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Techniques for Accurate Sizing of Gold Nanoparticles Using Dynamic Light Scattering with Particular Application to Chemical and Biological Sensing Based on Aggregate Formation Tianyu Zheng,† Steven Bott,‡ and Qun Huo*,† †
NanoScience Technology Center and Department of Chemistry, University of Central Florida, 12424 Research Parkway Suite 400, Orlando, Florida 32826, United States ‡ Nano Discovery Inc., 3259 Progress Drive Suite 141, Orlando, Florida 32826, United States
ABSTRACT: Gold nanoparticles (AuNPs) have found broad applications in chemical and biological sensing, catalysis, biomolecular imaging, in vitro diagnostics, cancer therapy, and many other areas. Dynamic light scattering (DLS) is an analytical tool used routinely for nanoparticle size measurement and analysis. Due to its relatively low cost and ease of operation in comparison to other more sophisticated techniques, DLS is the primary choice of instrumentation for analyzing the size and size distribution of nanoparticle suspensions. However, many DLS users are unfamiliar with the principles behind the DLS measurement and are unware of some of the intrinsic limitations as well as the unique capabilities of this technique. The lack of sufficient understanding of DLS often leads to inappropriate experimental design and misinterpretation of the data. In this study, we performed DLS analyses on a series of citrate-stabilized AuNPs with diameters ranging from 10 to 100 nm. Our study shows that the measured hydrodynamic diameters of the AuNPs can vary significantly with concentration and incident laser power. The scattered light intensity of the AuNPs has a nearly sixth order power law increase with diameter, and the enormous scattered light intensity of AuNPs with diameters around or exceeding 80 nm causes a substantial multiple scattering effect in conventional DLS instruments. The effect leads to significant errors in the reported average hydrodynamic diameter of the AuNPs when the measurements are analyzed in the conventional way, without accounting for the multiple scattering. We present here some useful methods to obtain the accurate hydrodynamic size of the AuNPs using DLS. We also demonstrate and explain an extremely powerful aspect of DLSits exceptional sensitivity in detecting gold nanoparticle aggregate formation, and the use of this unique capability for chemical and biological sensing applications. KEYWORDS: gold nanoparticle, dynamic light scattering, particle sizing, chemical sensing, biosensing
1. INTRODUCTION Gold nanoparticles (AuNPs), also known as gold colloids, are attracting considerable interest in the nanotechnology field due to their unique chemical and optical properties, and their application in chemical and biological sensing, biomolecular imaging, in vitro diagnostics, cancer therapy, catalysis, and many other areas.1−8 One of the best known optical properties of AuNPs is their enhanced light absorption and scattering properties in the surface plasmon resonance (SPR) wavelength region. The surface plasmon resonance wavelength of AuNPs is strongly dependent on the size of individual AuNPs and the aggregation state of the AuNPs.9 The enhanced surface plasmon resonance effect of AuNPs results in greatly increased scattered light intensities compared to particles of the same size and shape but composed of materials not exhibiting surface © 2016 American Chemical Society
plasmon resonance. Under white light illumination, AuNPs change color from burgundy red to purple or blue with increased nanoparticle size, or with AuNPs aggregate formation. Because of the intense color (due to enhanced absorption) of AuNPs, the color change of AuNPs has been used widely as a sensing platform for chemical and biological target detection.10−13 The enhanced light absorption and scattering of AuNPs has also been applied to in vivo imaging and cancer therapy.4,14−17 After being delivered into biological cells, the large amount of heat released from the AuNPs upon absorption of light can lead to the disruption of cell membranes Received: June 8, 2016 Accepted: July 29, 2016 Published: July 29, 2016 21585
DOI: 10.1021/acsami.6b06903 ACS Appl. Mater. Interfaces 2016, 8, 21585−21594
Research Article
ACS Applied Materials & Interfaces
results, some important considerations concerning DLS experimental design are presented and discussed. Additionally, using suspensions of mixed 20 and 100 nm AuNPs with different number concentrations as illustrative examples, we demonstrate the exceptional power of DLS for detecting gold nanoparticle aggregate formation, and the significant potential of this capability for sensing applications. Although this study is focused on AuNPs, the knowledge and methods discussed here may be applied to the study of and application to other nanoparticle materials.
