R E S E A R C H P A P E R
Semi-synthetic biotin imprinting onto avidin crosslinked
gold–silver nanoparticles
Ayc¸a Atılır O¨ zcan•Arzu Erso¨z• Deniz Hu¨r• Filiz Yılmaz• Aytac¸ Gu¨ltekin•Adil Denizli• Rıdvan Say
Received: 20 January 2012 / Accepted: 22 May 2012 / Published online: 10 June 2012 Ó Springer Science+Business Media B.V. 2012
Abstract This study is a different and new applica-tion of molecular imprinted polymers (MIPs) based on sensor technologies. In this study, semi-synthetic biotin imprinted polymeric shell has been decorated onto the surface of avidin crosslinked Au/Ag nanocl-usters using bis (2-20-bipyridyl) MATyr-MATrp-ruthenium(II) (MATyr-Ru-MATrp) as photosensitive monomer. The synthesized nanoclusters have been used the recognition of biotin by flourometric method. Synthesis of the photosensitive monomers has been realized by AmiNoAcid (monomer) Decorated and Light Underpinning Conjugation Approach (AN-ADOLUCA) method. This method provides a strategy for the preparation of photosensitive ruthenium based aminoacid monomers and oligomers, aminoacid
monomer-protein crosslinking using photosensitation and conjugation approach on micro and nano-struc-tures by ruthenium-chelate based monomers. The affinity constant (Ka) of biotin imprinted Au/Ag nanoclusters has been determined using the Scatchard method and found to be 3.89 9 105M-1. The obtained calibration graph is linear for the range of 0.051 and 2.50 lM of biotin. The detection limit of biotin has been found to be 15 nM. Also, the reusability of these nanoclusters has been investigated and it has been observed that the same clusters could be used 10 times during a long period without any binding capacity decreasing.
Keywords Molecular imprinting Sensor Biotin Nanocluster Conjugation method
Introduction
Photoimmobilization demands the presence of medi-ating photosensitive reagents, generally activated by incident light of an appropriate wavelength. After the light activation, the reagents undergo distinct chem-ical processes that finally lead to the formation of covalent bonds between the photogenerated interme-diates and the biomolecules (Rusmini et al.2007). The underlying chemistry is proposed in the literature to involve the formation of radicals allowing tyrosine residues to give covalent bonds with another tyrosine
Electronic supplementary material The online version of this article (doi:10.1007/s11051-012-0945-y) contains supplementary material, which is available to authorized users. A. Atılır O¨ zcan (&) A. Erso¨z D. Hu¨r
F. Yılmaz R. Say
Department of Chemistry, Anadolu University, Fen Faku¨ltesi, Kimya Bo¨lu¨mu¨, Yunus Emre Kampu¨su¨, 26470 Eskis¸ehir, Turkey
e-mail: aatilir@anadolu.edu.tr A. Gu¨ltekin
Department of Engineering of Energy Systems, Karamanog˘lu Mehmetbey University, Karaman, Turkey A. Denizli
Department of Chemistry, Hacettepe University, Ankara, Turkey
and use a strategy to oxidative protein–protein cross-linking using photosensitization (Fancy and Kodadek 1999; Brown and Kodadek 2001; Duroux-Richard et al.2005).
Certain nanomaterials are ideal probe candidates because of their (i) small size (1–100 nm) and correspondingly large surface area-to-volume ratio, (ii) chemically tailorable physical properties, (iii) unusual target binding properties, and (iv) overall structural robustness. Tailorable physical properties and oriented surface modification are very important aspects of nanomaterials. Indeed, in this regard, nanomaterials and biology have a sustained history while nanoparticles have been used as bio-conjugation and cellular labeling agents for the past four decades (Rosi and Mirkin 2005; Wang et al. 2006). At this point in time, the use of nanobio-conjugates for life sciences and biotechnology applications is one of the fastest moving fields of nanobiotechnology (Jain 2007; Mazumder et al.2009). To apply nanoparticles in biological systems or aqueous environment, it is essential to modulate the chemical nature of nanopar-ticle surfaces to alter their biocompatibility and add additional biochemical functionalities and stabilities. By employing different conjugation technologies they can not only be rendered biocompatible, but also, to fulfill tasks. These embody receptors are used as targeting, sensing, imaging, catalysis, or preconcen-trator (Walther et al.2008; Choi et al.2006,2008; Ali et al. 2008; Huh et al. 2005). To achieve this goal, different monomeric or polymeric coatings are applied to provide biocompatibility and additional bioconju-gation (also for multivalent interaction) for targeting those particular drugs which prevent the disease spreeding and other applications (Hezinger et al. 2008).
