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Influence of adsorption on proteins and amyloids detection by silicon nitride nanopore Sebastien Balme, Pierre-Eugene Coulon, Mathilde Lepoitevin, Benoit Charlot, Naresh Yandrapalli, Cyril Favard, Delphine Muriaux, Mikhael Bechelany, and Jean-Marc Janot Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02048 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 13, 2016
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Influence of adsorption on proteins and amyloids detection by silicon nitride nanopore Sébastien Balme1*, Pierre Eugène Coulon2, Mathilde Lepoitevin1, Benoît Charlot3, Naresh Yandrapalli4, Cyril Favard4, Delphine Muriaux4, Mikhael Bechelany1, Jean-Marc Janot1 1
Institut Européen des Membranes, UMR5635, Université de Montpellier CNRS ENSCM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France 2
Laboratoire des Solides Irradiés, École polytechnique, Université Paris-Saclay, Route de Saclay, 91128 Palaiseau Cedex, France
3
Institut d’Electronique et des Systèmes, Université de Montpellier, 34095 Montpellier Cedex 5, France 4
Centre d'Études d'Agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS UMR5236, Montpellier, France
KEYWORDS : Nanopore, protein adsorption, amyloid
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ABSTRACT
For the two last decades the emerging single nanopore technologies have opened the route to multiple sensing applications. Beside the DNA, the identification of proteins and amyloids is a promising field for early diagnosis. However the influence of the interactions between the nanopore surface and the proteins should be taken into account. In this work, we have selected 3 proteins (avidin, lysozyme and IgG) because they exhibit different affinity with SiNx surface and the lysozyme amyloid. Our results show that the piranha treatment of SiNx decreases significantly the proteins adsorption. Moreover we have successfully detected all proteins (pore diameter 17 nm) and shown the possible discrimination of denatured lysozyme and amyloids. For all proteins, the capture rates are lower than expected and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that only proteins interacting with the nanopore surface wall are staying enough time to be detected. For the lysozyme amyloid, we show that the use of the nanopore is suitable to determine the number of monomer unit even if only the proteins in interaction with the nanopore are detected.
1. INTRODUCTION Recent advances in single nanopore technologies have opened opportunities to develop new single molecule1, 2, 3 or nanoparticle4, 5 sensors. In this domain, the main efforts focused on single molecule detection and especially of DNA for sequencing application.6, 7 However beside DNA sensing, nanopore-based sensors are also promising to analyze proteins. Biological nanopores such as α-hemolysin8 and aerolysin9 were used to detect the unfolding of proteins,10 proteinDNA complexes11 or peptide cleavage12. The ability to design solid-state nanopores with
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diameters larger than 10 nm increases their interest for protein sensing. Indeed they make possible the detection of (i) both native and unfolding proteins,13,
14, 15, 16, 17, 18, 19
(ii) their
complexation,20, 21 (iii) DNA-protein complexes22, 23, 24 and (iv) proteins self-assembly such as amyloid25,
26, 27
or fibril28. Solid state nanopores permit also to work using a large range of
voltage compared to the biological ones. This is convenient in order to investigate deeply the influence of high voltages29 or electro-osmotic flux30 on protein translocation. In most cases, the solid-state nanopores are used without a functionalization in order to limit the nonspecific adsorption. Even if the protein detection using SiNx nanopore was proven, several questions are not totally elucidated. The first one concerns the protein dwell time inside the nanopore. Indeed, the experimental dwell time is several orders longer than the expected one assuming that a protein is subject to electrophoretic mobility or diffusion.16, 31 The second one is related to the capture rate. Indeed, the latter is lower than the calculated one assuming that the protein entrance inside nanopore is only due to a diffusion process.15, 18 The low capture rate has been interpreted as coming from the protein adsorption and/or the missing events. In order to investigate the origin of the missing events, Plesa et al.15 have adsorbed the BSA on the