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Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams
Optics and Lasers in Engineering ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams G. Marinaro a,b, A. Accardo b, N. Benseny-Cases a,1, M. Burghammer a,c, H. Castillo-Michel a, M. Cotte a,d, S. Dante b, F. De Angelis b, E. Di Cola a,2, E. Di Fabrizio e,f, C. Hauser g, C. Riekel a,n a
The European Synchrotron (ESRF), CS 40220, F-38043 Grenoble Cedex 9, France Istituto Italiano di Tecnologia, Via Morego 30, Genova 16163, Italy c Department of Analytical Chemistry, Ghent University, Krijgslaan 281, S12B-9000 Ghent, Belgium d LAMS (Laboratoire d'Archéologie Moléculaire et Structurale), UMR-8220, 3 rue Galilée 94200 Ivry-sur-Seine, France e Physical Science and Engineering Divisions, KAUST (King Abdullah University of Science and Technology), Jeddah, Saudi Arabia f BIONEM lab University of Magna Graecia, Campus Salvatore Venuta, Viale Europa Germaneto, Catanzaro 88100, Italy g Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos 138669, Singapore b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 September 2014 Received in revised form 10 February 2015 Accepted 3 March 2015
Droplets with colloidal biological suspensions evaporating on substrates with defined wetting properties generate confined environments for initiating aggregation and self-assembly processes. We describe smart micro- and nanostructured surfaces, optimized for probing single droplets and residues by synchrotron radiation micro- and nanobeam diffraction techniques. Applications are presented for AcIVD and β-amyloid (1–42) peptides capable of forming cross-β sheet structures. Complementary synchrotron radiation FTIR microspectroscopy addresses secondary structure formation. The high synchrotron radiation source brilliance enables fast raster-scan experiments. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Biological colloids Digital microfluidics Micro-/nano X-ray diffraction MicroFTIR Synchrotron radiation
1. Introduction The evaporation of droplets of colloidal biological suspensions results in various types of patterns depending on the nature of surface interactions [1]. Indeed, coffee-ring type residues are formed on wetting surfaces [2]. More complex patterns are observed on superhydrophobic surfaces (SHS) ranging from spherical to collapsed coffee-ring type residues depending on the solute concentration [3]. The formation of these “confined environments” is due to convective flow and diffusion mediated mass transport to the droplet interface resulting in the formation of gelated layers [4] which are at the origin of aggregation and self-assembly processes. Microscopic evidence for such processes can be obtained by probing droplets or residues by Xray micro- and nanobeam scattering techniques at high-brilliance synchrotron radiation (SR) sources [3]. Wide-angle X-ray scattering and small-angle X-ray scattering techniques (summarized here as
micro X-ray diffraction: μXRD) in combination with raster-scan probing reveal structural features from atomic to macroscopic scales [3,5–7]. Spectroscopy probes with raster-scan capability, such as Fourier transform infrared microspectroscopy (μFTIR), can provide complementary information. This text will review methodological advances in fabricating structured substrates with tailored wetting capabilities, optimized for μXRD raster-scan probing. The use of such substrates will be demonstrated for selected short peptides capable of forming nanofibrillar cross-β sheet phases [8]. We will also discuss μFTIR experiments addressing secondary structures formed during peptide self-assembly. All experiments reported were performed at the European Synchrotron Radiation Facility (ESRF), a state-of-the-art 3rd generation SR source.
2. Methods n
Corresponding author. E-mail address: [email protected] (C. Riekel). 1 Current address: The Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, Great Britain. 2 Current address: Laboratoire Interdisciplinaire de Physique (LIPhy UMR5588 CNRS/UJF), 140 rue de la Physique, BP87 38402 Saint Martin d'Hères Cedex.
2.1. Substrate technologies Structured substrates for μXRD probing of droplets should -ideally- have the following properties: (i) low X-ray absorption, (ii) low X-ray background scattering, (iii) none or weak
http://dx.doi.org/10.1016/j.optlaseng.2015.03.004 0143-8166/& 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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shadowing-effects, (iv) tailored wetting behaviour and (v) predetermined aggregation or self-assembly points. Pillared Si-SHSs (Fig. 1A), based on Si-wafers of 500 μm thickness, are a well-established technology [9] but pose problems as supports for weakly X-ray scattering biological specimens due to high X-ray absorption (e.g. 82% at λ 1 Å [10]). The signal/noise ratio of X-ray signals is, however, high thanks to the low X-ray scattering background of single-crystalline silicon. As alternative way, a more Xray transparent substrate such as 500 μm thick polymethylmethacrylate (PMMA) sheets can be chosen to fabricate PMMA-SHSs [11]. The strong diffuse X-ray scattering background from the PMMA sheet requires, however, precise background subtraction techniques [11]. Although experimental considerations usually prevail, reducing the number of steps in producing a structured substrate can be a valuable economic argument. Indeed, creating a SHS from PMMA by surface roughening via plasma etching (Fig. 1C; left) requires fewer fabrication steps than a pillared PMMA-SHS [11]. It is also interesting noting that by reducing the nanofibrillar PMMA density and in the absence of a Teflon layer, the surface wetting properties can be tailored from superhydrophobic to superhydrophilic (Fig. 1C; right) [12,13]. A thin PMMA film can also be spin-coated and structured on highly X-ray or IR transparent substrates such as Si3N4 membranes, [13] allowing reducing absorption and diffuse X-ray scattering. Depending on the position of the X-ray beam on a droplet, the edge of a SHS will more or less shadow the μXRD pattern for a SRbeam aligned parallel to the surface (“horizontal geometry”: HG; see Figs. 2 and 3). Shadowing problems can be an issue when probing a droplet or residue close to the surface. Highly X-ray transparent, thin Si3N4 substrates with SU-8 pillars are better suited for HG-mode probing provided that the surrounding Si-frame is also thin (Fig. 1E) [10]. Moreover, it has the intrinsic advantage that the residue can be easily detached from the substrate and posed to a thin capillary tip (Fig. 4A) [16]. This is, however, not possible for fragile morphologies such as nanofilaments which have to be probed on the substrate in NG-mode. In order to reduce and locally avoid absorption in NG, one can use a pillared Si-SHS based on a thin silicon substrate (o50 μm) with etched holes (Fig. 1D) [15]. The variation of X-ray absorption across the substrate requires, however, elaborate data treatment for
absorption corrections which can be avoided in practice by using a SHS based on a thin Si3N4 membrane with SU-8 pillars (Fig. 1E) [10]. The radial pillar-gradient shown in Fig. 1E generates in addition a pinning centre which allows keeping the droplet at a constant position during evaporation. An alternative is provided by fabricating a cone in a forest of silicon pillars via ion-beam milling (Fig. 1E) [9]. Pillar gradients and cones are of particular interest for concentrating ultradilute solution droplets at predetermined points for probing experiments [9,17,18]. In summary, pillared SHSs based on Si3N4 membranes and SU-8 pillars [10] provide a significant advantage for probing weakly scattering organic or biological materials with SR scattering techniques. PMMA-SHSs based on PMMA thin films with nanofibrillar surface roughness have a considerable potential, in particular for FTIR applications [13]. Pillared Si-SHSs based on standard Si-wafers remain of interest for stronger scattering materials in view of a well-established nanofabrication technology. Pillared Si-SHSs with holes are used for TEM applications [9] but are not optimal for X-ray scattering in view of local absorption variations.
