Solvent-Responsive Molecularly Imprinted Nanogels for Targeted

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Solvent-responsive molecularly imprinted nanogels for targeted protein analysis in MALDI-TOF mass spectrometry. Maddalena Bertolla, Lucia Cenci, Andrea Anesi, Emmanuele Ambrosi, Franco Tagliaro, Lia Vanzetti, Graziano Guella, and Alessandra Maria Bossi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16291 • Publication Date (Web): 02 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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ACS Applied Materials & Interfaces

Solvent-responsive Molecularly Imprinted Nanogels for Targeted Protein Analysis in MALDITOF Mass Spectrometry. Maddalena Bertolla,a‡ Lucia Cenci,b‡ Andrea Anesi,a Emmanuele Ambrosi,c Franco Tagliaro,d Lia Vanzetti,e Graziano Guellaa and Alessandra Maria Bossib* a. b.

University of Trento, Dept. of Physics, Via Sommarive 14, 38123 Trento, Italy; University of Verona, Dept. of Biotechnology, Strada Le Grazie 15, 37134 Verona, Italy;

c.

University Cà Foscari Venezia, Dept. of Molecular Sciences and Nanosystems, Via Torino 155/b, 30173 Venice, Italy;

d.

University of Verona, Dept. of Diagnostics and Public Health, Unit of Forensic Medicine, P.le L.A. Scuro 10, 37134 Verona, Italy; e.

Fondazione Bruno Kessler CMM-MNF, Via Sommarive 18, 38123 Trento, Italy.

1 Environment ACS Paragon Plus


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ABSTRACT: Molecular imprinted poly-acrylamido-derivative nanogels have shown their selectivity to bind the protein human serum transferrin (HTR), and also showed their capability for instantaneous solvent-induced modification upon the addition of acetonitrile. Integrated to MALDI-TOF mass analysis the HTR imprinted solvent-responsive nanogels permitted the determination of HTR straight from serum and offered novel perspectives in targeted protein analysis.

KEYWORDS: molecularly imprinted polymers, nanogels, polyacrylamide, responsive material, MALDI, mass spectrometry, serum transferrin, targeted protein analysis.


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1. INTRODUCTION Biomimetic materials with entailed recognition properties, prepared through template assisted synthesis, are referred to as molecularly imprinted polymers (MIPs).1,2 MIPs can target a variety of organic compounds, including those with high molecular weight and complex structures, such as peptides and proteins.3,4 Down-sizing the MIPs to the nano-dimension, i.e. preparing nanoMIPs, results in homogeneous nanoparticulate of 20-100 nm, with affinity, selectivity and binding kinetics on the par of natural antibodies.5-7 Among the nanoMIPs, polyacrylamide (PA) and -acrylamido-derivative (PAD) nanoMIPs represent an interesting class of nanomaterials, not only in terms of affinity and selectivity5,7, but also for the additional advantages of (i) being synthesized in water, guaranteeing compatibility with protein targets7, and (ii) depending on the solvent conditions and owing to their hydrogel nature,8,9 they can exhibit the ability to shrink and expand the hydrogel network, a property that would find use in the modulation of the binding and release of the target-molecule, in analogy to intelligent materials.10,11 Indeed, in the present work, we evaluated the solvent responsivity of protein-selective PADnanoMIP material, its compatibility to and the potential use in Matrix Assisted Laser Desorption Ionization (MALDI) Time of Flight (TOF) mass spectrometry (MS)12 analysis, with the final aim to contribute to the methodological advances in targeted protein analysis. MALDI-TOF-MS technique offers the advantages to detect macromolecules with high mass accuracy, femtomol sensitivities and fast read-out systems, further scalable in high-throughput testing.13,14 Experimentally, the analyte is deposited on a metal target plate and co-crystallized with an organic energy-absorbing matrix, which, hit by the laser light at a suitable wavelength, both desorbs and ionizes the analyte.15,16 In the case of complex biological samples, such as



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serum, a significant gain in the sensitivity of the MALDI-TOF-MS analysis has been demonstrated by the introduction of selective target plate surfaces, in order to enrich a particular protein, while reducing contaminants (for a comprehensive review see