As it is known, biotin has many important roles in the biological functions. For example; biotin is the critical cofactor in biotin-dependent carboxylase (BDC) enzymes, whose chemical function is the fixation of carbon dioxide from bicarbonate (Knowles 1989; Jitrapakdee and Wallace1999; Attwood1995). Also, it is well known that avidin has high affinity toward biotin molecule and forms a complex ion with biotin in a ratio of 1:4. This high affinity between avidin and biotin has used in many studies to develop selective detection systems in the literature (Pe´rez-Luna et al.1999; Anzai et al.1998; Morpurgo et al. 2004; Wilchek and Bayer 1988,1999; Livnah et al.
1993). The alternative methods for biotin recognition are metal–biotin interactions (Sanchez et al. 2002) and biotin imprinted polymers (Piletska et al. 2004; Izenberg et al. 2009). Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. Target mole-cules (i.e., biotin) can be used as templates for imprinted crosslinked polymers (Say et al. 2009). When they are compared with natural recognition products such as antibodies, molecular imprinted polymers (MIPs) offer advantages such as durability, specificity and ease of mass production, that have previously not been offered by alternative techniques (Kriz et al.1997; Hawkins et al.2005). In this study, biotin imprinted Au/Ag nanoclusters have been synthesized using bis (2-20-bipyridyl) MATyr-MATrp-ruthenium(II) (MATyr-Ru-MATrp) as a photosensitive monomer, avidin as a ligand monomer, and biotin as a template molecule applying AmiNoAcid (mono-mer) Decorated and Light Underpining Conjugation Approach (ANADOLUCA) method (Say 2011; Say et al.2011). Then, these nanoclusters have been used for the biotin recognition as an alternative and unique imprinting method.
ANADOLUCA method provides a strategy for the preparation of photosensitive ruthenium based aminoacid monomers and haptens, aminoacid mono-mer-protein crosslinking using photosensitation and conjugation approach on micro and nano-structures by ruthenium-chelate based monomers. The photosensi-tive, covalent and crosslinking conjugation methods based on aminoacid and ruthenium-chelate based monomers provide accurately antibody orientation and prevent denaturation during bonding and after bonding. Indeed, they provide efficiency of bounded proteins as well as many uses such as reusable enzymes, reusable separation solid phase systems based affinity cromatography, theranostics, nanopro-tein carrier, receptor targeted nanocargoes, manage-able imaging, and detection technologies.
Experimental
Reagents and apparatus
The chemicals were purchased from Aldrich and Sigma and used without further purification. All water used in the experiments was purified using a Barnstead
(Dubuque, IA) ROpure LP reverse osmosis unit with a high-flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANO pure organic/colloid removal and ion exchange packed-bed system.
The melting points were determined on Sanyo Gallenkamp. Elemental analyses were performed by Vario ELIII. NMR spectra were recorded on a Bruker 500 MHz NMR spectrometer Ultra-shield FT-NMR spectrometer at room temperature. All matrix-assisted laser desorption ionisation time-of-flight mass spec-trometry (MALDI-TOF/MS) mass spectra were acquired on a Voyager Biospectrometry STR Work-station. The system utilizes a pulsed nitrogen laser and emitting at 337 nm. The acceleration voltage was set to 20 kV and the delay time was 100 ns. Mass analysis was carried out in positive reflector mode and delayed extraction mode. a-cyano-4-hydroxycinnamic acid (CHCA) was used as a matrix solution. 10 mg CHCA was solved in 1:1, 1 mL of 0.3 % trifluoroacetic acid (TFA) solution and acetonitrile. 2 lL of sample solution was mixed with 23 lL of 10 mg mL-1 solution of CHCA in acetonitrile/0.3 % TFA. This preparation (1 lL) was placed onto a MALDI-TOF/ MS sample plate and allowed to dry.
