Molecularly imprinted polymers coupled to mass spectrometric detection for metallothionein sensing

Molecularly imprinted polymers coupled to mass spectrometric detection for metallothionein sensing VANĚČKOVÁ, T.; POMPEIANO VANÍČKOVÁ, L.; TVRDOŇOVÁ, M.; POMORSKI, A.; KRĘŻEL, A.; VACULOVIČ, T.; KANICKÝ, V.; VACULOVIČOVÁ, M.; ADAM, V. Talanta 2019, vol. 198, June 2019, pp. 224-229 ISSN: 0039-9140 DOI: https://doi.org/10.1016/j.talanta.2019.01.089 Accepted manuscript © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0/), doi: https://doi.org/ 10.1016/j.talanta.2019.01.089 Final version available from https://www.sciencedirect.com/science/article/pii/S0039914019301109 dspace.vutbr.cz 1 Molecularly imprinted polymers coupled to mass spectrometric detection for metallothionein sensing Tereza Vaneckova1,2, Lucie Pompeiano Vanickova1,2, Michaela Tvrdonova3, Adam Pomorski4, Artur Krężel4, Tomas Vaculovic3,5, Viktor Kanicky3,5, Marketa Vaculovicova1,2, Vojtech Adam1,2* 1Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ613 00 Brno, Czech Republic 2Central European Institute of Technology, Brno University of Technology, Purkynova 123, CZ-612 00 Brno, Czech Republic 3 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 753/5, CZ-625 00 Brno, Czech Republic 4Department of Chemical Biology, Faculty of Biotechnology, University of Wroclaw, JoliotCurie 14a, 50-383 Wrocław, Poland 5 Central European Institute of Technology, Masaryk University, Kamenice 753/5, CZ-625 00 Brno, Czech Republic * Corresponding author: Vojtech Adam, Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic; E-mail: [email protected]; phone: +420-5-4513-3350; fax: +420-5-4521-2044 2 Abstract We report a facile method for detection of metallothionein (MT), a promising clinically relevant biomarker, in spiked plasma samples. This method, for the first time, integrates molecularly imprinted polymers as purification/pretreatment step with matrix assisted plasma desorption/ionization time-of-flight mass spectrometric detection and with laser ablation inductively coupled plasma mass spectrometry for analysis of MTs. The prepared MTimprinted polydopamine layer showed high binding capacity and specific recognition properties toward the template. Optimal monomer (dopamine) concentration was found to be 16 mM of dopamine. This experimental setup allows to measure µM concentrations of MT that are present in blood as this can be used for clinical studies recognizing MT as marker of various diseases including tumour one. Presented approach not only provides fast sample throughput but also avoids the limitations of methods based on use of antibodies (e.g. high price, cross-reactivity, limited availability in some cases, etc.). Keywords Metallothionein; molecularly imprinted polymers; polydopamine; MALDI-TOF-MS; LAICP-MS 3 Introduction Metallothioneins (MTs) are low molecular mass (< 7kDa), cysteine-rich proteins, ubiquitously present in practically all eukaryotes [1-3]. MTs perform a wide range of functions in an organism including essential metal homeostasis, i.e. Zn2+ and Cu+, heavy metal detoxification, i.e. Cd2+, scavenging of reactive oxygen species and regulation of transcription [4-8]. The elevated concentration of MTs has been observed in blood and/or tissues coming from patients with various tumour diseases (e.g., colon, breast, liver, kidney, lung, nasopharynx, ovary, salivary gland, prostate, thyroid, and urinary bladder cancer) as it was reviewed [9, 10], demonstrating that MTs are promising oncosuppressors [11-13]. Numerous analytical approaches have been suggested for detection and determination of MTs as reviewed elsewhere [14, 15]. More recently, a method based on the enzyme-linked immunosorbent assay and the real time polymerase chain reaction was used [16]. Another study reported a microfluidic MT electrochemical immunosensor utilizing superparamagnetic agarose beads [17], where a curve fitting approach was be used for voltammograms of various isoforms of MTs [18]. Mass spectrometry coupled with matrix assisted laser desorption/ionization (MALDI) or inductively coupled plasma (ICP) ionization was also used for determination of MTs [19]. Recently, bottom-up mass spectrometry-based approach for human MT isoforms quantification has been developed [20], nevertheless combinations of these types of detection with biological molecule recognition elements are needed. Molecularly imprinted polymers (MIPs) are biorecognition surfaces with high affinity towards desired template. Named as natural receptor mimics, MIPs are being considered as an alternative to biological receptors, such as enzymes, antibodies or aptamers [21, 22]. Their major advantages cover predictable specific recognition, low cost, ease of preparation, good mechanical/chemical stability, and reusability [23]. Although MIPs have been successfully applied using wide range of small molecules [24], imprinting of biomacromolecules, such as 4 proteins, faces challenges. Macromolecular templates have a tendency to adsorb to polymers, where it is not trivial to remove them from the polymer matrix, as they produce heterogeneous sites, and may be sensitive to denaturation or presence of organic solvents, which are usually essential for formation of polymers [22, 25]. To date, only few studies reported combination of molecular imprinting technology together with MALDI time-of-flight mass spectrometry (MALDI-TOF-MS) [26-33]. However, determination of MT using molecularly imprinting technology combined with mass spectrometric detection (neither MALDI nor ICP) has not been reported yet. In the present study, we developed an easy method for MT purification from a complex matrix using MT-selective PDA layer combined followed with MALDI-TOF-MS and LAICP-MS detection techniques (Fig. 1). The analytical performance of the sensor was evaluated. The obtained results reveal a new perspective in recognition and separation of this template, which is a potential marker of diseases. Material and methods Materials Dopamine hydrochloride, albumin from human serum (≥ 97%), Trizma® (TRIS base), and lysozyme from chicken egg white were purchased from Sigma-Aldrich company (St. Louis, MO, USA). Acetic acid (99.8%) and hydrochloric acid (reagent grade, 35%) was obtained from PENTA (Chrudim, Czech Republic). Amicon® Ultra 0.5 mL 50K Centrifugal Filters were purchased from Merck Millipore (Billerica, MA, USA). Tris-HCl buffer (20 mM, pH 8.5) was prepared from Trizma® and hydrochloric acid was used for pH adjustment. Aqueous solution of acetic acid (3%, v/v) was used as a washing buffer. Deionized water used during the experiments was prepared with a Milli-Q water purification system (Millipore, Milford, MA, USA). 5 Preparation of MT-imprinted polydopamine layer Polydopamine (PDA) MIP was prepared by self-polymerization inspired by works of [34, 35]. In brief, dopamine was dissolved in 1 mL Tris-HCl buffer (20 mM, pH 8.5). Template molecules of MT-1, MT-3 or lysozyme, for initial optimization, were then mixed with the stock solution of DA at 1:1 (v/v) ratio. The target concentration was varied depending on the experiment. Next, 1 µL of the polymerization mixture was applied on the MALDI target plate (Bruker MTP AnchorChip 384BCTM) or, in case of initial experiments, bottom of the 96 wellplate and let to polymerize and dry at the room temperature for 24 h. Subsequently, the prepared polymeric layer was overlaid with a sample solution and the incubation was carried for 1 hour at room temperature. The resulting self-assembled polymer was then washed with 3% acetic acid to remove the bound MT or lysozyme. Final wash was performed with Milli-Q water. Control, non-imprinted polymer (NIP), was prepared under the same conditions without adding the template (MT/lysozyme). Each of the analysed polymers was prepared in triplicate. Sample preparation Standard solutions The coding sequences of human metallothionein-1 (MT-1) (UniProt:P13640-2) and metallothionein-3 (MT-3) (UniProt:P25713-1) were purchased from Genscript (Piscataway, NJ, USA) and inserted into the pTYB21 vector (New England Biolabs, UK). Prepared plasmid transformed into BL21(DE3)pLysS E. coli cells. Protein production and purification was conducted as previously stated [36]. The obtained recombinant protein MT-1 used in the MALDI-MS experiments had seven bound Zn(II) ions. For LA-ICP-MS experiments were prepared MT-1 with seven bound Cd(II) ions and MT-3 with seven bound Zn(II) ions. 6 Plasma sample preparation Whole blood was collected from a healthy volunteer and then the whole blood was centrifuged for 10 min at 2000 rcf and the plasma was further centrifuged for 30 min at 22000 rcf. The plasma was diluted 50× with Tris-HCl buffer (20 mM, pH 8.5) and spiked with MT-1 to its final concentration of 5 μM. All subjects gave their informed consent for inclusion, before participating in the study. The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Ethics Committee of Masaryk University. MALDI-TOF-MS MIP/NIP were analysed using MALDI-TOF-MS (ultrafleXtreme instrument, Bruker Daltonik GmbH, Bremen Germany) equipped with a laser (operating at wavelength of 355 nm with an accelerating voltage of 25 kV, a maximum energy of 43.2 μJ, and a repetition rate of 2000 Hz) in linear positive ion mode for data acquisition. Three different organic matrix solutions were tested, namely α-cyano-4-hydroxycinnamic acid, sinapinic acid and 2,5dihydroxybenzoic acid (DHB) (Bruker Daltonik, Bremen, Germany), with DHB diluted in 0,1% trifluoroacetic acid (Sigma-Aldrich) being considered the optimal solution, since less background was produced in the final spectrum [18]. Matrix (0.5 µL) was applied on the prepared MIP and/or NIP polymerized layer (as previously described in section 2.2) and dried under atmospheric pressure and ambient temperature (25 °C). The laser frequency was set to 1000 Hz and laser energy was optimized prior to each measurement. Calibration was done externally using a protein standard mixture I and II (Bruker Daltonics, Bremen, Germany) in the range of m/z 1–90 kDa. A total of 500 spectra were summed for each spot using the Random Walk raster pattern, with no evaluation criteria and were analysed with the Flex Analysis software (Version 3.4). 