17

). Within this

framework, MIP-based enrichment upstream to MALDI-TOF-MS analysis was also proposed.1824

Magnetic-MIP core-shell materials, directed at lysozyme recognition and followed by elution

and MS, allowed lysozyme analysis in egg white samples.18 Phosphate-imprinted mesoporous silica nanoparticles, demonstrated to be ideal sorbents for the selective enrichment in phosphopeptides, yielding to highly efficient off-line (i.e. elution-based) analysis of protein phosphorylation.19 An array of MIP nano-columns, called integrated selective enrichment target (ISET), was specifically designed to quickly screen a large number of conditions for the optimization of SPE conditions for phosphopeptide enrichment prior to MALDI-TOF-MS analysis.20 Whereas the direct analysis onto an imprinted self-assembled monolayer MALDItarget plate of a large protein, such as bovine serum albumin, did not result in success.25 Yet, in the present work we investigated the possibility of the use of the solvent-responsive PAD-MIP nanomaterials as potential candidates for the advancements in protein targetedMALDI-TOF-MS analysis, which will permit a controlled release of the bound analyte straight at the MALDI target-plate. Beside the recognized role of MIPs in the enrichment, an envisaged gain in analyte pre-concentration can be anticipated by the in-situ solvent-controlled release of the bound species, together with the simplification of the handling protocol. As a model for the MALDI-TOF-MS analysis of the PAD-nanoMIP material performance was proposed to investigate the binding of the protein human serum transferrin (HTR), given its key importance in serum clinical samples26 and its high molecular weight (78 KDa). This allowed us to test our approach towards large, macromolecular analytes.


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2. RESULTS AND DISCUSSION Synthesis and characterization of the Responsive PAD-nanoMIPs. The HTR targeted PAD-nanoMIPs were synthesized by using as template the peptide-epitope27 of sequence CGLVPVLAENYNK, called herein CK13, rationally selected according to the protocol earlier proposed by us.28 Considering the amphipathic nature of the peptide-template, neutral acrylamide, hydrophobic tert-butylacrylamide and charged itaconic acid monomers were mixed, to allow both electrostatic and hydrophobic interactions.5 The 80% mol/mol crosslinker (N,N’-methylene-bisacrylamide) ensured rigidity and anisotropy29 to the PAD-nanoMIPs. The synthesis was performed in water supplemented with 0.02% (w/v) SDS, a condition favoring tert-butylacrylamide solvation.8 Control non imprinted nanoparticles, called “PAD-nanoNIPs”, were prepared in an identical manner but in the absence of template. The overall yield of the syntheses was >95%. The size of the nanoparticles, suspended at 1 mg/ml in PBS and measured by Dynamic Light Scattering (DLS), resulted with Z-average of 42 ± 5 nm (SI). The polydispersity index (PDI) was ≤ 0.2 indicating a homogeneous population, in accordance with literature.30 Scanning Electron Microscopy (SEM) analysis (Figure 1A-B) offered morphological insights over the synthesized materials, showing regular quasi-spherical nanoparticles with a mean diameter of 41 ± 7 nm, in reasonable agreement with DLS (SI). Static light scattering (SLS) allowed to estimate the mean molecular weight (Mn) of 3.3 106 Da for the nanoparticles. X-ray Photoelectron Spectroscopy (XPS) analysis confirmed equal elemental composition for PAD-nanoMIPs and -NIPs (details in SI); the zeta-potential at pH 7.4 was -29 mV, indicating colloidal stability.