Photo-luminescence spectra were obtained using a Carry Eclipse Varian Model Fluorescence Spectrom-eter. Transmission electron microscopy (TEM) image of Au/Ag nanoclusters was recorded on a FEI 120 kV electron microscope.
Synthesis of chlorobis (2-20-bipyridyl) MATyr-ruthenium(II) and bis (2-20-bipyridyl) MATyr-MATrp-ruthenium(II) [MATyr-Ru-MATrp]
Aminoacid monomers methacryloyl tyrosine (MA-Tyr), methacryloyl tryptophan (MATrp), and methac-ryloylamidocystein (MACys) were prepared and characterized according to following previously pub-lished method (Hur et al. 2007). Dichlorobis (2-20 -bipyridyl) ruthenium(II) (RuCl2(bipyr)2) was synthe-sized according to the procedure that has published by Evans et al. (1973).
For the synthesis of chlorobis (2-20-bipyridyl) MATyr-ruthenium(II); 1 eq RuCl2(bipyr)2 was dis-solved in methanol. The solution was cooled to 0°C and ethylenetriamine was added into this solution. 1.2 eq MATyr in methanol solution was added dropwise into that solution and the mixture refluxed
at 55°C for 48 h. At the end of the reaction time, the solvent was removed under reduced pressure and the residue was dissolved in dichloromethane. The solu-tion was washed with 39 H2O and dried with MgSO4. After the evaporation of the solvent, the product (Fig.1a) washed with ether and dried under vacuum. M.p.: 125–128°C.
Chlorobis (2-20-bipyridyl) MATyr-ruthenium (II) (1 eq) was dissolved in methanol. MATrp solution (1.2 eq) in methanol was added by dropwise into that solution at room temperature and the mixture was refluxed at 80°C for 24 h. At the end of this period, the solvent was removed under reduced pressure and the residue was dissolved in dichloromethane. The solution was washed with water and dried with MgSO4. After the solvent was evaporated, the product (MATyr-Ru-MATrp) (Fig.1b) washed with ether and dried under vacuum. M.p.: 110–112°C.
Synthesis of biotin imprinted avidin crosslinked Au/Ag nanoclusters
In this study, the gold–silver (Au–Ag) nanoclusters were preferred as supporting material because of their optical properties. The Au–Ag nanoclusters were prepared in a two-phase (water/toluene) system using a modified Brust method (Brust et al.1998). The TEM image of nanoclusters was investigated and (Fig. S1— Supplemental information) as can be seen from Fig. S1, the shape of nanoclusters is close to spherical, aggregated and average size is about 40 nm.
To prepare biotin imprinted Au/Ag nanocluster, 1.5 mg MACys activated Au/Ag nanocluster (Dilte-miz et al.2008) was dispersed in 5 mL of phosphate buffer and modified with 20 lL of MATyr-Ru-MATrp (5.0 mg mL-1). Then, the pre-organized complex solution which includes 200 lL of avidin (100 lg mL-1) and 200 lL of biotin (400 lg mL-1) was added into above crosslinking solution. After the addition of 0.25 mL of N,N0-Methylenebisacrylamide (0.1 M) and 50 lL of 100 mM ammonium persulfate (APS) mix-ture, the solution was stirred for 10 h in daylight. Then, the particles were isolated with centrifugation and mixed with 5 mL of 0.1 M NaOH for 24 h for the removal of biotin template and washed several times with deionized water, and alcohol. Finally, the biotin imprinted nanostructures were dispersed in 3 mL of phosphate buffer and stored. The estimated schematic representation of prepared Au/Ag nanocluster sensor
was shown in Fig.2. For the fluorescence measure-ments first, 50 lL of dispersed biotin imprinted nanocluster was mixed with 3.0 mL of biotin solution (3.47 9 10-7M). Then, 50 lL of dispersed biotin imprinted nanoclusters was individually interacted with 3.0 mL of 8.19 9 10-7, 1.23 9 10-6, 1.64 9 10-6, 2.05 9 10-6, and 2.46 9 10-6M biotin solu-tions, and all interactions were measured by fluorim-etry at 280 nm (excitation). The experiments were performed in replicates of three. On the other hand, two different types of non-imprinted (NIP) Au–Ag nano-cluster sensors which were used as reference material were prepared in a similar way as described above without using biotin template and without using both avidin and biotin templates.