2.2. Synchrotron radiation scattering techniques High brilliance SR is produced in a so-called storage ring by electron bunches moving close to the speed of light through periodic magnetic devices (e.g. undulators) [19]. The main elements of a μXRD beamline are shown in Fig. 2. SR emitted in a narrow cone from an undulator is monochromated (ΔE/E 10 4) by a double Si-crystal to -typically- E 13 keV (λ 0.95 Å) and focused at sample position by refractive, reflective or diffractive optical elements. μXRD experiments reported below were performed at the ESRFID13 beamline which uses currently refractive lenses made of Be or Si providing routinely beam sizes from a few μm down to 100 nm with a flux up to 9 1011 photons/s in an about 2 3 μm2 focal spot at 13 keV [7,20–22]. Experiments are generally performed in air although the control of humidity would allow modulating the droplet evaporation rate by up to about factor 7 [3]. Droplets are generally probed in HG-mode but a grazing-incidence geometry is
Fig. 1. SEM images for selected microfabricated surfaces (A): Si-SHS with a forest of Si-pillars. (B): PMMA-SHS with micropillars showing nanofibrillar roughness. (Adapted from: [11]) (C): Left: PMMA-SHS with a high-density nanofibrillar PMMA surface. (Adapted from: [14]); Right: Superhydrophilic Si3N4 substrate with a low-density nanofibrillar PMMA surface (same scale). (D): Pillared Si-SHS with 6 μm diameter, etched holes developed for transmission electron microscopy. (Adapted from: [9]) (E): SHS based on a Si3N4 membrane and a gradient of SU-8 pillars. (Adapted from: [10]) (F): Pillared Si-SHS with central nanocone. (Adapted from: [15]) The pillar-gradient (E) and the nanocone provide an attraction potential for an evaporating droplet.
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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Fig. 2. Schematic design of μXRD beamline with focusing optics. The origin (source point) of the cone of emitted X-rays is located at the centre of an undulator. The beam is monochromated by a double Si-crystal and focused by refractive or other X-ray optical systems to the sample position. (a): Probing a residue attached to a substrate in “normal geometry” (NG-mode). (b): Probing a droplet in “horizontal geometry” (HG-mode). The absorption of part of the scattered X-rays by the substrate is schematically shown for scattering from a droplet volume element close to the substrate. The XRD patterns are collected by a CCD or pixel detector.
Fig. 3. μXRD of evaporating Ac-IVD peptide solution droplet on a PMMA-SHS. Successive raster-scans of the droplet through a 1 μm X-ray beam were performed in HGmode with 100 μm step-increments and 0.5 s/pattern. Each “image” corresponds to 17 18 “pixels”, composed of individual diffraction patterns. The circles are guides-tothe-eye. The times indicated correspond to the middle of each raster-scan after the droplet deposition. At t 40 min short-range order water scattering and two Bragg peaks (d¼ 5.4 Å and 4.7 Å) are observed at the droplet rim (left zoomed pattern). The zoomed pattern from the residue reveals a cross-β sheet structure which is shown schematically with hydrogen-bonded peptide chains symbolized as arrows [8].
Fig. 4. (A) Optical image of coffee-ring type Ac-IVD residue detached from a PMMA-SHS and posed to a glass support [16]. (B): Transmission μXRD raster-scan with 20 μm step-increments in horizontal and vertical directions. (C): Zoom into rectangular, dashed area in (B). The orientations of the fibre-axes of the cross-β sheet patterns at the rim are indicated by white arrows. (Adapted from: [16]).
also possible [6,10]. Raster-scan experiments are performed by stepscanning (1D or mesh) the sample through the beam and recording after each step a pattern by a charge-coupled detector (CCD) or pixel detector [7]. Depending on the sample, the time/pattern can be in the second or sub-second range. Evaporation times in the order of about an hour for 4–5 μL droplets provide the experimenter enough time
for droplet deposition by a manual pipette. The high evaporation rate of nL and subnL droplets [3] requires, however, motorized pipettes, integrated in the beamline control system. μFTIR experiments were performed at the ESRF-ID21 beamline which is extracting infrared radiation from a continuous bending magnet spectrum reaching diffraction-limited resolution and up to
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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Fig. 5. (A) Optical image of Ac-IVD peptide residue from 2.15 mg/ml solution on 1 μm thick Si3N4 membrane. (B): μXRD raster-scan of whole residue with 0.15 0.15 μm2 focal spot, 100 μm step-increments and 2 s/pattern. A zoom of a single cross-β sheet pattern is shown as inset. The fibre axis is indicated in all patterns by an arrow. (C): High resolution μXRD raster-scan with 1.0 h*1.5 v μm2 step-increments and 3 s/pattern. The composite diffraction image composed of 10201 pixels (patterns) shows the outline of the coffee-ring type residue and a crack which is also visible in the optical image. (D): Zoom into crack-region of composite image (dashed rectangle in (C)). The average of 300 patterns is shown as inset.
factor 50 higher intensity in a o10 μm spot as compared to a Globar source [23]. The experimental station comprises a Nicolet NEXUS spectrometer and a confocal Nicolet Continuum microscope. μFTIR experiments are performed in a vertical TG, typically based on a 6 n 6 μm2 beam and an MCT detector. Decomposition of spectra was performed using the Omnic 8.1 software from Thermo Fisher Scientific Inc. 2.3. Materials For details on Ac-IVD, see: [16]. β-amyloid (1 42) (Bachem, Germany) was first dissolved in trifluoro acetic acid (TFA) at a concentration of 1 mg/mL to eliminate the presence of seeds and to obtain a monomeric peptide dispersion. TFA was gently dried under nitrogen, and the peptide was successively dissolved in Milli-Q water (1 mg/mL), sonicated for 5 min, and centrifuged at 1000 rpm for 10 min. A few μL droplet from the pH 7.0 supernatant was used for sample preparation.