and death of the cancer cells. Because the light absorption of AuNPs is size-dependent, the therapeutic effect of AuNPs is also nanoparticle size-dependent. Recently, our group and others have developed an AuNP-enabled sensing platform which is based on AuNP aggregate formation using dynamic scattered light (DLS) in an assay suspension.18,19 Upon binding with specific chemical or biological targets, AuNP probes can form clusters and aggregates which, consequently, lead to an increased average particle size that is readily measurable by DLS. This platform has so far been applied successfully to quantitative detection and analysis of a wide range of chemical and biological targets, including proteins, DNAs and RNAs, viruses and viral pathogens, small chemicals, and toxic metal ions, with excellent sensitivity.20−34 In these applications, the quantitative detection of target chemical and biological agents is based on measuring the size of individual and clustered AuNPs. Knowledge of techniques for obtaining accurate measurements of AuNP size is critical in guiding the design of improved sensors, probes and assays to obtain optimum performance and quality. DLS is an analytical technique used routinely to analyze the size and size distribution of particles with diameters ranging from a few nm to a few microns.35 DLS detects the scattered light intensity fluctuations caused by the Brownian motion of the particles suspended in a liquid. From an analysis of the autocorrelation function of the scattered light intensity fluctuations, the diffusion coefficient of suspended particles is calculated and the hydrodynamic size of the particles is determined by applying Stokes−Einstein equation.36 DLS reveals not only the average size but also the size distribution of the particles.37 Modern commercial DLS instruments are relatively low cost, easy to operate, and output the size data directly from the raw measurement without the need for further mathematical processing. The size range of most nanoparticle materials falls exactly within the ideal detection range of DLS, from a few nanometers to a few micrometers. DLS has become an essential tool in nanotechnology research. However, if not used properly, DLS can lead to confusing and misleading results. There are several problems that researchers using DLS often encounter: (1) the DLS-measured average size of a nanoparticle material is not the same and may even differ widely from the size determined by other techniques such as scanning or transmission electron microscopy; (2) the size distribution of a multi modal nanoparticle sample may not agree with that determined by other methods; (3) the DLSreported average size of a nanoparticle suspension may have poor reproducibility, and finally (4) the way in which nanoparticle aggregate formation affects the average size and size distribution is not straightforward. The resolution of these problems lies in developing a better understanding of the physical principles of DLSespecially the difference between the scattered light intensity-weighted particle size measurements of DLS versus mass or number-weighted particle size measurements used by other common measurement techniquesand in learning the particular experimental design and sample preparation techniques required for DLS measurements. In this study, we performed DLS measurements of citratestabilized AuNPs with diameters of 10, 20, 40, 60, 80, and 100 nm. We demonstrate how the scattered light intensity of the AuNPs can vary substantially with particle size, and how the particle concentration and incident laser beam power can affect the reported hydrodynamic diameter. On the basis of these
2. MATERIALS AND METHODS 2.1. Chemicals and Materials. Citrate ligand-capped AuNPs with various diameters (10, 20, 40, 60, 80, and 100 nm) were purchased from Ted Pella Inc. (Redding, CA). The AuNPs were manufactured by British Biocell International (BBI), and have been adopted by the National Institute of Standard and Technology (NIST) as reference materials. Corresponding concentrations of each AuNP sample are 5.70 × 1012, 7.00 × 1011, 9.00 × 1010, 2.60 × 1010, 1.10 × 1010, and 5.60 × 109 particle/mL for the 10, 20, 40, 60, 80, and 100 nm AuNPs, respectively. For concentration-dependent studies, each original AuNP sample was diluted to 1/2, 1/5, and 1/10 fold by mixing an appropriate volume of nanopure water with the AuNP suspension. The nanopure water has a resistivity of 18 MΩ·cm (Barnstead Nanopure Diamond Purifier: model # D11931). Each AuNP sample was also concentrated 2×, 5×, and 10× fold by centrifuging followed by removal of appropriate amount of supernatant water. 100 uL of AuNP suspension with different sizes and concentrations was placed in a cuvette (Cat. No. 67.758, Sarstedt, Germany) for DLS measurement. The cross section of the cuvette where the incident light beam enters and the scattering light is detected has a dimension of 2 × 10 mm. The incident light beam enters the center of the cuvette through the longer dimension, 10 mm, and the scattered light is detected from the side of the cuvette at 90°. The optical path length for the scattered light is about 1 mm. 2.2. DLS Measurements. DLS analysis of all AuNP suspensions were performed using a Zetasizer Nano ZS90 DLS system equipped with a green laser (532 nm, 4 mW) and an avalanche photodiode detector (APD) (quantum efficiency >50% at 532 nm) (Malvern Instruments Ltd., England). The measured scattering light intensity is displayed as photon count rate with a unit of kilo count per second (kcps). Due to the different scattered light intensities of AuNPs