Results and discussion
Characterizations of chlorobis (2-20-bipyridyl) MATyr-ruthenium(II) (C34H33ClN5O4Ru) and MATyr-Ru-MATrp (C49H49N7O7Ru) monomers
The elemental analysis of C34H33ClN5O4Ru and C49H49N7O7Ru were performed to determine the elemental compositions of the synthesized monomers.
The elemental compositions of C34H33ClN5O4Ru and C49H49N7O7Ru were determined as C: 56.75 %; H: 5.01 %; N: 10.23 % and C: 61.56 %; H: 4.9 %; N: 11.67 %, respectively. The obtained results were consistent with the theoretical values of C34H33ClN5 O4Ru (C: 57.34 %; H: 4.67 %; N: 9.83 %) and C49H49N7O7Ru (C: 62.01 %; H: 5.2 %; N: 10.33 %).
1
H-NMR data for C34H33ClN5O4Ru were the following: 1H-NMR (500 MHz, CDCl3): 8.56 (d, 4H, 3J = 7.92 Hz), 8.42 (d, 4H, 3J = 7.5 Hz), 7.92 (t, 4H,3J = 7.82 Hz), 7.3 (t, 4H,3J = 7.4 Hz), 5.6 (d, 1H, 2J = 2.4 Hz), 5.3 (d, 1H, 2J = 2.4 Hz), 3.28 (t, 2H,3J = 7.2 Hz,), 1.8 (s, 3H) ppm. 1 H-NMR data for C49H49N7O7Ru (500 MHz, CDCl3), ppm: 9.4 (s, 3H), 8.52 -7.07 (HAryl), 7.24 (s, 1H), 5.6 (d, 2H, 2J = 2.45 Hz), 5.3 (d, 2H, 2J = 2.6 Hz), 3.28 (t, 4H,3J = 7.21 Hz,), 1.9 (s, 6H) ppm. MALDI-TOF MS analysis were also performed in order to characterize the C34H33ClN5O4Ru and C49H49N7O7Ru molecules. The mass spectra of C34H33ClN5O4Ru gave the following ion peaks: m/e 79, 101, 250, 351, 413, and 448.
These peaks were related with the bipyridyl, Ru, MATyr, Ru-MATyr, Ru(bpy)2, and Ru(bpy)2Cl groups in the C34H33ClN5O4Ru structure, respectively. In the same way, the following ion peaks were observed for C49H49N7O7Ru: m/e 250, 257, 272, 351, 413, 529, and Fig. 1 Synthesis of
achlorobis(2-20-bipyridyl) MATyr-ruthenium(II) photosensitive chelate and bMATyr-Ru-MATrp photosensitive chelate
622 indicating the presence of MAT, Ru-bpy, MATrp, Ru-MAT, Ru(bpy)2, Ru-bpy-MATrp, and Ru-MATyr-MATrp groups in the structure, respectively.
Biotin recognition by biotin imprinted avidin crosslinked Au/Ag nanoclusters
The effect of concentration on the adsorption of biotin using biotin imprinted avidin crosslinked Au/Ag nanoclusters, non-biotin imprinted avidin crosslinked Au/Ag nanoclusters and only MACys activated Au/Ag nanoclusters has been investigated (Fig.3). As shown in Fig. 3, when the biotin imprinted avidin crosslinked Au/Ag nanoclusters were used as a sensor system, the higher interaction was observed compared to non-biotin imprinted avidin crosslinked Au/Ag nanoclus-ters sensor system. Also, it was observed that MACys activated Au/Ag nanoclusters sensor system has no effect on the biotin recognition.
In Fig.3, the emission intensity linearly increased with the increasing concentration (8.2 9 10-7 –2.5 9 10-6M) of biotin after the interaction with biotin imprinted avidin crosslinked Au/Ag nanoclus-ters. The detection limit was found to be 15 nM from the standard deviation of blank. The linear range was established between 0.051 and 2.50 lM with the coefficient (R2) of 0.987. There are many different methods like as radioisotopic and non-radioisotopic binding assay, spectrophotometric, chromatographic, and polorographic methods for biotin detection in the literature and these methods have different detection limits (Livaniou et al. 2000; Gregory and Bachas 2001; Hu et al.2009) in a large scale. Therefore, this
result can be an acceptable detection limit for the determination of biotin.