3. Probing peptide self-assembly Studying self-assembly of short peptides is of interest for unravelling pathways resulting in amyloidic nanofibrillation. We will discuss the use of SR microprobe techniques for the ultrashort, hydrogel forming designer peptide Ac-IVD [16] and β-amyloid (1– 42) peptide which is one of the main components of the amyloid plaques found in the brains of Alzheimer patients. In-situ studies of droplet evaporation address transient phases and nanofibrillar ordering, mediated by flow effects. Indeed, Fig. 3 shows the evaporation of a 4 μL droplet of 4.5 mg/ml Ac-IVD peptide solution on a PMMA-SHS. The 5.4 Å reflection appearing at
40 min after droplet deposition is attributed to an α-helical transient phase [24] which is supported by circular dichroism (CD) spectroscopy [16]. The 4.8 Å reflection corresponds to a coexisting nanofibrillar cross-β sheet phase [8] which is the only phase remaining in the residue. We note the high orientational ordering of the nanofibrils in the residue with the fibre-axis normal to the rim (Fig. 4A–C) [16]. Self-assembly into a cross-β sheet amyloidic phase with a fibreaxis orientation normal to the residual rim is also observed for an Ac-IVD residue on a wetting Si3N4 membrane (Fig. 5A and B). μXRD with a nanobeam allows obtaining a high-resolution diffraction image revealing domains with a homogeneous fibre axis orientation and with the presence of cracks formed during the drying process. (Fig. 5C and D) This allows averaging a large amount of patterns to improve the counting statistics (Fig. 5D, inset). One can relate the orientational ordering of the cross-β-type nanofilaments at the rim of the residue to an arrested flow pattern. Highly anisotropic nanoscale objects such as cylindrical tobacco mosaic virus particles have been shown to align with their long axis parallel to the rim of the residue due to capillary flow effects [6]. The observed orientation of the cross-β type nanofilaments suggests rather a nucleation/growth process from the interface into the gelated layer at the rim of the evaporating droplet. We note that a layer of colloidal calcium carbonate at the rim of an evaporating droplet serves also for nucleation and growth of CaCO3 crystallites [25]. Optical images of a droplet of β-amyloid (1–42) solution on a pillared Si-SHS and its detached residue are shown in Fig. 6A and B. A zoom into the rim reveals filamentous extensions which are formed as the retracting viscous interface starts wetting the pillars. μXRD probing of the filamentous extension reveals a cross-β sheet amyloidic structure with the fibre axis aligned along the filamentous direction suggesting self-assembly in an extensional flow (Fig. 6D and E)
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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Fig. 6. (A): β-amyloid (1–42) solution (1 mg/ml) droplet on pillared Si-SHS. (B): Detached residue composed of a thin membrane surrounded by a coffee-ring type rim, posed to a glass capillary support. (C): Zoom into dashed rectangle in (B). (D): μXRD raster-scan with 1.5 h*0.5 v μm2 beam and 2 μm step-increments of dashed area indicated in (C). (E): Details of μXRD raster-scan (dashed rectangle in (D)). The local fibre axes of the cross-β structure are indicated by arrows. An average of several patterns from the filament is shown as inset.
Fig. 7. (A): β-amyloid (1–42) μFTIR spectrum for the Amide I/II band range obtained in about 45 s with a 6 μm beam from the inner membrane of the residue (Fig. 6B). (B): FTIR raster-scan with 5 μm step-increments plotted on image of residue. The intensity is scaled to the absorbance at the centre of the Amide I band β-sheet peak (1633 cm 1).
Simulation of the β-amyloid (1–42) X-ray fibre diffraction pattern indicates a parallel cross-β sheet structure [8]. We note that the structures of several peptides with cross-β sheet structures have been determined from single crystals to atomic resolution [26–29]. Obtaining such structures are, however, in many cases impossible in view of crystallization problems and polymorphism [30]. Complementary techniques providing information on secondary structures are therefore of interest. Indeed, one can use raster-scan μFTIR for localizing βtype secondary structures based on the comparison with published spectral features [31]. As an example we show the μFTIR spectrum collected within the membrane of the β-amyloid (1–42) residue (Fig. 6B). The strong peak at 1633 cm 1 is characteristic of a β-sheet vibration (Fig. 7A) [32]. Raster-scan μFTIR reveals a homogeneous
distribution of β-sheet secondary structure across the residue (Fig. 7B). The absorbance -and in particular of the rim- is, however, generally too high for a quantitative spectral analysis excluding the possibility of probing for differences in secondary structure across the rim. Droplet solute concentrations have therefore to be lower for μFTIR than for μXRD experiments. It is also advantageous expanding coffee-ring type residues by using superhydrophilic substrates [12] (Fig. 1C) and using D2O as solvent for in-situ experiments during droplet evaporation. Indeed, a droplet of Ac-IVD in D2O solution forms a coffee-ring type residue on a superhydrophilic substrate (Fig. 8A). The μFTIR spectrum from the rim reveals a strong β-sheet vibration peak at 1628 cm 1 (Fig. 8B).[32,33] The spectrum has been resolved into individual peaks by decomposition and Gaussian fits (Fig. 8B). The peak at 1690 cm 1 is
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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Fig. 8. (A): SEM image of coffee-ring formation from 0.1 mg/ml Ac-IVD in a D2O solution droplet on Si3N4 with a nanofibrillar superhydrophilic PMMA layer. (B): μFTIR spectrum from the rim of the residue (red line) and fitted spectrum (blue line) based on a decomposition and Gaussian peak fits. The Amide II band position corresponds to nanofibrils from Ac-IVD in aqueous solution [33]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
at the position of an antiparallel β-sheet B1 vibration [32]. The broad band at 1645 cm 1 indicates the presence of other components such as random, α-helical and β-hairpin structures. We note that a spatial separation into α- and β-phases across a coffee-ring type residue was observed for the β-amyloid (25–35) fragment [13]. The absence of a strong Amide II band, as observed for Ac-IVD nanofibrils formed in H2O [33], is tentatively attributed to H/D exchange [34]. 4. Summary and outlook The brilliance of 3rd generation SR sources enables μXRD and μFTIR raster-scan imaging experiments. The drive for faster image generation, smaller beam sizes and smaller sample volumes is related to a continuously increasing SR source brilliance, more efficient X-ray optics and detection systems. This has to be matched by sample environments allowing confining processes in small volumes and at defined positions. The development of smart surfaces described in this article is an answer to this quest. With the upcoming upgrade of the ESRF (and other 3rd generation SR-sources) to a diffraction-limited (4th generation) SR-source, [35] the brilliance of the emitted SR will increase by about factor 30 for photon energies410 keV [36]. This and the availability of ultrafast pixel detectors [37] enabling a continuous scanning mode will significantly reduce scanning times. We exemplify this by the 101n101 pixel raster-scan diffraction image shown in Fig. 5C which took 11 h including 22% overheads due to detector readout and motor movements. For the -conservative- assumption of a linear correlation of increase in SR-source brilliance and flux in the focal spot, the total scan-time will be reduced to about 17 min assuming the same counting statistics and no overheads. Scan-times will even be smaller due to the absence of readout noise of pixel detectors.
Acknowledgements The superhydrophobic substrates were fabricated at the Istituto Italiano di Tecnologia (IIT) in Genova. IIT and ESRF provided support for the thesis projects of A.A. and G.M. References [1] Berthier J. Microdrops and digital microfluidics. Norwich N.Y.: William Andrew; 2008. [2] Deegan RD, Bakajin O, Dupont TF, Huber G, Nagel SR, Witten TA. Capillary flow as the cause of ring stains from dried liquid drops. Nature 1997;389:827–9. [3] Accardo A, Di Fabrizio E, Limongi T, Marinaro G, Riekel C. Probing droplets on superhydrophobic surfaces by synchrotron radiation scattering techniques. J Synchrotron Radiat 2014;21:643–53. [4] Sobac B, Brutin D. Structural and evaporative evolutions in desiccating sessile drops of blood. Phys Rev E 2011;84:011603. [5] Gebhardt R, Vendrely C, Burghammer M, Riekel C. Characterization of the boundary zone of a cast protein drop: fibroin β-sheet and nanofibril formation. Langmuir 2009;25:6307–11.
