Article pubs.acs.org/Langmuir
Sizing of Metallic Nanoparticles Confined to a Microfluidic Film Applying Dark-Field Particle Tracking Christoph Haiden,*,†,‡ Thomas Wopelka,‡ Martin Jech,‡ Franz Keplinger,† and Michael J. Vellekoop§ †
Institute of Sensor and Actuator Systems, Vienna University of Technology, Gusshausstrasse 27-29, A-1040 Vienna, Austria Austrian Center of Competence for Tribology, Viktor Kaplan-Strasse 2, A-2700 Wiener Neustadt, Austria § Institute for Microsensors, -Actuators & -Systems, University of Bremen, MCB, Otto-Hahn-Allee 1, D-28359 Bremen, Germany ‡
S Supporting Information *
ABSTRACT: We present Brownian motion-based sizing of individual submicron and nanoparticles in liquid samples. The advantage of our approach is that particles can freely diffuse in a 10 μm thin liquid film and are therefore always within the focal depth of a low numerical aperture objective. Particles are visualized with darkfield microscopy, and the resulting diffraction-limited spots are tracked over a wide field of view of several hundred micrometers. Consequently, it is ascertained that long 2D trajectories are acquired, which leads to significantly increased particle sizing precision. The hydrodynamic diameters of metal particles with nominal sizes ranging from 70 to 200 nm in aqueous solution were determined by tracking for up to 2 min, and it was investigated if the diffusion characteristics were influenced by the proximity of substrates. This was not the case, and the estimated diameters were in good agreement with the values obtained by electron microscopy, thus validating the particle sizing principle. Furthermore, we measured a sample mixture to demonstrate the distinction of close particle sizes and performed the conjugation of a model protein (BSA) on the nanoparticle surface. An average increase in the radius of 9 nm was determined, which corresponds to the size of the BSA protein.
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INTRODUCTION Detection and characterization of micro- and nanoparticles (NPs) in fluids plays an important role in many industrial, environmental, and scientific areas.1,2 Classical analytical methods like electron microscopy, atomic force microscopy, or small-angle X-ray scattering3 are well-established but require complex and expensive setups and are, with few exceptions,4 usually not suitable for measurements in liquids or online particle detection. Therefore, extensive work has been carried out over the last years to develop and improve a wide variety of sensing principles and sensor systems for particle detection purposes. Many devices are based on electrical, mechanical, and magnetic principles,5−7 but depending on the properties of the particles or the liquid medium it is not always possible to apply one of them. Optical detection methods, however, can be used to detect and characterize particles in liquids over a wide range of sizes and materials. Bright-field video microscopy and subsequent 2D tracking of individual particles have become very popular and are often used for the acquisition of directed or random particle movement.8−10 With high magnification objectives and, therefore typically high numerical aperture (NA), it is possible to optically detect and track particles down to the diffraction limit.11 To characterize smaller particles in liquid, principles based on the scattering of light are routinely applied, particularly static and dynamic light scattering (SLS, DLS).12 Unfortunately, these ensemble methods cannot directly provide the number of particles and suffer from a reduced peak © 2014 American Chemical Society
resolution, which complicates the measurements of multimodal samples.13 More recently, nanoparticle tracking analysis (NTA) was introduced by NanoSight (Amesbury, U.K.) as a cost-efficient alternative to ensemble light-scattering instruments.2,13−17 It is based on the visualization of particles in the 10−1000 nm range using dark-field illumination and subsequent 2D tracking of their Brownian motion to determine the size. Besides NanoSight instruments, also standard microscopes in the dark-field configuration have been used for video microscopy and 2D tracking of particles.18−20 However, in sample cells with a thickness along the optical axis much larger than the focal depth, particles can easily diffuse out of focus. Therefore, only a small number of trajectory positions can be acquired, in turn yielding broader size distributions. For instance, in a typical NTA measurement, the vast majority of tracks comprised fewer than 20 displacements.16 As a consequence, particle sizing based on shorter tracking times has to rely on an ensemble average of many short trajectories to provide meaningful results for the size distribution.2,14,16,18 Despite some mathematical efforts to improve short trajectory measurements,16,21,22 it would be beneficial to get the complete trajectory information. In our setup, we utilize dark-field video microscopy to visualize plasmonic NP freely diffusing in a 10 μm thick water Received: April 29, 2014 Revised: July 10, 2014 Published: July 18, 2014 9607
dx.doi.org/10.1021/la5016675 | Langmuir 2014, 30, 9607−9615
Langmuir
Article
Figure 1. (a) Schematic of the dark-field arrangement and the nanoparticle sample confined to a thin film between the glass substrates. (b) Snapshots of scattering 90 nm AuNP confined to a 10 μm film, recorded with 10×/0.3 and 50×/0.45 objectives (top, middle). The magnification changer of the microscope was used to adjust the resulting image magnification so that pixel sizes were 2.5 px/μm and 6.1 px/μm, respectively. An image of NP confined to a 200 μm film shows both in-focus and out-of-focus particles (bottom). (c) Enlarged image of NPs after bandpass filtering as employed in the particle-tracking process.
compare the diffusive characteristics, we used thicker films as well (200 μm). Furthermore, a mixture of two particle samples (bimodal size distribution) and AuNP with surface-conjugated bovine serum albumin (BSA) were sized.
film between two glass substrates (Figure 1). Unlike for large sample chambers, this small film height keeps all particles in the focal depth of a low-NA objective over extended observation times. The diffusive particle movement is then acquired by tracking, and individual sizes can be calculated. The width of a size distribution, and hence the uncertainty in size measurement, is narrowing down proportionally to 1/√N, where N is the number of displacements.23−25 Although tracking of extraordinarily long trajectories of diffusing fluorescent or scattering NP has been done previously,25,26 it is limited to the investigation of only a single particle at a time and requires a much more sophisticated setup. While in this case only a single, true diffusion coefficient is determined, in typical particle sizing applications, and also in this work, the size distribution of multiple particles in a sample is estimated. With a precise measurement method, the true particle size distribution of finite width can be reproduced, while a less precise method would yield a broader distribution. Importantly, narrow distributions permit the discrimination of peaks in polydisperse samples that would otherwise overlap. There are only few diffusion studies of samples constrained to a liquid film of several micrometers. They have dealt with microparticles27 or rod-shaped species with lengths on the order of the film height,28,29 where particles were subject to rather strong hydrodynamic interactions because of their size and distance to the substrates. Even more pronounced effects on diffusion have been observed in extremely thin films compared with NP dimensions (