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The effectiveness of the imprinting process onto the PAD-nanoMIPs and the thermodynamic parameters associated to the binding (Kd, ∆H0, ∆G0) were investigated by isothermal titration calorimetry (ITC) (details in SI). High affinity was reported for the binding of the peptide CK13 to the PAD-nanoMIPs, exhibiting a Kd = 7 ± 1 nM. The PAD-nanoMIPs demonstrated also the ability to bind the whole protein HTR with Kd = 57 ± 8 nM, confirming the validity of the epitope imprinting approach.27,28 The kinetics of the binding, assessed by batch-incubation experiments followed by quantitation of the bound analyte, was on the timescale of minutes (Figure 2A); thus, a 30 minutes incubation time was set for all ensuing experiments. After the incubation, high ionic strength (500 mM NaCl) and detergent (0.2% Tween-20) washings were used to remove the non-specific-bound species.31 The binding capacity of the PAD-nanoMIPs for HTR was estimated as 6.40 ± 0.66 µg/mg, whereas the HTR bound to PAD-nanoNIP was 1.19 ± 0.31 µg/mg. The imprinting factor (IF), calculated as the ratio between the HTR bound to PAD-nanoMIP respect to that bound to nanoNIP, was IF = 5.4 (Figure 2B; details in SI). The selectivity of PAD-nanoMIPs was assessed by batch incubation followed by quantitative gel-electrophoresis of the bound species. Serum protein competitors were chosen. In particular, albumin was selected for being the most abundant protein in serum (clinical reference range: 3555 mg/mL) and the main competitor to HTR in the binding to PAD-nanoMIPs (estimated ratio albumin/HTR: 10-35). Moreover, albumin and HTR share a high degree of physico-chemical similarity: HTR has a molecular weight (MW) of 78 KDa and pI = 7.2, albumin has a MW of 68 KDa and pI = 6.9. In contrast, cytochrome c (MW 11.7 KDa; pI = 10.6) was chosen for being a small unrelated protein. The binding of albumin was estimated to be: 4.18 ± 0.61 µg/mg to PAD-


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nanoMIP and 4.78 ± 0.83 µg /mg to PAD-nanoNIP, indicating lack of specificity (Figure 2B). No binding was observed between cytochrome c and the nanomaterials, as a confirmation of the lack of attractive coulombic forces (Figure 2B). PAD-hydrogels are reported to swell and shrink with kinetics depending on the anisotropy of the matrix, as well as on the monomer composition32 and on the nature and type of the crosslinker.33,34 Here, the swelling ratio (SR) of the PAD-nanoMIPs, determined as SR = V/V0 = [d3/d03], was calculated from the ratio of their diameters. The diameters of PAD-nano-MIPs solvated in water (d0) and in acetonitrile/water (d) at the ratio: 3:7, 1:1 and 7:3 (v/v) were measured by DLS. Figure 1C showed that upon acetonitrile addition, an instantaneous shrinking of PAD-nanoMIPs was observed at all the acetonitrile concentrations tested. Acetonitrile 1:1 and 7:3 (v/v) resulted in the shrinking of the PAD-nanoMIPs to about one third of the initial size, then followed by a partial re-swelling with a gain of about the 80% of the initial size, as the solvent diffusion equilibrium was reached within the nanogel-network. The observed effect can be hypothesized to impact on the molecular recognition of the PAD-nanoMIPs: in buffer the imprinted binding sites retain the 3D structure complementary to their template and do bind HTR with high affinity, whereas upon the solvent change (i.e. from buffer to acetonitrile) the polymeric network shrinks, with consequent deformation of the imprinted sites and loss of recognition. Whether the solvent-responsivity of the PAD-nanoMIPs produces effects on the exposure and/or release of the bound species, was the fact to prove, given the implications of a greater accessibility to solvent of the bound species to the quality of the mass analysis.15,16 To examine more in details the acetonitrile-responsivity of the PAD-nanoMIPs and its effect to the bound HTR, a quantitative determination of the affinity for the pair HTR/PAD-nanoMIPs in