[6] Gebhardt R, Teulon JM, Pellequer JL, Burghammer M, Colletier JP, Riekel C. Virus particle assembly into crystalline domains enabled by the coffee ring effect. Soft Matter 2014;10:5458–62. [7] Riekel C, Burghammer M, Davies R, Gebhardt R, Popov D. Fundamentals of non-crystalline diffraction with microfocus techniques. In: García-Gutiérrez M, Nogales A, Gómez M, Ezquerra TA, editors. Applications of Synchrotron Light to Non-Crystalline Diffraction in Materials and Life Sciences. Heidelberg: Springer; 2008. [8] Jahn TR, Makin OS, Morris KL, Marshall KE, Tian P, Sikorski P, et al. The Common architecture of cross-β amyloid. J Mol Biol 2010;395:717–27. [9] De Angelis F, Gentile F, Mecarini F, Das G, Moretti M, Candeloro P, et al. Breaking the diffusion limit with super-hydrophobic delivery of molecules to plasmonic nanofocusing SERS structures. Nat Photonics 2011;5:683–8. [10] Marinaro G, Accardo A, Angelis FD, Dane T, Weinhausen B, Burghammer M, et al. A superhydrophobic chip based on SU-8 photoresist pillars suspended on a silicon nitride membrane. Lab Chip 2014;14:3705–9. [11] Accardo A, Gentile F, Mecarini F, De Angelis F, Burghammer M, Di Fabrizio E, et al. In situ X-ray scattering studies of protein solution droplets drying on micro- and nanopatterned superhydrophobic PMMA surfaces. Langmuir 2010;26:15057–64. [12] Accardo A, Gentile F, Mecarini F, De Angelis F, Burghammer M, Di Fabrizio E, et al. Ultrahydrophobic PMMA micro- and nano-textured surfaces fabricated by optical lithography and plasma etching for X-Ray diffraction studies. Microelectron Eng 2011;88:1660–3. [13] Accardo A, Shalabaeva V, Cotte M, Burghammer M, Krahne R, Riekel C, et al. Amyloid β peptide conformational changes in the presence of a lipid membrane system. Langmuir 2014;30:3191–8. [14] De Angelis F, Das G, Candeloro P, Patrini M, Galli M, Bek A, et al. Nanoscale chemical mapping using three-dimensional adiabatic compression of surface plasmon polaritons. Nat Nanotechnol 2010;5:67–72. [15] Gentile F, Moretti M, Limongi T, Falqui A, Bertoni G, Scarpellini A, et al. Direct Imaging of dna fibers: the visage of double helix. Nano Lett 2012;12:6453–8. [16] Hauser CAE, Deng RS, Mishra A, Loo YH, Khoe U, Zhuang FR, et al. Natural trito hexapeptides self-assemble in water to amyloid beta-type fiber aggregates by unexpected alpha-helical intermediate structures. Proc Natl Acad Sci USA 2011;108:1361–6. [17] Gentile F, Das G, Coluccio ML, Mecarini F, Accardo A, Tirinato L, et al. Ultra low concentrated molecular detection using super hydrophobic surface based biophotonic devices. Microelectron Eng 2010;87:798–801. [18] Miele E, Malerba M, Dipalo M, Rondanina E, Toma A, De Angelis F. Controlling wetting and self-assembly dynamics by tailored hydrophobic and oleophobic surfaces. Adv Mater 2014;26:4179–83. [19] Als-Nielsen J, McMorrow D. Elements of modern X-ray physics. Chichester, UK: John Wiley & Sons, Ltd; 2011. [20] Riekel C. New avenues in X-ray microbeam experiments. Rep Prog Phys 2000;63:233–62. [21] Riekel C, Burghammer M, Popov D. Protein micro- and nanocrystallography using synchrotron radiation. In: Nicolini C, editor. Synchrotron Radiation and Structural Proteomics. Singapore: Pan Stanford Publishing; 2012. p. 3–30. [22] Schroer CG, Boye P, Feldkamp JM, Patommel J, Schropp A, Schwab A, et al. Hard X-Ray scanning microscopy with fluorescence and diffraction contrast. J Phys Conf Ser 2009;186. [23] Carr GL. Resolution limits for infrared microspectroscopy explored with synchrotron radiation. Rev Sci Instrum 2001;72:1613–9. [24] Crick FHC. The packing of α-helices: Simple coiled-coils. Acta Crystallogr 1953;6:689–97. [25] Accardo A, Burghammer M, Di Cola E, Reynolds M, Di Fabrizio E, Riekel C. Calcium carbonate mineralization: X-ray microdiffraction probing of the interface of an evaporating drop on a superhydrophobic surface. Langmuir 2011;27:8216–22. [26] Colletier JP, Laganowsky A, Landau M, Zhao M, Soriaga AB, Goldschmidt L, et al. Molecular basis for amyloid-β polymorphism. PNAS 2011;108: 16938–16943. [27] Nelson R, Sawaya MR, Balbirnie M, Madsen AØ, Riekel C, Grothe R, et al. Structure of the cross- β spine of amyloid-like fibrils. Nature 2005;435:773–8.
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
G. Marinaro et al. / Optics and Lasers in Engineering ∎ (∎∎∎∎) ∎∎∎–∎∎∎ [28] Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Siever SA, Apostol MI, et al. Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature 2007;447:453–7. [29] Wiltzius JJ, Sievers SA, Sawaya MR, Cascio D, Popov D, Riekel C, et al. Atomic structure of the cross-β spine of islet amyloid polypeptide (amylin). Protein Sci 2008;17:1467–74. [30] Paravastu AK, Leapman RD, Yau WM, Tycko R. Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils. PNAS 2008;105:18349–54. [31] Miller L, Bourassa MW, Smith RJ. FTIR spectroscopic imaging of protein aggregation in living cells. Biochim Biophys Acta 2013;1828:2339–46. [32] Chirgadze YN, Nevskaya NA. Infrared spectra and resonance interaction of amide-I vibration of the antiparallel-chain pleated sheet. Biopolymers 1976;15:607–25. [33] Mishra A, Chan KH, Reithofer MR, Hauser CAE. Influence of metal salts on the hydrogelation properties of ultrashort aliphatic peptides. RSC Adv 2013; 3:9985–93.
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[34] Dzwolak W, Loksztejn A, Smirnovas V. New insights into the self-assembly of insulin amyloid fibrils: an H–D exchange FT-IR study. Biochemistry 2006;45:8143–51. [35] Eriksson M, van der Veen F, Quitman C. Diffraction-limited storage rings—a window to the science of tomorrow. J Synchrotron Radiat 2014;21:837–42. [36] ESRF, White Paper: Upgrade Programme, Phase II (2015–2019), 〈www.esrf.fr/ files/live/sites/www/files/about/upgrade/documentation/whitepaper-upgra de-phaseII.pdf〉; 2014. [37] Denes P, Schmitt B. Pixel detectors for diffraction-limited storage rings. J Synchrotron Radiat 2014;21:1006–10.