nanopore prior to the protein detection. Since the capture rate is constant, the authors have concluded that the protein adsorption effect is not relevant to explain the low event rates. This indirect proof should be supported by a study of protein adsorption. Indeed, the protein adsorption could influence the dynamic of the protein inside the nanopore31 as well as the real protein concentration close to the nanopore. The understanding of the protein adsorption process at the solid-liquid interface is crucial for the development of the membranes used in dialysis or in biosensing applications.32, 33 In the latter area, the nonspecific adsorption is at the origin of the difficulties to upscale many devices for
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real life applications. From a fundamental point of view, the protein adsorption on solid surfaces is a very complicated phenomenon which involves electrostatic interactions, hydrogen bonds and Van der Wall forces.33, 34 Even if there is an attempt to classify the protein in two groups (soft and hard proteins) in order to predict their behavior and affinity with the surface, the experiments show that it is difficult to anticipate this interaction. The quantification of the nonspecific adsorption is commonly achieved by the radiolabeling technique (typically 127I)35, ellipsometry36 or quartz crystal microbalance37. Here, we use another method, developed in our group, based on confocal spectroscopy. This allows a real-time quantification and localization of the adsorbed protein, under similar conditions to the nanopore experiments (same surface and protein concentration).35 For many years, different strategies were investigated to design surface which exhibits low nonspecific adsorption. Among those, the most used are the surface PEGylation, either by PEGSH assembly on gold surface38, 39, or PLL-g-PEG40 adsorption on negative charged surface or chemical grafting of PEG-NH2 or PEG-COOH41. The adsorption of surfactant tween 20 on hydrophobic SiNx have been also considered to prevent the unspecific adsorption of αSynuclein.42 The most efficient one is the functionalization with phospholipids (PL), even if it strongly depends on their head group composition36. The PL can be deposited, as a bilayer on hydrophilic surfaces37 or as a monolayer chemically grafted or by self-assembly using modified PL.43, 44, 45 Some strategies to limit the nonspecific adsorption of proteins have been reported in the case of nanopore such as PEGylation after gold deposition46 or PL bilayer self-assembly on hydrophilic SiNx25. The latter permits the anchoring proteins to the lipid coating. Based on that, the authors of this work have proposed an elegant method to differentiate proteins which is based on their 5D-finger printing.47
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This present work aims to investigate the influence of proteins adsorption on their translocation through a solid-state SiNx single nanopore. We have selected 3 proteins (IgG, avidin and lysozyme) which exhibit a global positive charge at pH 7. Among those proteins, the choice of avidin was also motivated in order to discuss further the work reported by Firnkes et al.30 about the EOF effect on protein translocation. The lysozyme was chosen since it is a small protein usually considered as a model for hard proteins.48, 49 This protein is also able to form amyloids which can be detected through the nanopore without applying a coating to prevent its clogging
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as opposed to αβ-amyloid25. Finally, the IgG has an interesting electrical response
since its relative current blockade follows a bimodal distribution when it is anchored onto the nanopore inner wall surface.25 For all proteins, we have investigated the proteins translocation through a SiNx single nanopore, previously treated with a piranha solution. As we cannot assume the same behavior for all of our proteins, we studied the kinetics of adsorption of each of them on SiNx surface. Furthermore, we investigated the lysozyme amyloid. Indeed, the amyloid detection presents a real interest for early diagnosis of many diseases. 2. EXPERIMENTAL SECTION 2.1. Material Sodium chloride (S753), TRIS tabs (5030), PBS tabs (P4417), Avidin from egg white (A9275), γ-Globulins from bovine blood (G7516), Sulfuric acid ACS reagent 95%- 98% (32051), hydrogen peroxide wt 30% (216763) were purchased from Sigma-Aldrich. Lysozyme (62971) was purchased from Fluka. Alexa-fluor 594® succinimidyl-ester tri(ethylamine) salt (A37572) was purchased from Molecular Probes. SiNx grids (thickness 10 nm, windows 50 µm, NT005Z) were purchased from Norcada. Deionized water was obtained from a Milli-Q system
