 nm. These plots used for estimating the amounts of the components absorbed onto MIPs and non-imprinted polymer (NIPs).The extent of uptake of the template molecule by the polymer were estimated by measuring the absorbance (concentration) of the washed solution. All the measurements were made in triplicate. The infrared spectra of the materials were recorded using Jasco FT/IR5300 from 400–4,000 cm−1.
Modification of silica particles Activated silica particles (5.0 g) were treated with 5 % solution of APES (150 mL toluene), refluxed for 8 h, and centrifuged, and thus, amino-terminated silica particles were extracted with toluene, dried in vaccuo at 110 °C, and stored in vaccuo at room temperature. Estimation of amino groups on silica surface Amino groups introduced on the silica surface were determined by titration. To 0.10 g silica particles, 20 ml of 0.01 N
Author's personal copy Water-compatible ‘aspartame’-imprinted polymer
HCl was added and charged the mixture on magnetic stirrer at 65 °C for 2 h. The solution was filtered, and filtrate was titrated with 0.01 N NaOH using phenolphthalein as an indicator. Grafting step Silica particles with initiator sites were washed several times with toluene and dried in oven at 120 °C for 5 h before use. The prepared silica particle was treated with maleimide (0.005 mol), 2-aminoethyl piperazine (0.005 mol) in toluene and 1,3 propane sultone (0.4387 mL) in a UV chamber in the presence of AIBN used as a initiator for photopolymerisation. The mixture was filtered in sintered crucible and washed with toluene three to four times and dried in air. Furthermore, adduct was prepared by the similar procedure as NIP, except the use of aspartame as the template molecule. A homogeneous mixture of aspartame was prepared. MIP was prepared by extracting aspartame at optimised parameters. The corresponding structures of MIP and NIP are shown in Fig. 1. The absorbance scan of aspartame solution (shows a maximum absorption wavelength at 208 nm). The technique thus developed was validated by determining its performance characteristics regarding linearity, repeatability and precision. The extent of uptake of the template molecule by 100 mg of grafted polymers was estimated by measuring the absorbance of the washed Fig. 1 Schematic representation of synthesis of NIP and adduct on the silica surface
solution. All the measurements were made in triplicate. Linearity of calibration plots was good (r2 >0.946). The spectrophotometric technique was validated by determining linearity in the concentration range 0.005– 0.015 M of aspartame solution (λ=208 nm, r2 (correlation coefficient)=0.906, detection limit=29.2×10 − 3 μgmL−1, limit of quantification=96.36×10−3 μgmL−1).
Results and discussion Preparation and characterisation of surface imprinted polymer Aspartame in the buffer solutions is most stable over the pH range 4–5, becoming less stable as the pH increased or decreased [59]. Aspartame bears a net negative charge at neutral pH and no charge at pI as the zwitterionic character of the amino acids cancels out the positive charge of –NH3+ and negative charge of –COO− moiety at their respective pI. Thus, we chose a zwitterionic polymeric format to imprint aspartame, which could provide electrostatic interactions with respective counter charges along with additional Hbonding and other hydrophobic interactions to generate specific recognition sites in the imprinting network. Silica having silanol groups react with silane coupling agent to produce an initiator site at the surface. Successive surface reactions, the first being the reaction of the free silanol groups
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on the surface with APES followed by reaction of amino group via Michael addition reaction with comonomers completes the grafting procedure. Many examples of Michael addition on surfaces via amino group are found in literature [60]. The introduction of amino groups as an initiator site is achieved by treatment of silica with APES. Infrared spectra of silica charged with initiator group shows a new absorption at 1,656 cm−1 in comparison to untreated silica surface, thereby suggesting the introduction of amino group on silica surface. The content of amino group charged on the surface of silica as an initiator site as per the titration is 0.27 mmol/g whereas the grafting of polyelectrolyte on silica surface leaves behind 6.70×10−3 mmol/g of amino centres. The amino-charged silica surface acts as an electron donor and maleimide behaves as an electron acceptor. Subsequent reaction steps are propagated by means of Michael-type addition reaction as detailed in earlier publication from our group [61] on silica surface. Maleimide is highly reactive toward primary amine groups to perform Michael addition reactions. Here, the –NH2 groups of amino ethylpiperazine acts as nucleophile which was condensed with maleimide. Furthermore, the polymer chain was modified by a sulfoalkylating agent, 1,3-propane sultone. Asapartame is used as an imprinting template on the