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Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams G. Marinaro a,b, A. Accardo b, N. Benseny-Cases a,1, M. Burghammer a,c, H. Castillo-Michel a, M. Cotte a,d, S. Dante b, F. De Angelis b, E. Di Cola a,2, E. Di Fabrizio e,f, C. Hauser g, C. Riekel a,n a
The European Synchrotron (ESRF), CS 40220, F-38043 Grenoble Cedex 9, France Istituto Italiano di Tecnologia, Via Morego 30, Genova 16163, Italy c Department of Analytical Chemistry, Ghent University, Krijgslaan 281, S12B-9000 Ghent, Belgium d LAMS (Laboratoire d'Archéologie Moléculaire et Structurale), UMR-8220, 3 rue Galilée 94200 Ivry-sur-Seine, France e Physical Science and Engineering Divisions, KAUST (King Abdullah University of Science and Technology), Jeddah, Saudi Arabia f BIONEM lab University of Magna Graecia, Campus Salvatore Venuta, Viale Europa Germaneto, Catanzaro 88100, Italy g Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos 138669, Singapore b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 September 2014 Received in revised form 10 February 2015 Accepted 3 March 2015
Droplets with colloidal biological suspensions evaporating on substrates with defined wetting properties generate confined environments for initiating aggregation and self-assembly processes. We describe smart micro- and nanostructured surfaces, optimized for probing single droplets and residues by synchrotron radiation micro- and nanobeam diffraction techniques. Applications are presented for AcIVD and β-amyloid (1–42) peptides capable of forming cross-β sheet structures. Complementary synchrotron radiation FTIR microspectroscopy addresses secondary structure formation. The high synchrotron radiation source brilliance enables fast raster-scan experiments. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Biological colloids Digital microfluidics Micro-/nano X-ray diffraction MicroFTIR Synchrotron radiation
1. Introduction The evaporation of droplets of colloidal biological suspensions results in various types of patterns depending on the nature of surface interactions [1]. Indeed, coffee-ring type residues are formed on wetting surfaces [2]. More complex patterns are observed on superhydrophobic surfaces (SHS) ranging from spherical to collapsed coffee-ring type residues depending on the solute concentration [3]. The formation of these “confined environments” is due to convective flow and diffusion mediated mass transport to the droplet interface resulting in the formation of gelated layers [4] which are at the origin of aggregation and self-assembly processes. Microscopic evidence for such processes can be obtained by probing droplets or residues by Xray micro- and nanobeam scattering techniques at high-brilliance synchrotron radiation (SR) sources [3]. Wide-angle X-ray scattering and small-angle X-ray scattering techniques (summarized here as
micro X-ray diffraction: μXRD) in combination with raster-scan probing reveal structural features from atomic to macroscopic scales [3,5–7]. Spectroscopy probes with raster-scan capability, such as Fourier transform infrared microspectroscopy (μFTIR), can provide complementary information. This text will review methodological advances in fabricating structured substrates with tailored wetting capabilities, optimized for μXRD raster-scan probing. The use of such substrates will be demonstrated for selected short peptides capable of forming nanofibrillar cross-β sheet phases [8]. We will also discuss μFTIR experiments addressing secondary structures formed during peptide self-assembly. All experiments reported were performed at the European Synchrotron Radiation Facility (ESRF), a state-of-the-art 3rd generation SR source.
2. Methods n
Corresponding author. E-mail address: [email protected] (C. Riekel). 1 Current address: The Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, Great Britain. 2 Current address: Laboratoire Interdisciplinaire de Physique (LIPhy UMR5588 CNRS/UJF), 140 rue de la Physique, BP87 38402 Saint Martin d'Hères Cedex.
2.1. Substrate technologies Structured substrates for μXRD probing of droplets should -ideally- have the following properties: (i) low X-ray absorption, (ii) low X-ray background scattering, (iii) none or weak
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Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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shadowing-effects, (iv) tailored wetting behaviour and (v) predetermined aggregation or self-assembly points. Pillared Si-SHSs (Fig. 1A), based on Si-wafers of 500 μm thickness, are a well-established technology [9] but pose problems as supports for weakly X-ray scattering biological specimens due to high X-ray absorption (e.g. 82% at λ 1 Å [10]). The signal/noise ratio of X-ray signals is, however, high thanks to the low X-ray scattering background of single-crystalline silicon. As alternative way, a more Xray transparent substrate such as 500 μm thick polymethylmethacrylate (PMMA) sheets can be chosen to fabricate PMMA-SHSs [11]. The strong diffuse X-ray scattering background from the PMMA sheet requires, however, precise background subtraction techniques [11]. Although experimental considerations usually prevail, reducing the number of steps in producing a structured substrate can be a valuable economic argument. Indeed, creating a SHS from PMMA by surface roughening via plasma etching (Fig. 1C; left) requires fewer fabrication steps than a pillared PMMA-SHS [11]. It is also interesting noting that by reducing the nanofibrillar PMMA density and in the absence of a Teflon layer, the surface wetting properties can be tailored from superhydrophobic to superhydrophilic (Fig. 1C; right) [12,13]. A thin PMMA film can also be spin-coated and structured on highly X-ray or IR transparent substrates such as Si3N4 membranes, [13] allowing reducing absorption and diffuse X-ray scattering. Depending on the position of the X-ray beam on a droplet, the edge of a SHS will more or less shadow the μXRD pattern for a SRbeam aligned parallel to the surface (“horizontal geometry”: HG; see Figs. 2 and 3). Shadowing problems can be an issue when probing a droplet or residue close to the surface. Highly X-ray transparent, thin Si3N4 substrates with SU-8 pillars are better suited for HG-mode probing provided that the surrounding Si-frame is also thin (Fig. 1E) [10]. Moreover, it has the intrinsic advantage that the residue can be easily detached from the substrate and posed to a thin capillary tip (Fig. 4A) [16]. This is, however, not possible for fragile morphologies such as nanofilaments which have to be probed on the substrate in NG-mode. In order to reduce and locally avoid absorption in NG, one can use a pillared Si-SHS based on a thin silicon substrate (o50 μm) with etched holes (Fig. 1D) [15]. The variation of X-ray absorption across the substrate requires, however, elaborate data treatment for
absorption corrections which can be avoided in practice by using a SHS based on a thin Si3N4 membrane with SU-8 pillars (Fig. 1E) [10]. The radial pillar-gradient shown in Fig. 1E generates in addition a pinning centre which allows keeping the droplet at a constant position during evaporation. An alternative is provided by fabricating a cone in a forest of silicon pillars via ion-beam milling (Fig. 1E) [9]. Pillar gradients and cones are of particular interest for concentrating ultradilute solution droplets at predetermined points for probing experiments [9,17,18]. In summary, pillared SHSs based on Si3N4 membranes and SU-8 pillars [10] provide a significant advantage for probing weakly scattering organic or biological materials with SR scattering techniques. PMMA-SHSs based on PMMA thin films with nanofibrillar surface roughness have a considerable potential, in particular for FTIR applications [13]. Pillared Si-SHSs based on standard Si-wafers remain of interest for stronger scattering materials in view of a well-established nanofabrication technology. Pillared Si-SHSs with holes are used for TEM applications [9] but are not optimal for X-ray scattering in view of local absorption variations.