The affinity constants (Ka) of biotin can be estimated from the thermodynamic analysis of the fluorescence intensity as a function of biotin concen-tration based on Scatchard plot. The Scatchard plot of biotin rebinding to avidin crosslinked Au/Ag nanocl-usters includes two different lines (Fig. 4). This situation suggests that the Au/Ag nanoclusters have two binding sites for biotin. The Kavalues for the first and second binding of biotin to the avidin crosslinked Au/Ag nanoclusters were found to be 7.21 9 105and 5.79 9 104M-1, respectively. The mean of affinity constant is 3.89 9 105M-1. In the natural systems, the affinity constant of binding of biotin by an isolated avidin is approximately 107M-1 (Green and Toms 1973; Green 1975; Wilchek et al. 2006). Therefore,
Fig. 2 Schematic representation of biotin imprinted avidin crosslinked Au/Ag nanoclusters: a before the removal of biotin template and b after the removal of biotin template
-100 0 100 200 300 400 500 600 700 800
0 5E-07 1E-06 1,5E-06 2E-06 2,5E-06 3E-06
Biotin concentration (M)
I (a.u)
Biotin imp Au/Ag NC Non biotin imp Au/Ag NC MAC activated Au/Ag NC
Fig. 3 The effect of the concentration of biotin on the biotin imprinted avidin crosslinked Au/Ag nanoclusters, non-biotin imprinted avidin crosslinked Au/Ag nanoclusters and MACys activated Au/Ag nanoclusters
it can be said that this interaction can be acceptable for the synthetic recognition systems. R2values of these plots were 0.9990 and 0.9980 for the first and second binding of biotin to biotin imprinted avidin cross-linked Au/Ag nanoclusters, respectively. The Kavalue of non-biotin imprinted avidin crosslinked Au/Ag nanoclusters was calculated by the same way and found to be 1.71 9 104M-1.
Reusability of biotin imprinted avidin crosslinked Au/Ag nanoclusters
The biotin-bound nanoclusters were washed with 0.1 M glycine–HCl to get free biotin and then washed with 50 mM NaOH solution in order to regenerate and sterilize these nanoclusters. After this procedure, biotin imprinted Au/Ag nanoclusters were washed with distilled water for 10 min, then equilibrated with the phosphate buffer at pH 7.0. After the washing steps, the clusters were interacted with biotin solution and the fluorescence intensity was measured. In order to show the stability and reusability of the nanoclus-ters, the adsorption–desorption cycle was repeated 10 times using the same nanoclusters and 1.6 9 10-6M biotin solution in all cycles.
As shown in Fig.5, the biotin imprinted avidin crosslinked Au/Ag nanoclusters can be used repeat-edly without loosing their recognition capacities for biotin. This is the main advantage of biotin imprinted avidin crosslinked Au/Ag nanoclusters according to natural recognition systems. Non-biotin imprinted avidin crosslinked Au/Ag nanoclusters are also reus-able but its affinity constant has relatively lower value then the biotin imprinted avidin crosslinked Au/Ag nanoclusters.
Conclusion
In this study, we have developed a new method for the biotin recognition applying photosensitation, conju-gation approach (ANADOLUCA) and molecular imprinting technique on Au/Ag nanoclusters using ruthenium-chelate based monomers. These avidin crosslinked bioconjugates provide accurate biotin orientation, specificity and photostability. The Ka values for the binding of biotin to biotin imprinted avidin crosslinked Au/Ag nanoclusters and non-biotin imprinted avidin crosslinked Au/Ag nanoclusters were found to be 3.89 9 105and 1.71 9 104M-1, respec-tively. As seen from the results, the biotin binding capacity was increased by biotin imprinting and the value of Kafor biotin imprinted Au/Ag nanoclusters suggests that the affinity of the binding sites is very durable as well as biological receptors (105–107). In addition, we can say that, our method has acceptable detection limit and working range. Also, the prepared nanotraps are useful for the separation of biotin and can be used 10 times without significant decrease in their binding capacities.
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