with different sizes, the power of the incident laser beam needs to be adjusted to obtain an optimum photon count rate. The incident laser power was adjusted to specific levels as needed by using a built-in attenuator. The attenuation level is indexed by an attenuation number corresponding to a particular attenuation level. The attenuation numbers 11, 10, 9, and 8 correspond to laser powers 4, 1.2, 0.4, and 0.12 mW, respectively. The Malvern DTS 5.10 software was applied to process and analyze the data. All average particle sizes reported here are based on scattered light intensity weighted averages. For each sample suspension, two DLS measurements were made with a fixed run time of 20 s. The scattering angle was set at 90°. In addition to the Zetasizer Nano ZS90 system, we also used a Zetasizer Nano ZS DLS system (Malvern Instruments Ltd., England) for a comparison study. The Nano ZS system is equipped with a 4 mW red laser (633 nm) and a detection angle of 173°. Both systems use the same software for data collection and processing. 2.3. Transmission Electron Microscopy Study. The TEM images of 100 nm AuNPs were obtained by using JEOL JEM-1011 transmission electron microscopy with an accelerating voltage of 100 kV. TEM sample grids were prepared by depositing 2 uL AuNPs suspension onto carbon coated copper grids and vacuum-dried. The average diameter was determined by analyzing 150 particles from the TEM images using ImageJ software developed by the National Institutes of Health. 21586
DOI: 10.1021/acsami.6b06903 ACS Appl. Mater. Interfaces 2016, 8, 21585−21594
Research Article
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3. RESULTS AND DISCUSSIONS Before discussing the results of individual experiments, a few clarifications need to be made on the DLS data presented here. From a DLS measurement, one can obtain the size distribution curve as well as the average particle size of the sample.37 There are three types of size distributions commonly used in DLS: intensity-, volume-, and number-weighted size distributions. The direct result of DLS measurement is expressed as an intensity-weighted size distribution. Then on the basis of the refractive index of the particle being studied, the intensityweighted size distribution can be converted to number- or volume-weighted distribution. However, this conversion can be problematic with AuNPs. Because the refractive index of AuNPs are dependent on size and also on surface chemistry, if the precise size dependence of the refractive index of the AuNPs and size and refractive index of the surface ligands is unknown, the conversion of intensity-weighted to volume or number-weighted distributions will sometimes result large errors. For this reason, we suggest that for AuNP DLS analyses, only intensity-weighted size distributions and average particle sizes be reported and compared from study to study. Another important parameter obtained from DLS measurement is the zaverage particle size (Dz).38 The z-average particle size is obtained from the intensity-weighted distribution curve according to the following formula: Dz =
nm. Because citrate ligand is a very small molecule, we can assume that the core diameter determined from TEM images is very close to the diameter of the AuNPs. Overall, the TEM imaging confirmed the high quality and monodispersity of the AuNP-100 nm product. The Malvern ZS90 DLS system is equipped with a 4 mW He−Ne green laser (532 nm). The power of the incident laser beam can be adjusted using different attenuator numbers.39 For each sample, the appropriate attenuation number of the laser was selected to bring the scattered light intensity, i.e., the photon count rate, into the appropriate range suggested in the Malvern user’s manual (a few hundred kcps, kilo counts per second). This adjustment, which is sometimes overlooked, is essential to bring the scattered photon level into the range that prevents saturation of the APD detector. If the detector is saturated, then a size measurement result will still be reported by the instrument, but the saturation causes the detector to miss counts (photons) and results in systematic errors in the reported particle size. In fact, even if the scattered light level is within the suggested range, it is good practice to vary the attenuation level and check that the reported kcps responds linearly to the level: for example, cutting the laser power from 4 mW to 1.2 mW (attenuation number 11 to 10) should result in a 3.3X reduction in photon count rate. This quick test ensures that the scattered light level is in the linear range of the detector. If this test is omitted, particularly with many highly scattering AuNP samples, then the detector may be highly saturated and therefore reporting a wildly erroneous count level which may appear to fall in the suggested count range. However, varying the attenuation level to check for linear response will easily detect the detector saturation problem and the attenuation level should be increased until the APD responds linearly to laser power indicating that it is out of the detector saturation range. When we placed the as-received AuNP-100 nm suspension for DLS measurement using attenuation number 11, the photon count rate exceeds 6000 kcps. This count rate greatly exceeds the threshold (1000 kcps according to the user’s manual) of the detector, leading to saturation of the detector. To avoid this problem, an attenuation number 8 was selected. With this attenuation number, the laser power is reduced to 3% (0.12 mW) of the full laser power, and the scattered light intensity of the nanoparticle suspension is