polyelectrolyte grafted silica bed. Both the polymer and template exist in zwitterionic state anchoring each other via electrostatic and other non-covalent forces including hydrogen bonding, wherever possible. A close comparison of Fourier transform infrared (FTIR) spectra of NIP, adduct and MIP has been shown in Fig. 2. Absorption at ∼1,100 cm−1 in the curves a, b and c overlaps the absorption due to Si-O-Si stretching with sulphonate group absorption from sulfobetaine moiety. The imidyl group (O=C–N–C=O) of maleimide absorption is seen at 1,715 cm−1 as a sharp peak in NIP (curve c) and as a small shoulder in adduct as it merges with >C=O absorptions of aspartame and again reappears in MIP (curve a) as a merged peak with >C=O absorption of maleimide. The presence of aspartame in adduct (curve b) is characterised by a sharp absorption at 1,184 cm−1 [C–C(=O)–O of esters absorb in 1,210–1,163 cm−1 range] and at 1,044 cm−1due to O–C–C bond of esters of primary alcohols. It is to be mentioned here that aspartame is an ester of phenylalanine (L-aspartyl-L-phenylalanylmethyl ester). Aromatic stretch from phenyl ring of aspartame is observed as a broad hump in the range 1,500– 1,560 cm−1 in curve b of adduct spectra. The absence of characteristic ester absorption and aromatic ring absorptions in curve a of MIP spectra ensures the complete removal of aspartame from adduct on extraction with methanol as discussed below. Optimisation of analytical parameters Several analytical parameters like extracting solvent, its temperature, volume, extraction time, number of washes,
Fig. 2 Comparative study of FTIR of silica grafted MIP (a), adduct (b) and MIP (c)
reusability, etc., under which binding of aspartame to the aspartame-imprinted polymer is best and much higher than binding of the NIP have been optimised to obtain the paramount condition to extract aspartame from the adduct. Among the solvents chosen (deionised water, ethanol/water (1:1), acetic acid, water, ethanol, THF, water, methanol, etc.) methanol at room temperature was found to be best suited. The optimised extraction conditions are 10 ml methanol at room temperature (32 °C). Aliquots of extracting solvent in different portions were evaluated for efficient extraction. Portions of 8–18 mL were undertaken, but volumes lesser than 10 mL were insufficient, and volumes larger than 10 mL probably allowed unnecessary dilution which lowered the extraction efficiency (Fig. 3). Three 10-mL portions of extracting solvent were optimised for best extraction as lesser than three washes were inadequate, and more than three washes slightly lessened the efficacy of extraction. Three washes of optimised extracting solvent with continuous mechanical stirring (600 rpm) for 10 min were able to extract template completely. Another set of parameters for rebinding template to the imprinted cavities generated under the aforementioned experimental parameters like concentration, pH of aspartame solution and time required to rebind on the polymer surface were optimised. In rebinding
Author's personal copy Water-compatible ‘aspartame’-imprinted polymer Fig. 3 a Effect of the retention time to rebind the template on molecularly imprinted polymer (MIP); b effect of volume on recovery percent; c effect of concentration of template solution on recovery (percent) of the template from aqueous solution, d effect of pH of the template solution on recovery (percent)
experiments, as shown in Fig. 3a, 25 min were optimum for uptake of the template in MIP cavities. Figure 3 shows that MIP takes utmost of 25 min to rebind the template molecule. Longer period of contact might direct the equilibria in reverse direction. Figure 3c shows the concentration dependence of aspartame imprinted polymer. The uptake of aspartame by MIP was linearly dependent on concentration of the template with saturation of 5.9 ppm by 1 mg of MIP, being attained at 0.002 M of template aspartame in aqueous solution. The 0.002 M aspartame solution is found to be better suited for experimental study. Solution of template molecule with concentrations lower or higher than this concentration was not found to be suitable for further experiments. At lower concentration, the non-specific bindings are not able to override the specific bindings, but as the crowding begins at high concentrations of template in solution, the adherence of template to the non-imprinted polymer network could be noted as the competition for the specific sites in the imprinted network becomes high. As shown in Fig. 3d, pH 6.08 was found to be most suitable for recovery of template. Aspartame is a dipeptide of aspartic acid and phenylalanine (L-aspartyl-L-
Fig. 4 Representation of structural analogues of aspartame