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(Millipore). The buffer solution was filtered at 0.45 µm and degassed by sonication 5 minutes before to use. 2.2. Protein detection with SiNx Nanopore We used SiNx TEM grids (thickness 10 nm, window 50x50 µm) drilled by the electron beam of a TEM JEOL 2010F as single nanopore for the detection of the proteins. The final nanopore has a circular shape with a diameter of 16 ± 1 nm. (Figure SI-1) The grid was mounted in a Teflon cell containing a buffer solution 500 mM NaCl, 5 mM of TRIS at pH~7.4. The current was measured by two Ag/ AgCl electrodes. One electrode was plugged to the negative end of the amplifier (trans chamber) and the other electrode connected to the ground (cis chamber). Proteins (concentration 100 nM) were added inside the cis chamber and a negative current (typically -200 mV) was applied at the trans chamber. It can be noticed that we have not painted PDMS on SiNx substrate to reduce the noise.50,
51
This choice is motivated to control the surface which is in
contact of protein solution. The ion current was recorded using a patch-clamp post amplifier (EPC 800, HEKA electronics, Germany) at a sampling frequency 200 kHz; a filter of 10 kHz was applied. The data acquisition was performed by a HEKA LIH 8+8 acquisition card and the patch master software (HEKA electronics, Germany). The event analysis was performed using the Matlab (Matworks, USA) code developed by Plesa et al.52 The threshold to detected event is defined by the multiplying a peak detection factor and the rms noise level calculated by the global standard deviation methods. In this work, the peak detection factor has been fixed at 7. In addition we have considered only current blockade longer than 20 µs. 2.3. Kinetics of protein adsorption on SiNx surface The experiments to study the kinetics of the protein adsorption were performed using a labmade confocal fluorescence setup previously described (Figure SI-1).35,
53
To sum up, a
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fluorescence profile of a previously labeled protein solution is recorded step by step (Detector APD id100-50 from IDQ, electronics SPC-130EM Becker & Hickl) from the interface of interest until the bulk solution (at least 50 µm from interface) by normal scanning (100 nm to 1 µm steps, collection time 50 to 100 ms) of a focused beam laser (Laser DPSS 593 nm, 100 to 500 nW from Dream Lasers Technology, confocal volume about a femtolitre, objective UPlanaApo 60x/1.20 w from Olympus).
From the analysis of the profile, the amount of the adsorbed protein is
extracted.53 The kinetic is recorded by iterative collection of the emission profiles. The proteins were labeled using the Alexa-fluor 594 succinimidyl-ester reagent. The labeling ratios are 0.21, 0.28, 0.729 for lysozyme, avidin and IgG respectively; these ratios are calculated from the absorption spectra of the solution as well as controlled by fluorescence correlation spectroscopy (FCS) (ratio of free label against bonded label). The protein adsorption was performed on a SiNx layer (100 nm) deposited on a microscope cover-glass (diameter 25 mm). SiNx deposition was made with a Plasma Enhanced Chemical Vapor Deposition (PECVD) system. Glass slides were cleaned with acetone, rinsed with isopropyl alcohol and blow dried with nitrogen in a cleanroom. The deposition was made at 280°C in a PECVD (Corial D250) reactor during 60 s at a pressure of 1800 mTorr. A first sequence of 30 s surface cleaning was operated before the deposition using a mixture of NH3, N2 and Ar gazes ionized with a 200 W RF power under flows of 400, 500 and 400 sccm (Standard Cubic Centimeters per Minute), respectively. After the surface preparation, 50 sccm of Silane (SiH4) were introduced in the reactor in the same conditions to achieve a deposition rate of about 120 nm mn-1. Samples surfaces were then analyzed with both Dektak profilometer and atomic force microscopy (AFM); the roughness was measured between 0.3 nm and 0.4 nm (Figure SI-4). The adsorption of proteins was investigated on SiNx surfaces subjected to 2 different treatments, (i) only washed with ethanol and milliQ water (raw-SiNx)