reduced to 800 kcps, within the acceptable threshold of the detector. Figure 2 is the DLS analysis results of AuNP-100 nm suspensions at different concentrations. All the measurements were made at the attenuation level 8 and in each case the laser power was varied, as described above, to check that the count level was below detector saturation. Figure 2A is the intensity-weighted size-distribution curve. Figure 2B,C shows the reported z-average hydrodynamic diameters and the average scattered light intensities, respectively, of the AuNP-100 nm suspensions of varying concentration. At a concentration of 5.6 × 109 per mL or lower, the AuNPs show a relatively uniform and monodisperse size distribution. However, at the next two higher concentrations measured, the distribution curves split into two peaks, one peak at larger size around 100 nm, and another peak at around 15 nm. At the highest concentration, only a single peak with a size around 8 nm was observed from the distribution curve. Upon dilution of the concentrated samples to the original concentrations, the average diameter and size distribution curve returns to what was observed from the original
∑ Si ∑
() Si Di
(1)
where Si is the scattering light intensity of particle i at the scattering angle used, and Di is the diameter of particle i. 3.1. Effect of Nanoparticle Concentration on a DLS Measurement. We first examined the DLS-measured size of an AuNP product with a nominal diameter around 100 nm (AuNP-100 nm). Before conducting DLS analysis, transmission electron microscopy (TEM) was used to confirm the size and size distribution of the AuNPs in dry state. Multiple sample areas were scanned, and a representative TEM image of AuNPs-100 nm is shown in Figure 1. From the direct analysis of approximately 150 particles individually, the actual average core diameter of these nanoparticles was determined to be 99
Figure 1. A representative transmission electron microscope image of AuNPs with an average diameter of 99 nm. 21587
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concentration of AuNP-100 nm indicating that no irreversible physical change (such as aggregate formation) occurred during the concentration and dilution process. These results illustrate the sometimes anomalous size distribution curves reported by DLS. In this case, the anomalous results were due to the multiple scattering effect as discussed further below. From Figure 2B, it can be seen that below a certain concentration, the measured average diameter is rather consistent, around 100 nm, in agreement with the TEM analysis. However, with increased nanoparticle concentration, the measured average diameter decreased from ∼100 nm to ∼50 nm, and further down to ∼3 nm for the most concentrated sample! This dramatic error in the measured hydrodynamic diameter of AuNP-100 nm from DLS is largely a result of the multiple scattering effect.36 DLS measurements are based on measuring the time scale of light fluctuations at the detector caused by the random diffusion or movement of suspended particles in a small “scattering volume” within a sample cuvette. The scattering volume is defined as that volume, within the sample and illuminated by the laser beam, containing particles whose scattered light is directly detected by the detector. Small particles diffuse rapidly and therefore result in rapid intensity fluctuations at the detector; large particles diffuse more slowly with resultant longer time scale scattered light fluctuations at the detector. Ideally in a DLS measurement, the concentration of the suspension being measured is dilute enough that light scattered from a diffusing particle in the scattering volume makes its way to the detector without interference. Multiple scattering refers to the process by which a photon of light is scattered from a diffusing particle in the scattering volume and then is rescattered by one or more particles before reaching the detector. The rescattering process changes the time scale of the light fluctuations at the detector, and generally results in underreporting of the true particle size. The amount of multiple scattering is proportional to a parameter called the photon mean free path (“MFP”). The MFP is the average distance that a scattered photon travels before being rescattered by another particle in a particular sample. In a dilute suspension the MFP is usually large but at high concentrations, the mean free path will be much shorter. If the MFP is much longer than the distance through the suspension from the scattering volume to the detector, then no multiple scattering will occur. As soon as the MFP is close to or less than that distance, multiple scattering becomes increasingly significant. The MFP is related to particle number concentration and a parameter called the particle “scattering cross section”, Csca.40−42 MFP = Figure 2. (a) Intensity-weighted size distribution curve of 100 nm AuNPs at different concentrations (Legends represents the dilution or concentration factor of the AuNP solutions relative to the original AuNP solution). (b) The corresponding z-average hydrodynamic diameter; (c) represents the experimentally measured and predicted using the Beer−Lambert lawscattered light intensity of AuNP-100 nm suspensions. All data were obtained under an attenuation of 8 at a 3% maximum laser power provided by the instrument. Each measurement was conducted in duplicate and the error bars in the plot represent the measurement standard deviation. (d) Shows the relative contributions of particle absorption and scattering to the overall extinction cross section of the particles.