phenylalanylmethyl ester). On scrutinising the behaviour of two amino acids at varying pH of aqueous solution, it is found that pI of phenylalanine is ∼6 [62], and this aa is in zwitterionic form in the range 4.41–7.76, whereas aspartic acid is fully deprotonated beyond pH 6. It is expected that the molecules of template will be in zwitterionic form at pH∼6. As shown in Fig. 3d, pH 6 was found to be suitable for uptake of template molecules by MIP. Hence, at this pH, the electrostatic interactions among the respective charge centres of template and polymeric chain are optimised to be best suited for binding in a highly polar solvent, i.e. water, bearing high dielectric constant enabling them to attain electrostatic interactions efficiently (Fig. 3). During network formation, increasing the potential for growing polymer chains with template binding complexes to reach a global energy minimum will lead to increased memorisation of the chain conformation and enhance template binding parameters in both highly and weakly crosslinked polymers. Frustrations between the template and polymer chains in forming complexes can be minimised by molecular imprinting [63, 64]. The imprinting effect of
Author's personal copy M. Singh et al. Fig. 5 Cross-selectivity study of adsorption of various interferrants on aspartame-MIP under optimized conditions [1: D-aspartic acid, 2: L-glutamic acid, 3: L-aspartic acid, 4: Lphenylalanine, 5: acetanilide]
MIP is frequently assessed by the imprinting factor (IF) defined as IF ¼
BMIP BNIP
ð1Þ
where BMIP is retention (percent) by MIP and BNIP is retention (percent) by NIP [64]. For this aspartame imprinted polymer, IF is found to 2.11, indicating the selectivity of MIP for aspartame compared with NIP. Such data verify the fact that the MIPs possessed the good selectivity for aspartame due to the fixed recognition cavities created during imprinting process which was propounded to be originating from ‘stoichiometric noncovalent interactions’ where the interaction during the polymerisation was stoichiometric in nature [64]. Cross-selectivity study The cross-selectivity of the prepared MIP is evaluated via several structural analogues of aspartame. The analogues chosen were having similar functional groups, etc. The interferrants such as L-aspartic acid, L-glutamic acid, acetanilide, L-phenylalanine and D-aspartic acid were chosen as they comprise of similar functional groups (Fig. 4). Figure 5 shows the response of MIP at optimised parameters towards interferrants. The template fits into the imprinted cavity driven by the electrostatic interaction in this study followed by aligning of the functional groups on the MIP around the template molecule’s conformational orientation. In NIP, although the functional groups are also present in the polymer, they are randomly arranged in such a manner that it is ineffective for correct binding with the template. Similarly, an interferrant is different in size and conformation and might lack key structural features responsible for fitting into the recognition cavities. This leads to much greater affinity for the template molecule by molecular imprinting, in comparison with the non-imprinted one. As the figure shows, MIP shows an imprinting effect for the template molecule.
The non-specific bindings are responsible for the template adsorption shown by the NIP as evident in the figure. This ascertains that MIP cavities are specific and selective for aspartame only, even in mixtures of template and nontemplate and/or interferrants also. Analytical applications The technique thus developed was validated by determining its performance characteristics regarding linearity, repeatability and precision. To test the UV response linearity, a series of standard solutions in the concentration range 0.005–0.015 M was analysed (at least 15 samples). Detection limit was estimated [65] as 29.2×10−3 μgmL−1 The MIP has been prepared from adduct as per the optimised parameters above and has been reused thrice with the same sample. Molecularly imprinted technique developed for aspartame was applied on various pharmaceutical samples available in Indian Market which contains aspartame as the major constituent. The pharmaceutical sample (Sugarfree gold) is reported to contain 35 mg of aspartame. The total aspartame recovered from the sample was 88 %.
Conclusions In this work, a surface MIP (water-compatible) was successfully developed for selective recognition of target molecule, i.e. aspartame, the industrially and clinically significant dipeptide aspartame in the presence of various interferrants without any pre-treatment or other such complicated pathological/clinical procedures. Polymerisation on the surface was propagated by means of Michael addition reaction on amino-grafted silica surface. Selectivity studies suggest that the MIPs show signs of good selective recognition when compared with NIPs. Experimental results obtained in this work demonstrate that surface MIPs can be used for the
Author's personal copy Water-compatible ‘aspartame’-imprinted polymer
selective detection of aspartame from aqueous solutions. The specific selectivity of the synthesised MIPs implied potential applications in analytical separation techniques. Acknowledgement The work was supported by UGC, new Delhi [F.no.41-331/2012(SR)].
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