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and (ii) immersed in piranha solution (H2SO4/H2O2 with a ratio 3/1) for 30 min and rinsed with milliQ water (hydrophilic SiNx). A typical experiment consists to add 200 µl of solution of the labeled proteins (final concentration 100 nM, NaCl 500 mM, Tris 5 mM, pH 7.5) in a Teflon cuvette (6 mm diameter) obstructed on its bottom by the studied glass interface. 2.4. Measurement of the diffusion coefficient of the protein The diffusion coefficients of the proteins have been measured by fluorescence correlation spectroscopy (FCS). These experiments have been performed using the confocal fluorescence setup previously described, except for the detector which was replaced by a more suitable HPM100-40 Becker& Hickl PM tube (the absence of after pulses for this detector permits a better resolution for the FCS studies). The measurements have been done for all the proteins labeled with the alexa-fluor 594 under buffer NaCl 500 mM, Tris 5mM, pH 7.5 at a concentration around 1 nM. Data were recorded for 400 s at a count rate of 2-4 kHz (6 µW laser excitation). The coefficients of diffusion were calculated from the autocorrelation curve using the quickfit54 software. The confocal volumes and the calibration of the coefficients are performed using the alexa fluor 594 as standard (D=370 µm2 s-1). 2.5. Native Gel electrophoresis. A 10% native-PAGE (5% stacking gel and 10% separating gel) was used in this study in order to avoid protein denaturation and the subsequent disassembly of oligomers. Specific amount (10µg) of lysozyme before and after heating treatment was added to the loading buffer (125 mM Tris-HCl, 20% (v/v) glycerol, 0.02% (w/v) bromophenol blue, pH 6.8 in Millipore water). Without any further heating, the samples were loaded into the gel. At room temperature, electrophoresis was run initially at 80 V for 30 min and then at 110 V for 90 min. At the end, the gel was stained with Coomassie Brilliant Blue solution and imaged with trans-illumination.
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Thermo-Scientific PageRulerTM plus pre-stained protein ladder was used as a molecular weight marker.
3. RESULTS AND DISCUSSION Before studying the adsorption of proteins onto SiNx surfaces as well as their detection through single nanopore transport, the diffusion coefficient of the proteins has to be known. This parameter can be deduced from the crystallographic structure or from the molecular weight of the proteins assuming they have spherical geometry. However these diffusion coefficients are roughly approximated since the protein conformation can change with the ionic strength and pH. One way to measure this diffusion coefficient is to use the light scattering measurements (DLS). This method gives acceptable results, nevertheless, it requires a high concentration of proteins (several mM) which is very far from the conditions used for the nanopore experiments (from nM to fM scale). In order to work under the same concentration range, the diffusion coefficient of the proteins was measured by fluorescence correlation spectroscopy (FCS). From this method we were also able to evaluate the amount of free fluorophore in the solution and thus to get information about the protein labeling. The fluorescence correlation spectra of the proteins are reported on Figure 1. For each protein the best fit is obtained for a combination of two components (i) a fast one around 370 µm2 s-1 which corresponds to the free alexa in solution and (ii) a slow one, 41.4, 67.6 and 106 µm2 s-1 which are assigned to the labeled IgG, avidin and lysozyme respectively. From the diffusion coefficient D, the size of the proteins has been estimated (Table 1). For avidin and lysozyme a spherical geometry is assumed. For IgG we assume a prolate ellipsoid where the ratio a/b is roughly fixed from the crystallographic structure.
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5 4 3 2 1 5 4 3 2 1 4
(a)
(b)
(c)
3 2 1 10-7
10-6
10-5
10-4
10-3
10-2
10-1
Time (s) Figure 1 : FCS curves for the lysozyme (a), avidin (b) and IgG (c) (points are the raw data and lines are the non linearfit). These experiments have been done with proteins labelled with alexa fluor 594.
Protein
D (µm2 s-1)
*Rh (nm) **a/b (nm)
Volume (nm3)
Avidin
67.6
3.30*
150.5*
Lysozyme
106
2.09*
38.2*
IgG
41.4
9.5/13.4**
356**
Table 1 : diffusion coefficient (D) for the proteins extracted from the FCS results. Rh is the hydrodynamic radius calculated assuming a spherical geometry* for the protein (avidin and lysozyme). a/b are the diameters assuming a oblate ellipsoid geometry** of IgG (a/b