k concentration × Csca
(2)
where k is a proportionality constant which depends on the experimental geometry. Csca is a scattered light attribute of a particle which depends of the particle size, shape and refractive index. Csca has units of area and quantifies the degree to which the particle is likely to scatter a nearby photon. For spherical particles and for a few other shapes, it is readily calculable via on line programs such as Mieplot.exe.43,44 As noted earlier, the refractive index of AuNPs is a function of particle size because of the surface plasmon resonance phenomenon.9,44,45 Nevertheless, for these purposes, use of the bulk material refractive 21588
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quantifies the likelihood a photon of light near a particle will be absorbed or scattered by the particle. The exponential falloff in scattered light intensity at higher concentrations dominates over the linear rise with concentration at lower concentrations, leading to the observed form of the measured scattered intensity versus concentration for the 100 nm AuNPs in Figure 2C. If we assume that α is small enough at the lowest concentration measured that Io equals Isa good assumption since Is increases almost linearly between the two lowest concentration measurementsthen α can be found by using the scattered light intensity at the lowest concentration and at any higher concentration by taking advantage of the fact that both Io and α are directly proportional to concentration. Using the scattered light intensities versus concentration shown in the “Measured” data points in Figure 2C an average value of α was found, based on the discussion above. Then, using the Beer− Lambert law, a theoretical scattered intensity versus concentration graph was calculated as shown in 2C. The agreement between the measured and calculated scattered intensities lends credence to this simple Beer−Lambert model. Plots of scattered intensity versus concentration, like those of hydrodynamic diameter, Dz, versus concentration give an indication of where the multiple scattering might start becoming a problem with increasing concentration. However, since Dz starts falling only when multiple scattering is becoming significant, but scattered intensity is affected also by particle light absorption and/or multiple scattering, Dz is a more definitive indication of multiple scattering. Figure 2D shows the relative contribution to the total extinction cross section of the particles of light scattering and absorption for AuNPs as a function of particle size, calculated by Mieplot, again using the bulk material refractive index for gold at the 532 nm laser wavelength. For the 100 nm AuNPs, the scattering and absorption have about equal contributions; for smaller AuNPs, the absorption cross section is much larger than the scattering cross section. Because of this, for smaller AuNPs, the scattered light versus concentration curve may peak substantially before multiple scattering becomes a problem, while with larger particles, the scattered light peak versus concentration may be a good indicator of multiple scattering. In any case, at concentrations at which the scattered intensity starts rising less than linearly with increased concentration, concern about multiple scattering may be prudent. 3.2. Multiple Scattering Effect of AuNPs of Different Sizes. The amount of light scattered from a particle is strongly dependent on its size. Within Rayleigh scattering range, the scattering light intensity is proportional to the sixth power of the diameter of the particle.44 Smaller particles scatter light less intensely than larger particles. In addition to the 100 nm AuNPs, we also performed DLS measurements of a series of AuNPs with smaller diameters as a function of different concentrations. Figure 3A,B show the average particle size and scattered light intensity from AuNP with diameters of 20, 40, 60, 80, and 100 nm. It should be noted that due to decreasing scattered light intensity of smaller nanoparticles, the laser power (via attenuation number) must increase accordingly to obtain reliable DLS measurements. The attenuation numbers used for 20, 40, 60, and 80 nm particles were 11, 10, 9, and 8 respectively. From 3A and 3B, it can be seen that multiple scattering effect appears with the 80 and 100 nm particles, but there is no clear, consistent decline in particle diameter versus concentration for the smaller AuNPs over the concentration
index for the AuNPs in the 10−100 nm size range provides a reasonably good estimate of their scattering properties. For a system of particles of different sizes, such as AuNPs, eq 2 is useful in estimating the concentration at which multiple scattering will occur for particles of different sizes. First, the multiple scattering limit at one particular size, usually the largest size of interest, must be empirically determined by measuring the apparent hydrodynamic size as a function of number concentration. The proportionality constant, k, will be independent of particle size when the particle sizes are substantially smaller than the wavelength of the light used in the scattering measurement. MFP is set to equal to the known distance through the cuvette from the scattering volume to the detector. For the Malvern ZS90 system used in our study, this distance is 1 mm. Csca for the known particle size is calculated using Mieplot. With the number concentration at which the multiple scattering becomes significant, k is determined from a plot such as that shown in Figure 2B. As is evident from the figure, the reported hydrodynamic diameter starts falling at a concentration of 1.12 per μm3 (1.12 × 10−12 per mL). Using Mieplot.exe, Csca is found to be 1.93 μm2 for the 100 nm AuNPs, and k is determined to be 0.22 (dimensionless). k can then be used to estimate the multiple scattering limit for other AuNP particle sizes as is discussed further below. Although many of the commercial DLS instruments are not designed specifically to make accurate scattered light intensity measurements, some important information can be gleaned from the intensity measurements about why the 100 nm AuNP samples are producing such anomalous results. With this in mind, the scattered light intensities of the AuNP-100 nm samples of various concentrations were recorded (Figure 2C). Below 5.60 × 109 particle/mL, the scattered light intensity increases almost linearly with increasing nanoparticle concentration. After reaching a peak point, the scattering light intensity actually decreases with increasing nanoparticle concentration. The