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acetonitrile 7:3 (v/v) was attempted (Figure S11): ITC measures indicate the complete loss of interaction consequent to the change in polarity of the environment. The effect of acetonitrile in modulating the HTR/PAD-nanoMIP interaction was then investigated by DLS. As shown in Figure 3, the size of HTR (0.2 µM) in buffer was 8 ± 1 nm, whereas upon acetonitrile addition an instantaneous unfolding was observed (extended protein chain and aggregation; ≤ 210 nm), as supported by theoretical and practical studies on protein folding.35-37 The same trend was observed for HTR-loaded PAD-nanoMIPs, whose dimensions were 25 ± 3 nm in buffer, but underwent to immediate size increment to 59 ± 5 nm upon acetonitrile addition. As a plausible explanation, the occurrence of the PAD-nanoMIP solventinduced shrinking can be accompanied by HTR exposure to the external milieu and unfolding. These results were also supported by the gel electrophoresis estimation of the acetonitrileinduced release of HTR from HTR-loaded PAD-nanoMIPs, (Figure S12). It was observed that in acetonitrile, the size increment reported for HRT bound to PAD-nanoMIPs was significantly smaller than that of free HTR. This might find explanation in a recent demonstration over the protective role played by polymers towards the unfolding and loss of activity of their bound protein.38

Integration of the Responsive PAD-nanoMIPs to MALDI-TOF Mass Spectrometry. The compatibility between the PAD-nanoMIP material and the ionization-desorption process39 typical of the MALDI-TOF-MS analysis was then evaluated. In MALDI-TOF-MS, the analyte ionization is reported to be mainly produced by its protonation/deprotonation upon collision with matrix ions which, in turn, are generated by photo- or cluster-ionization processes induced by the laser pulse.34 Moreover, the desorption/ionization process shows strong correlation to the


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structure of the analyte-matrix co-crystal:16,40 Inhomogeneous sample distribution during the crystallization causes the so-called “sweet spots”,41 confined zones at high ionization efficiency, but surrounded by vast areas of scarce to none ionization, that limit the reproducibility of analyses. The possible restrain of analyte by the nanomaterial and the consequent effects on its desorption/ionization efficiency were taken into consideration in a comparative analysis of the mass spectra of the sole analyte (CK13; HTR) or of the analyte bound to the PAD-nanoMIPs. The signal to noise ratio (S/N) of CK13 and of HTR ion-peaks in the corresponding mass spectra was used as a semi-quantitative parameter.42,43 PAD-nanoMIPs, respectively pre-incubated with CK13 (500 fmol) or with HTR (2 pmol), were spotted on the MALDI target plate, co-crystallized with the matrix and mass analyzed. For a comparison, the same quantities of sole CK13 and of HTR were spotted on the target, co-crystallized with the matrix and analyzed. As shown in Figure 4, similar S/N values were measured for the same molar amount of analyte ionized either just from the matrix or from the PAD-nanoMIP/matrix blend. These results proved minimal to no influence of the PAD-nanoMIP material to the ionization/desorption process. It can be assumed that the addition of the matrix, solvated in acetonitrile, induces the shrinking of the nanogel network, as a consequence the PAD-nanoMIP expels both water and analyte. In a mechanism that resembles that described for sol-gel-assisted laser desorption/ionization (SGALDI) MS,44 the voids are progressively filled with matrix (see Figure 1C, acetonitrile 7:3 (v/v), time > 5 minutes), the analyte comes in close contact to the matrix molecules, thus ready for desorption/ionization. Such anticipated advantage in the use of the responsive PAD-MIP nanomaterial in MALDI-TOF-MS was verified with an example based on the determination of HTR straight from serum.



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Serum, despite its utmost clinical importance, poses significant analytical challenges, being composed of about a million of proteins, whose dynamic range of concentrations spans about 9 orders of magnitude. Clinically referred as “normal” HTR in serum encompasses the values 1.53.5 mg/mL while albumin is 10 to 35-fold more concentrated. As shown in Figure 5, the mass spectrum of serum (diluted 1:10) let identify the sole major component albumin (Figure 5A). When optimizations, based on sample dilution to 1:400, were implemented the mass spectrum (Figure 5B) showed the ion peak of albumin (S/N = 30 ± 4) and traces of HTR, though notmeasurable (S/N < 3). Yet, when PAD-nanoMIPs were incubated with serum (diluted 1:10 in 5 mM Tris pH 7.4; time = 30 minutes), washed to remove non-specific binding and transferred to the MALDI target plate for the analysis, the results (Figure 5C) showed a drop in the albumin signal (S/N = 14 ± 3) and the enrichment to a measurable value of HTR (S/N = 6 ± 1). These results, despite the still uncomplete removal of the non-specifically bound contaminant protein, demonstrated the significant HTR enrichment promoted by the entailed nanomaterial.