2.2. Synchrotron radiation scattering techniques High brilliance SR is produced in a so-called storage ring by electron bunches moving close to the speed of light through periodic magnetic devices (e.g. undulators) [19]. The main elements of a μXRD beamline are shown in Fig. 2. SR emitted in a narrow cone from an undulator is monochromated (ΔE/E 10 4) by a double Si-crystal to -typically- E 13 keV (λ 0.95 Å) and focused at sample position by refractive, reflective or diffractive optical elements. μXRD experiments reported below were performed at the ESRFID13 beamline which uses currently refractive lenses made of Be or Si providing routinely beam sizes from a few μm down to 100 nm with a flux up to 9 1011 photons/s in an about 2 3 μm2 focal spot at 13 keV [7,20–22]. Experiments are generally performed in air although the control of humidity would allow modulating the droplet evaporation rate by up to about factor 7 [3]. Droplets are generally probed in HG-mode but a grazing-incidence geometry is
Fig. 1. SEM images for selected microfabricated surfaces (A): Si-SHS with a forest of Si-pillars. (B): PMMA-SHS with micropillars showing nanofibrillar roughness. (Adapted from: [11]) (C): Left: PMMA-SHS with a high-density nanofibrillar PMMA surface. (Adapted from: [14]); Right: Superhydrophilic Si3N4 substrate with a low-density nanofibrillar PMMA surface (same scale). (D): Pillared Si-SHS with 6 μm diameter, etched holes developed for transmission electron microscopy. (Adapted from: [9]) (E): SHS based on a Si3N4 membrane and a gradient of SU-8 pillars. (Adapted from: [10]) (F): Pillared Si-SHS with central nanocone. (Adapted from: [15]) The pillar-gradient (E) and the nanocone provide an attraction potential for an evaporating droplet.
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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Fig. 2. Schematic design of μXRD beamline with focusing optics. The origin (source point) of the cone of emitted X-rays is located at the centre of an undulator. The beam is monochromated by a double Si-crystal and focused by refractive or other X-ray optical systems to the sample position. (a): Probing a residue attached to a substrate in “normal geometry” (NG-mode). (b): Probing a droplet in “horizontal geometry” (HG-mode). The absorption of part of the scattered X-rays by the substrate is schematically shown for scattering from a droplet volume element close to the substrate. The XRD patterns are collected by a CCD or pixel detector.
Fig. 3. μXRD of evaporating Ac-IVD peptide solution droplet on a PMMA-SHS. Successive raster-scans of the droplet through a 1 μm X-ray beam were performed in HGmode with 100 μm step-increments and 0.5 s/pattern. Each “image” corresponds to 17 18 “pixels”, composed of individual diffraction patterns. The circles are guides-tothe-eye. The times indicated correspond to the middle of each raster-scan after the droplet deposition. At t 40 min short-range order water scattering and two Bragg peaks (d¼ 5.4 Å and 4.7 Å) are observed at the droplet rim (left zoomed pattern). The zoomed pattern from the residue reveals a cross-β sheet structure which is shown schematically with hydrogen-bonded peptide chains symbolized as arrows [8].
Fig. 4. (A) Optical image of coffee-ring type Ac-IVD residue detached from a PMMA-SHS and posed to a glass support [16]. (B): Transmission μXRD raster-scan with 20 μm step-increments in horizontal and vertical directions. (C): Zoom into rectangular, dashed area in (B). The orientations of the fibre-axes of the cross-β sheet patterns at the rim are indicated by white arrows. (Adapted from: [16]).
also possible [6,10]. Raster-scan experiments are performed by stepscanning (1D or mesh) the sample through the beam and recording after each step a pattern by a charge-coupled detector (CCD) or pixel detector [7]. Depending on the sample, the time/pattern can be in the second or sub-second range. Evaporation times in the order of about an hour for 4–5 μL droplets provide the experimenter enough time
for droplet deposition by a manual pipette. The high evaporation rate of nL and subnL droplets [3] requires, however, motorized pipettes, integrated in the beamline control system. μFTIR experiments were performed at the ESRF-ID21 beamline which is extracting infrared radiation from a continuous bending magnet spectrum reaching diffraction-limited resolution and up to
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Fig. 5. (A) Optical image of Ac-IVD peptide residue from 2.15 mg/ml solution on 1 μm thick Si3N4 membrane. (B): μXRD raster-scan of whole residue with 0.15 0.15 μm2 focal spot, 100 μm step-increments and 2 s/pattern. A zoom of a single cross-β sheet pattern is shown as inset. The fibre axis is indicated in all patterns by an arrow. (C): High resolution μXRD raster-scan with 1.0 h*1.5 v μm2 step-increments and 3 s/pattern. The composite diffraction image composed of 10201 pixels (patterns) shows the outline of the coffee-ring type residue and a crack which is also visible in the optical image. (D): Zoom into crack-region of composite image (dashed rectangle in (C)). The average of 300 patterns is shown as inset.
factor 50 higher intensity in a o10 μm spot as compared to a Globar source [23]. The experimental station comprises a Nicolet NEXUS spectrometer and a confocal Nicolet Continuum microscope. μFTIR experiments are performed in a vertical TG, typically based on a 6 n 6 μm2 beam and an MCT detector. Decomposition of spectra was performed using the Omnic 8.1 software from Thermo Fisher Scientific Inc. 2.3. Materials For details on Ac-IVD, see: [16]. β-amyloid (1 42) (Bachem, Germany) was first dissolved in trifluoro acetic acid (TFA) at a concentration of 1 mg/mL to eliminate the presence of seeds and to obtain a monomeric peptide dispersion. TFA was gently dried under nitrogen, and the peptide was successively dissolved in Milli-Q water (1 mg/mL), sonicated for 5 min, and centrifuged at 1000 rpm for 10 min. A few μL droplet from the pH 7.0 supernatant was used for sample preparation.