7 Fluorescence spectrometry Fluorescence spectrometric measurements were performed using Infinite M200 fluorescence microplate reader (Tecan, Männedorf, CH). Polymerization mixture (50 µL) was deposited on the bottom of the well of Corning® 96 Well Clear Flat Bottom UV-Transparent Microplate (Corning, NY, USA). Fluorescence emission of lysozyme was recorded at λex = 280 nm and λem = 330 nm with gain of the detector set to 100. LA-ICP-MS The analysis of MIP was performed by LA-ICP-MS setup that consists of LA system UP213 (NewWave Research, USA) emitting laser radiation with a wavelength of 213 nm with a pulse width of 4.2 ns. The ablated material was carried out from an ablation cell by a flow of a He (1.0 l/min) into ICP-MS Agilent 7500CE (Agilent Technologies, Japan) with quadrupole analyzer. The MIP and NIP were ablated with following ablation parameters: laser beam diameter of 110 μm, the repetition rate of 10 Hz; laser beam fluence of 6 J/cm2, the scan speed of 400 μm/s and distance between individual lines of 100 μm. The analytes MT-1 and MT-3 were monitored by measuring of isotope 111Cd and 66Zn, respectively. Results and discussion Optimization of MIPs preparation To address limitations of protein imprinting, several strategies have been investigated including metal ion-coordination polymerization, protein epitope approach, and surface imprinting [22, 25, 37]. The latter approach utilizes polydopamine (PDA), one of the most favourable polymers considering its green chemistry status and facile preparation. Dopamine 8 (DA), a functional monomer, can form a thin, self-polymerizing film on a wide variety of materials in a weak alkaline environment (pH > 8) [38]. It has been shown that PDA forms thin films by the spontaneous polymerization in the presence of oxygen; however some other polymerization methods involving radicals formation have been developed [39]. DA, commonly involved in human body as a neurotransmitter, is also a small-molecule mimicking the adhesive proteins. Its multifunctional groups and properties of hydrophilicity and biocompatibility make it suitable for imprinting of proteins. It can be self-polymerized under mild conditions (room temperature, pH 8.0) resulting in formation of an adherent polydopamine film. For example, approach for imprinting proteins using PDA coating of Fe3O4 nanoparticles has been reported [40]. Xia et al. suggested an approach for protein recognition and separation using PDA-coated molecularly imprinted silica nanoparticles [41]. Therefore, it is believed that polydopamine MIPs are expedient and appropriate materials applicable in the identification of proteins. The simple oxidative polymerization was employed in this study. For non-covalent imprinting, the optimal ratio of template to functional monomer (T/M) has to be achieved empirically [42]. Therefore, the concentration of monomer (dopamine) was tested in concentrations of 16, 32, 65, and 130 mM (data not shown). The functionality of the imprinted polymer was initially tested utilizing lysozyme as a template. The globular glycoprotein lysozyme (Lys) plays and important role in living organisms. Considering its excellent antibacterial property, Lys is widely used in medical and food industry. Thus, the development of an effective purification method for Lys is broadly valuable [43]. Based on the intrinsic fluorescence of Lys, the efficiency of the polymeric layer preparation was evaluated. According to the fluorescence spectrometry measurements, the dopamine concentration of 16 mM was chosen as the most appropriate due to the highest binding yield. 15 Figures Figure 1 Overall workflow of molecularly imprinted polymer (MIP) formation, sampling and MALDIMS detection of MT-1. 16 Figure 2 LA-ICP-MS quantification of 66Zn of different MT-1 concentrations in the sample extracted by MIP (red) and NIP (grey). 66Zn quantification in MIP after template removal is shown in blue. 17 Figure 3 LA-ICP-MS analyses of MIP and NIP using the MT-1 (Cd) and MT-3 (Zn) templates. (A) Sample containing MT-3(Zn) was applied to MIP/NIP created with template MT-1(Cd). 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