linear increase and then decrease in scattered light intensity with increasing concentration is primarily caused by two opposing factors. The increase in concentration means that the number of particles in the scattering volume increases and the amount of scattered light generated in that volume increases linearly with concentration (at least until very high concentrations are reached at which point interference effects make the scattering pattern more complex). However, with increasing concentration, some of the laser light is absorbed or scattered by the sample before the beam reaches the scattering volume, thereby decreasing the laser intensity at the scattering volume and reducing the amount of light available for scattering in the scattering volume. Likewise, light scattered from the scattering volume is absorbed or rescattered by the medium on the way to the detector, thereby also decreasing the amount of scattered light reaching the detector. The reduction in laser intensity at the scattering volume due to the partial extinction of the beam by light scattering and absorption by the AuNPs will follow a simple Beer−Lambert law relationship:46 Is = Io × e−α
(3)
where Io and Is are the concentration-dependent laser intensities at the scattering volume and at the detector, respectively, and α is a coefficient which is proportional to the product of extinction cross section of the particle, the particle number concentration and the total distance the laser and scattered light travel through the sample to the detector. The extinction cross section is a particle characteristic which 21589
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scattering light intensity of the sample suspensions should be monitored to check for multiple scattering. The method, using eq 2 as discussed above, can be used to reduce the amount of measurements needed to characterize the nanoparticle system. As demonstrated in our study here, a most suitable sample for DLS measurement condition should be a sample that has the concentration that gives the highest scattering light intensity, but below the concentration that multiple scattering effect starts to occur. 3.3. Possible Use of Backscattering Angle Detection to Reduce the Multiple Scattering Effect on DLS Measurement. The multiple scattering effects may be reduced by changing the geometry of the DLS measurement to minimize the distance through the cuvette that light must travel from the scattering volume and the detector. Almost all commercially available DLS instruments in the market use either a 90 deg angle detection, or a quasi-backscattering angle detection.47 The difference between these two detection geometries is illustrated in Figure 4A,B. As shown in the
Figure 3. Hydrodynamic diameter (a) and scattered light intensity (b) of 20, 40, 60, 80, and 100 nm AuNPs at different concentrations. The attenuation numbers applied for 20, 40, and 60 nm AuNP measurement were 11, 10, and 9, respectively. Attenuation 8 was used for both 80 and 100 nm AuNP study. (c) Shows the predicted concentration limit beyond which multiple scattering occurs.
ranges tested. The measured average particle size remains relatively constant for these smaller particles. Figure 3C shows the estimated multiple scattering limit for the AuNPs of different sizes, based on eq 2 and the discussion above. As expected, the estimated multiple scattering limits for the 80 and 100 nm AuNPs correspond to the concentrations at which the multiple scattering appears in Figure 3A. The predicted concentration limits for the smaller particles fall beyond the tested concentration ranges. The scattered intensity versus concentration graphs for the smaller particles may provide an “early warning” indication of the onset of multiple scattering. Many researchers synthesize their own nanoparticles in laboratories. Without knowing the size and size-dependent scattered light properties and the specific concentrations of the samples, it is necessary to analyze the as-made nanoparticle materials at different relative concentrations to determine the multiple scattering limits. Both the average particle size and the
Figure 4. Illustration of (a) a 90° angle detection and (b) quasibackscattering angle detection (173°) in DLS instruments. (c) The zaverage hydrodynamic diameter and (d) the scattered light intensity of AuNP-100 nm at different concentrations measured using a Malvern Zetasizer ZS system with a backscattering detection geometry.
illustration, the scattered light path length can be significantly shortened in the case of backscattering detection compared to 90 deg detection. Malvern supplies two different DLS models: the Zetasizer Nano ZS90 system has a detection angle of 90°, and the Zetasizer Nano ZS uses a backscattering detection, with a detection angle of 173°.47 The Model ZS90 is the instrument used in most measurements reported in this study. For comparison purpose, we also conducted a DLS measurement of AuNPs-100 nm using the Zetasizer Nano ZS system. The measured z-average hydrodynamic diameters and the back21590
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ACS Applied Materials & Interfaces scattered light intensities of AuNP-100 nm at different concentrations are presented in Figure 4C,D. Comparing the backscattered plots to the equivalent 90° plots in Figure 2B,C, the primary difference, as expected because of the shorter scattered light path length, is that the apparent hydrodynamic diameter falls off less steeply at the higher concentrations using the backscatter geometry (Figure 4C) compared to that with the 90° scattering geometry (Figure 2B). However, the concentration at which the multiple scattering is first apparent, 5.6 × 109 particles/ml is about the same for both scattering geometries, contrary to naive expectations. This apparently counterintuitive result is a consequence of the other differences in the experimental setup of the Model ZS90 versus ZS/laser wavelength and possible different solid angles of collections in the 90° versus backscatter geometries. The laser wavelength affects the scattering cross sections (c.f. eq 2); the scattering cross section of the 100 nm AuNPs are larger at the 633 nm wavelength at the backscatter angle versus the 532 nm wavelength at the 90° scattering angle. All other things being equal, this increases the sensitivity of the backscattered angle in this instrument to multiple scattering with AuNP-100 nm. In addition, possible differences in the solid angle of collection between the two instruments may also affect the relative multiple scattering tendencies. So the problem still remains: at increased AuNP concentrations, multiple scattering effect leads to distorted average particle size data. Our conclusion is that regardless of the model and type of the DLS instrument, the multiple scattering effect of intensely scattering nanoparticle materials such AuNPs should always be carefully considered and examined in DLS measurement. 