3. CONCLUSIONS In conclusion, we prepared biomimetic, protein-imprinted, PAD-nanogels with a bi-functional character: specifically targeted at binding the protein HTR and responsive to solvent. Both characteristics proven compatible to MALDI-TOF-MS targeted protein analysis, in an example that demonstrates the detection of HTR straight from serum. Based on a simple solvent-induced shrinking of the hydrogel network, but effective to modulate the biomimetic polymeric structure, responsivity was achieved. The solvent induced deformation of the imprinted binding sites demonstrated to correlate with the loss of interaction between the PAD-nanoMIPs and the target protein HTR. Integrated into MALDI-TOF-MS analytical


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platforms, the PAD-nanoMIPs provided advantages in terms of: no restrain of the nanomaterial to the desorption/ionization process, fast and easy protocol, straight analysis of serum samples and ability to determine also large macromolecules. The possibility to scale PADnanoMIPs/MALDI-TOF-MS for high throughput analysis would offer further perspectives in targeted proteomics16,20 and clinical diagnostics, provided to introduce quantitative determination of the analytes.

4. EXPERIMENTAL PROCEDURES Synthesis of PAD-nanomaterials: Acrylamide (Aam), itaconic acid (IA), N-tertbutylacrylamide (TBAm) were added at 8, 8 and 4% mol/mol respectively, together with 80% mol/mol of N,N’-methylenebisacrylamide (BIS) in water supplemented with SDS 0.02% (w/v) (see Table S1 for detailed composition). The solutions were filtered with a 0.45 µm filter. The template CK13, defined as indicated by the protocol described in

24

, was added to the PAD-

nanoMIP vials at the final concentration of 32 µM. Vials were closed with rubber caps, sonicated for 10 minutes and bubbled with N2 for 30 minutes. Then APS (0.04% w/v) and TEMED (0.03% w/v) were added and the polymerization was carried out at 20°C for 20 hours. Control, non-imprinted nanoparticles (PAD-nanoNIPs) were synthetized using the same protocol but in absence of the template. After the synthesis the PAD-nanomaterials were suspended in 250 mL of 50 mM Tris and then extensively dialyzed against 3 L of pure water using the Vivaflow 50 system (100000 MWCO) (Sartorius Stedim Italy, Firenze, Italy), lyophilized and stored at 4°C. After lyophilization, the yield of the polymerization was



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obtained by comparing the weighed materials respect to the weight calculated for a complete polymerization. The lyophilized PAD-nanoMIPs kept at 4°C were stable for several months.

Evaluation of the effectiveness of the template removal process on PAD-nanoMIPs: 10.5 mg of PAD-nanoMIPs were subjected to the washing protocol: 1) dispersed in 10 mL of 5 mM Tris free base, 2) washed with 3L of water, concentrated to the starting volume. Then to remove the template still present in the nanoMIPs the 70% acetonitrile was added, sonicated for 15 minutes and incubated for 2 hours at room temperature under mild shaking. 10% TFA was added and PAD-nanoMIPs were precipitate by centrifuging them at 12000 x g for 5 minutes. The supernatant was lyophilized and then re-dissolved in 100 µL of water; L-histidylL-tyrosine was added as internal standard to a final concentration of 0.025 mg/ml and the sample was analyzed on a reverse-phase HPLC Ascentis C18 column (250 mm x 4.6 mm) (Sigma-Aldrich, Darmstadt, Germany), using water with 0.1% trifluoroacetic acid (TFA) (solvent A) and acetonitrile with 0.1% TFA (solvent B) (gradient: 5-50%B in 20 minutes). The flow rate was set at 1 ml/min and the detection wavelength was 214 nm. No CK13 leakage was detected from PAD-nanoMIPs; it can assumed that the residual template is

Solvent-Responsive Molecularly Imprinted Nanogels for Targeted

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