3. Probing peptide self-assembly Studying self-assembly of short peptides is of interest for unravelling pathways resulting in amyloidic nanofibrillation. We will discuss the use of SR microprobe techniques for the ultrashort, hydrogel forming designer peptide Ac-IVD [16] and β-amyloid (1– 42) peptide which is one of the main components of the amyloid plaques found in the brains of Alzheimer patients. In-situ studies of droplet evaporation address transient phases and nanofibrillar ordering, mediated by flow effects. Indeed, Fig. 3 shows the evaporation of a 4 μL droplet of 4.5 mg/ml Ac-IVD peptide solution on a PMMA-SHS. The 5.4 Å reflection appearing at
40 min after droplet deposition is attributed to an α-helical transient phase [24] which is supported by circular dichroism (CD) spectroscopy [16]. The 4.8 Å reflection corresponds to a coexisting nanofibrillar cross-β sheet phase [8] which is the only phase remaining in the residue. We note the high orientational ordering of the nanofibrils in the residue with the fibre-axis normal to the rim (Fig. 4A–C) [16]. Self-assembly into a cross-β sheet amyloidic phase with a fibreaxis orientation normal to the residual rim is also observed for an Ac-IVD residue on a wetting Si3N4 membrane (Fig. 5A and B). μXRD with a nanobeam allows obtaining a high-resolution diffraction image revealing domains with a homogeneous fibre axis orientation and with the presence of cracks formed during the drying process. (Fig. 5C and D) This allows averaging a large amount of patterns to improve the counting statistics (Fig. 5D, inset). One can relate the orientational ordering of the cross-β-type nanofilaments at the rim of the residue to an arrested flow pattern. Highly anisotropic nanoscale objects such as cylindrical tobacco mosaic virus particles have been shown to align with their long axis parallel to the rim of the residue due to capillary flow effects [6]. The observed orientation of the cross-β type nanofilaments suggests rather a nucleation/growth process from the interface into the gelated layer at the rim of the evaporating droplet. We note that a layer of colloidal calcium carbonate at the rim of an evaporating droplet serves also for nucleation and growth of CaCO3 crystallites [25]. Optical images of a droplet of β-amyloid (1–42) solution on a pillared Si-SHS and its detached residue are shown in Fig. 6A and B. A zoom into the rim reveals filamentous extensions which are formed as the retracting viscous interface starts wetting the pillars. μXRD probing of the filamentous extension reveals a cross-β sheet amyloidic structure with the fibre axis aligned along the filamentous direction suggesting self-assembly in an extensional flow (Fig. 6D and E)
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Fig. 6. (A): β-amyloid (1–42) solution (1 mg/ml) droplet on pillared Si-SHS. (B): Detached residue composed of a thin membrane surrounded by a coffee-ring type rim, posed to a glass capillary support. (C): Zoom into dashed rectangle in (B). (D): μXRD raster-scan with 1.5 h*0.5 v μm2 beam and 2 μm step-increments of dashed area indicated in (C). (E): Details of μXRD raster-scan (dashed rectangle in (D)). The local fibre axes of the cross-β structure are indicated by arrows. An average of several patterns from the filament is shown as inset.
Fig. 7. (A): β-amyloid (1–42) μFTIR spectrum for the Amide I/II band range obtained in about 45 s with a 6 μm beam from the inner membrane of the residue (Fig. 6B). (B): FTIR raster-scan with 5 μm step-increments plotted on image of residue. The intensity is scaled to the absorbance at the centre of the Amide I band β-sheet peak (1633 cm 1).
Simulation of the β-amyloid (1–42) X-ray fibre diffraction pattern indicates a parallel cross-β sheet structure [8]. We note that the structures of several peptides with cross-β sheet structures have been determined from single crystals to atomic resolution [26–29]. Obtaining such structures are, however, in many cases impossible in view of crystallization problems and polymorphism [30]. Complementary techniques providing information on secondary structures are therefore of interest. Indeed, one can use raster-scan μFTIR for localizing βtype secondary structures based on the comparison with published spectral features [31]. As an example we show the μFTIR spectrum collected within the membrane of the β-amyloid (1–42) residue (Fig. 6B). The strong peak at 1633 cm 1 is characteristic of a β-sheet vibration (Fig. 7A) [32]. Raster-scan μFTIR reveals a homogeneous
distribution of β-sheet secondary structure across the residue (Fig. 7B). The absorbance -and in particular of the rim- is, however, generally too high for a quantitative spectral analysis excluding the possibility of probing for differences in secondary structure across the rim. Droplet solute concentrations have therefore to be lower for μFTIR than for μXRD experiments. It is also advantageous expanding coffee-ring type residues by using superhydrophilic substrates [12] (Fig. 1C) and using D2O as solvent for in-situ experiments during droplet evaporation. Indeed, a droplet of Ac-IVD in D2O solution forms a coffee-ring type residue on a superhydrophilic substrate (Fig. 8A). The μFTIR spectrum from the rim reveals a strong β-sheet vibration peak at 1628 cm 1 (Fig. 8B).[32,33] The spectrum has been resolved into individual peaks by decomposition and Gaussian fits (Fig. 8B). The peak at 1690 cm 1 is
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
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Fig. 8. (A): SEM image of coffee-ring formation from 0.1 mg/ml Ac-IVD in a D2O solution droplet on Si3N4 with a nanofibrillar superhydrophilic PMMA layer. (B): μFTIR spectrum from the rim of the residue (red line) and fitted spectrum (blue line) based on a decomposition and Gaussian peak fits. The Amide II band position corresponds to nanofibrils from Ac-IVD in aqueous solution [33]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
at the position of an antiparallel β-sheet B1 vibration [32]. The broad band at 1645 cm 1 indicates the presence of other components such as random, α-helical and β-hairpin structures. We note that a spatial separation into α- and β-phases across a coffee-ring type residue was observed for the β-amyloid (25–35) fragment [13]. The absence of a strong Amide II band, as observed for Ac-IVD nanofibrils formed in H2O [33], is tentatively attributed to H/D exchange [34]. 4. Summary and outlook The brilliance of 3rd generation SR sources enables μXRD and μFTIR raster-scan imaging experiments. The drive for faster image generation, smaller beam sizes and smaller sample volumes is related to a continuously increasing SR source brilliance, more efficient X-ray optics and detection systems. This has to be matched by sample environments allowing confining processes in small volumes and at defined positions. The development of smart surfaces described in this article is an answer to this quest. With the upcoming upgrade of the ESRF (and other 3rd generation SR-sources) to a diffraction-limited (4th generation) SR-source, [35] the brilliance of the emitted SR will increase by about factor 30 for photon energies410 keV [36]. This and the availability of ultrafast pixel detectors [37] enabling a continuous scanning mode will significantly reduce scanning times. We exemplify this by the 101n101 pixel raster-scan diffraction image shown in Fig. 5C which took 11 h including 22% overheads due to detector readout and motor movements. For the -conservative- assumption of a linear correlation of increase in SR-source brilliance and flux in the focal spot, the total scan-time will be reduced to about 17 min assuming the same counting statistics and no overheads. Scan-times will even be smaller due to the absence of readout noise of pixel detectors.
Acknowledgements The superhydrophobic substrates were fabricated at the Istituto Italiano di Tecnologia (IIT) in Genova. IIT and ESRF provided support for the thesis projects of A.A. and G.M. References [1] Berthier J. Microdrops and digital microfluidics. Norwich N.Y.: William Andrew; 2008. [2] Deegan RD, Bakajin O, Dupont TF, Huber G, Nagel SR, Witten TA. Capillary flow as the cause of ring stains from dried liquid drops. Nature 1997;389:827–9. [3] Accardo A, Di Fabrizio E, Limongi T, Marinaro G, Riekel C. Probing droplets on superhydrophobic surfaces by synchrotron radiation scattering techniques. J Synchrotron Radiat 2014;21:643–53. [4] Sobac B, Brutin D. Structural and evaporative evolutions in desiccating sessile drops of blood. Phys Rev E 2011;84:011603. [5] Gebhardt R, Vendrely C, Burghammer M, Riekel C. Characterization of the boundary zone of a cast protein drop: fibroin β-sheet and nanofibril formation. Langmuir 2009;25:6307–11.