3.4. Effect of Low Scattering Intensity on DLS Measurement. Samples with insufficient scattering intensity will also cause problems with DLS measurements since the lower counts do not allow the processed light scattering signal to accumulate sufficient data to provide a reliable analysis. Figure 5A−C shows the DLS analysis results of a 10 nm AuNP suspensions at different concentrations, including size distribution curves, Z-average diameter, and scattered light intensity. All three parameters exhibit poor reproducibility. The scattering intensity is only around 30−40 kcps at its original concentration and after 10× enrichment. The level of scattering intensity is close to the detection limit of the instrument and the analysis results show significantly increased error and poor measurement-to-measurement reproducibility. Significantly longer measurement times can be used in some cases to improve the quality of the measurements when the higher scattered light intensities are not attainable. 3.5. Using DLS for Quantitative Analysis of Scattered Light Intensity of Individual AuNPs. From the concentration and the average scattered light intensity of each of the AuNP sizes, we estimated the average scattered light intensity of individual AuNPs. In doing this, it is important that the sample used for this analysis be below the multiple scattering limit. Because different laser attenuation levels had to be used for AuNPs with different sizes as described earlier, the scattering intensity obtained at different attenuation numbers was normalized to the same laser power according to a linear relationship between the scattering light intensity and the laser power. Figure 6 is the measured scattered light intensity per particle for the different sized AuNPs. From this curve, it can be clearly seen that the scattered light intensity of individual
Figure 5. Intensity-weighted size distribution curve (a), hydrodynamic diameter (b), and corresponding scattered light intensity (c) of 10 nm AuNPs at different concentrations. Legends in (a) represent the dilution or concentration factor of the AuNP solutions relative to the original AuNP solution. The size distribution curve was obtained under an attenuation of 11 at the maximum laser power.
AuNPs increases dramatically with particle size. The curve based on the DLS measurements agrees very well with a theoretical prediction made using Mieplot.43 In the theoretical Mieplot calculations, the bulk material complex refractive index for gold was used for all particle sizes. The deviation of the predicted curve from the measured curve at larger AuNP size is likely due a combination of experimental precision and a small secondary effect, not accounted for in the calculation, related to the dependence of the complex refractive index on size for particles exhibiting surface plasmon resonance.48 The scattered light intensity of a 100 nm AuNP is nearly 1 million times stronger than that of a 10 nm AuNP. For applications that rely on the scattered light properties of AuNPs, for example, dark field optical microscope imaging of biological cells, we suggest AuNPs with a core diameter of at least 60 nm should be used for these purposes. 3.6. Impact of Nanoparticle Clusters or Aggregates Present in a Sample Suspension on Size Measurement and the Exceptional Sensitivity of DLS in AuNP 21591
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Figure 6. Measured and theoretical scattered light intensity for individual AuNPs of different sizes. The measured scattered light intensities at different laser powers (for varying particle sizes) were normalized to the maximum laser power; the scattering intensity per particle was calculated using the known concentration and sample volume (100 μL). The photon count rate was expressed as count per second (cps).
Aggregate Detection. The scattered light intensity of a particle increases with increasing particle size. It is clear that the z average size Dz reported by a DLS measurement of a nanoparticle suspension that contains a multimodel size distribution will be skewed toward larger average particle size. While this well-known characteristic of DLS measurements can be a problem in some particle size analyses, it also opens unique opportunities for other applications. To further illustrate the above points, we prepared a series of suspensions by mixing 20 and 100 nm AuNP at different particle number ratios, 100 000/1, 10 000/1, 1000/1, 100/1, and 10/1 (20 nm/100 nm). The 100 nm AuNPs were used here to serve as “simulated aggregates” in the 20 nm AuNP suspensions. Figure 7A,B shows the size distribution curves of each mixed suspension, and the z-average particle size versus the particle number ratio. When the number of the 100 nm AuNPs is very small in the mixed suspension, namely, at 100 000/1 ratio of 20 nm/100 nm AuNP, the distribution curve appears monodisperse, only one major intensity distribution peak close to 20 nm is detected. At a particle ratio of 10 000/1 and 1000/1, two peaks, i.e., the bimodel size distribution is resolved in the measurement. With increasing ratios of 100 nm AuNPs in the mixture, the distribution analysis reveals only a single peak again, with the average size very close to the pure AuNP-100 nm. Clearly, the average size and size distribution obtained from DLS is heavily skewed toward the larger particles, here the AuNP-100 nm. The z-average particle size Dz of the mixtures increases significantly with increasing proportion of the 100 nm AuNPs in the suspension. It is most striking to see that even at 0.001% (particle number ratio 100 000/1), the presence of AuNPs-100 nm caused Dz of the mixture suspension to increase from about 23 to 27 nm. According to the definition of the limit of detection (LOD) (σ + 3s, where σ is the measured z average diameter of the AuNP-20 nm sample, and s is the standard deviation of measurement), DLS can detect the presence of a single AuNP-100 nm in the presence of 100 000 20 nm AuNPs. In other words, if AuNP-20 nm forms an aggregate with a size roughly equivalent to one 100 nm AuNP, then the aggregate can be detected even it is present only at 0.001% of the total nanoparticle population.
Figure 7. Intensity-weighted size distribution curves (a) and z-average hydrodynamic diameter (b) of a series of AuNPs mixture suspensions made by mixing 20 and 100 nm AuNP at different particle number ratios. All measurements were made using an attenuation of 8 (3% of the maximum laser power). Under this condition, the count rate was determined to be around 250 kcps for the ctAuNP mixture suspension with particle number ratio of 100 000/1, i.e., within the count rate range which should produce reliable measurements.
For suspensions composed of a mixture of monodisperse nanoparticles of different sizes, the z average diameter of an arbitrary mixture can be calculated, based on the per particle scattered light intensity and the sizes and number concentrations of the constituent particle sizes, using Equation 1. For example, for a bimodal distribution of 20 and 100 nm AuNPs at the 1000:1 ratio of 20 nm:100 nm particles, the calculated z average diameter would be as follows: 1000 × Int 20nm + Int100nm Dz = Int 20nm Int100nm 1000 × 20nm + 100nm (4) where Int20 nm and Int100 nm can be obtained from the measurements shown in Figure 6. If this formula is applied to the 20 nm:100 nm AuNP mixtures, then the calculated z average diameters Dz of the mixtures can be compared to measured diameters, as shown in Figure 7. This unique characteristic of DLS measurements creates a tremendous opportunity for sensing applications based on particle aggregation. AuNPs can be surface functionalized with a wide range of chemical and biological ligands and receptor molecules. Upon binding with their target analytes, AuNPs can form aggregates. By detecting the AuNPs aggregate formation, the target analytes can be detected directly in the assay suspension. Indeed as demonstrated from the work published by us and other groups in the past few years, highly sensitive assays have been developed for a wide range of chemical and biological targets including proteins, DNAs, RNAs, viruses, small chemicals, and toxic metal ions.20−34 These assays comprise a single-step and washing-free process, easy to use, with results obtained typically within minutes. Once considered 21592
DOI: 10.1021/acsami.6b06903 ACS Appl. Mater. Interfaces 2016, 8, 21585−21594
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as a limitation of DLS technique for particle size analysis, the extraordinary sensitivity of DLS toward nanoparticle aggregate formation has turned out to be a significant advantage of DLS for sensing applications.
4. CONCLUSIONS In this study, by conducting DLS measurements on AuNPs with different concentrations and sizes, we demonstrated that the reported hydrodynamic diameters of AuNPs can vary significantly with nanoparticle concentration and the incident laser power. Either excessive or inadequate scattered light from the sample suspension can cause problems with DLS measurements. For AuNPs, the presence of multiple scattering at some concentrations can cause large errors in the measured average hydrodynamic size of the AuNPs. Using the technique described here, the concentrations at which multiple scattering becomes a problem for particles of different sizes can be easily detected or predicted using basic light scattering principles. To avoid the problem of multiple scattering, concentrations below that which causes multiple scattering must be used. Although this study is focused on the analysis of a citrate ligands-capped AuNP, the knowledge and methods discussed here may be applied to the study of and application to other nanoparticle materials. For example, silver nanoparticles also scatters light intensely, to a greater degree than even gold nanoparticles. Multiscattering effects are expected to occur on silver nanoparticles; therefore, when conducting DLS analyses on silver nanoparticles, care must be exercised to determine optimal concentrations for the measurements. In addition to spherical nanoparticles, nonspherical and multicomponent metallic nanoparticles such as nanorods, cubes, stars, core− shell particles have also been synthesized and reported. Because of the general strong light scattering properties of metallic nanoparticles, DLS analyses of all these nanoparticle materials should also be carefully conducted and verified using the techniques described here. Because the scattered light intensity of particles is strongly size-dependent, increasing with nearly the sixth power of the particle diameter, DLS’s intensity-weighted average diameter measurements offer exceptional advantages for nanoparticle aggregate detection. Learning the intrinsic limitations as well as the unique advantages of DLS will allow researchers to take best advantage of this versatile and widely applicable technique and to further extend its application to nontraditional areas.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 407-882-2845. E-mail:
[email protected] (Q.H.). Notes
The authors declare no competing financial interest.
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