[6] Gebhardt R, Teulon JM, Pellequer JL, Burghammer M, Colletier JP, Riekel C. Virus particle assembly into crystalline domains enabled by the coffee ring effect. Soft Matter 2014;10:5458–62. [7] Riekel C, Burghammer M, Davies R, Gebhardt R, Popov D. Fundamentals of non-crystalline diffraction with microfocus techniques. In: García-Gutiérrez M, Nogales A, Gómez M, Ezquerra TA, editors. Applications of Synchrotron Light to Non-Crystalline Diffraction in Materials and Life Sciences. Heidelberg: Springer; 2008. [8] Jahn TR, Makin OS, Morris KL, Marshall KE, Tian P, Sikorski P, et al. The Common architecture of cross-β amyloid. J Mol Biol 2010;395:717–27. [9] De Angelis F, Gentile F, Mecarini F, Das G, Moretti M, Candeloro P, et al. Breaking the diffusion limit with super-hydrophobic delivery of molecules to plasmonic nanofocusing SERS structures. Nat Photonics 2011;5:683–8. [10] Marinaro G, Accardo A, Angelis FD, Dane T, Weinhausen B, Burghammer M, et al. A superhydrophobic chip based on SU-8 photoresist pillars suspended on a silicon nitride membrane. Lab Chip 2014;14:3705–9. [11] Accardo A, Gentile F, Mecarini F, De Angelis F, Burghammer M, Di Fabrizio E, et al. In situ X-ray scattering studies of protein solution droplets drying on micro- and nanopatterned superhydrophobic PMMA surfaces. Langmuir 2010;26:15057–64. [12] Accardo A, Gentile F, Mecarini F, De Angelis F, Burghammer M, Di Fabrizio E, et al. Ultrahydrophobic PMMA micro- and nano-textured surfaces fabricated by optical lithography and plasma etching for X-Ray diffraction studies. Microelectron Eng 2011;88:1660–3. [13] Accardo A, Shalabaeva V, Cotte M, Burghammer M, Krahne R, Riekel C, et al. Amyloid β peptide conformational changes in the presence of a lipid membrane system. Langmuir 2014;30:3191–8. [14] De Angelis F, Das G, Candeloro P, Patrini M, Galli M, Bek A, et al. Nanoscale chemical mapping using three-dimensional adiabatic compression of surface plasmon polaritons. Nat Nanotechnol 2010;5:67–72. [15] Gentile F, Moretti M, Limongi T, Falqui A, Bertoni G, Scarpellini A, et al. Direct Imaging of dna fibers: the visage of double helix. Nano Lett 2012;12:6453–8. [16] Hauser CAE, Deng RS, Mishra A, Loo YH, Khoe U, Zhuang FR, et al. Natural trito hexapeptides self-assemble in water to amyloid beta-type fiber aggregates by unexpected alpha-helical intermediate structures. Proc Natl Acad Sci USA 2011;108:1361–6. [17] Gentile F, Das G, Coluccio ML, Mecarini F, Accardo A, Tirinato L, et al. Ultra low concentrated molecular detection using super hydrophobic surface based biophotonic devices. Microelectron Eng 2010;87:798–801. [18] Miele E, Malerba M, Dipalo M, Rondanina E, Toma A, De Angelis F. Controlling wetting and self-assembly dynamics by tailored hydrophobic and oleophobic surfaces. Adv Mater 2014;26:4179–83. [19] Als-Nielsen J, McMorrow D. Elements of modern X-ray physics. Chichester, UK: John Wiley & Sons, Ltd; 2011. [20] Riekel C. New avenues in X-ray microbeam experiments. Rep Prog Phys 2000;63:233–62. [21] Riekel C, Burghammer M, Popov D. Protein micro- and nanocrystallography using synchrotron radiation. In: Nicolini C, editor. Synchrotron Radiation and Structural Proteomics. Singapore: Pan Stanford Publishing; 2012. p. 3–30. [22] Schroer CG, Boye P, Feldkamp JM, Patommel J, Schropp A, Schwab A, et al. Hard X-Ray scanning microscopy with fluorescence and diffraction contrast. J Phys Conf Ser 2009;186. [23] Carr GL. Resolution limits for infrared microspectroscopy explored with synchrotron radiation. Rev Sci Instrum 2001;72:1613–9. [24] Crick FHC. The packing of α-helices: Simple coiled-coils. Acta Crystallogr 1953;6:689–97. [25] Accardo A, Burghammer M, Di Cola E, Reynolds M, Di Fabrizio E, Riekel C. Calcium carbonate mineralization: X-ray microdiffraction probing of the interface of an evaporating drop on a superhydrophobic surface. Langmuir 2011;27:8216–22. [26] Colletier JP, Laganowsky A, Landau M, Zhao M, Soriaga AB, Goldschmidt L, et al. Molecular basis for amyloid-β polymorphism. PNAS 2011;108: 16938–16943. [27] Nelson R, Sawaya MR, Balbirnie M, Madsen AØ, Riekel C, Grothe R, et al. Structure of the cross- β spine of amyloid-like fibrils. Nature 2005;435:773–8.
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i
G. Marinaro et al. / Optics and Lasers in Engineering ∎ (∎∎∎∎) ∎∎∎–∎∎∎ [28] Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Siever SA, Apostol MI, et al. Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature 2007;447:453–7. [29] Wiltzius JJ, Sievers SA, Sawaya MR, Cascio D, Popov D, Riekel C, et al. Atomic structure of the cross-β spine of islet amyloid polypeptide (amylin). Protein Sci 2008;17:1467–74. [30] Paravastu AK, Leapman RD, Yau WM, Tycko R. Molecular structural basis for polymorphism in Alzheimer's β-amyloid fibrils. PNAS 2008;105:18349–54. [31] Miller L, Bourassa MW, Smith RJ. FTIR spectroscopic imaging of protein aggregation in living cells. Biochim Biophys Acta 2013;1828:2339–46. [32] Chirgadze YN, Nevskaya NA. Infrared spectra and resonance interaction of amide-I vibration of the antiparallel-chain pleated sheet. Biopolymers 1976;15:607–25. [33] Mishra A, Chan KH, Reithofer MR, Hauser CAE. Influence of metal salts on the hydrogelation properties of ultrashort aliphatic peptides. RSC Adv 2013; 3:9985–93.
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[34] Dzwolak W, Loksztejn A, Smirnovas V. New insights into the self-assembly of insulin amyloid fibrils: an H–D exchange FT-IR study. Biochemistry 2006;45:8143–51. [35] Eriksson M, van der Veen F, Quitman C. Diffraction-limited storage rings—a window to the science of tomorrow. J Synchrotron Radiat 2014;21:837–42. [36] ESRF, White Paper: Upgrade Programme, Phase II (2015–2019), 〈www.esrf.fr/ files/live/sites/www/files/about/upgrade/documentation/whitepaper-upgra de-phaseII.pdf〉; 2014. [37] Denes P, Schmitt B. Pixel detectors for diffraction-limited storage rings. J Synchrotron Radiat 2014;21:1006–10.
Please cite this article as: Marinaro G, et al. Probing droplets with biological colloidal suspensions on smart surfaces by synchrotron radiation micro- and nano-beams. Opt Laser Eng (2015), http://dx.doi.org/10.1016/j.optlaseng.2015.03.004i