A rapid readout for many single plasmonic nanoparticles using dark-field microscopy and digital color analysis
Introduction
The trend in the development of next-generation biosensors is towards the detection of smaller and smaller amounts of materials, with the goal being the detection of a single molecule (Gooding and Gaus, 2016). The detection of single molecules has the potential to provide new information about biological systems by: i) uncovering the heterogeneity within samples, and ii) the potential to identify rare species or subtle changes in analyte concentration (Holzmeister et al., 2014). However, with the detection of single molecules comes the challenge of having collected sufficient single molecule events to be able to obtain quantitative information (Gooding and Gaus, 2016).
Owing to the characteristic of plasmonic nanoparticles (e.g. gold or silver nanoparticles), known as localized surface plasmon resonance (LSPR), the intense scattering of light by these nanoparticles can be directly imaged in the wide field of view of a dark-field microscope (Anker et al., 2008, Sriram et al., 2015). This widefield imaging of nanoparticles offers a unique opportunity to build a biosensor for single nanoparticle assays, capable of detecting and counting single molecules. Beuwer et al. (Beuwer et al., 2015) used a modified dark-field microscope to monitor the stochastic interactions of hundreds of single proteins on individual gold nanorods. Other approaches involve the use of single particle spectroscopy/spectrometry or laser imaging to obtain high resolution spectra from single nanoparticles, that monitor and analyse single nanoparticles in high spectroscopic detail (Chen et al., 2013, Taylor and Zijlstra, 2017). These methods are of great use in characterizing single nanoparticle interactions. However, for biosensing applications, such a high level of spectral detail is not as important as acquiring specific and relevant information from many single nanoparticles. Hence, an approach that is capable of rapid and high-throughput single nanoparticle analysis would be advantageous.
Recently, a new strategy to analyse and monitor the plasmonic properties of a nanoparticle known as colorimetric analysis has been developed. This approach analyses the color of scattered light from plasmonic nanoparticles images using dark-field microscopy. The wavelength of the scattered light is directly related to the LSPR of the nanoparticle and its size, shape and local environment. By analyzing the RGB (red, green and blue) of the light scattered by the nanoparticles, this technique has been used to identify the sizes of gold nanoparticles (Jing et al., 2012) and also detect single antibody-antigen binding events (Ungureanu et al., 2010, Verdoold et al., 2011). The technique was further modified to streamline the colorimetric detection, by measuring hue (a single color variable) to detect the formation of a DNA monolayer on gold nanoparticles (Zhou et al., 2016).
The recent development of single nanoparticle analysis using CCD detectors costing thousands of dollars for colorimetric analysis, has opened the possibility for high-throughput single nanoparticle assays to be conducted (Xie et al., 2017). However, in order to move towards achieving a portable device for point-of-care detection, it is important for the tools used in these approaches to be readily accessible and cost effective. Although consumer-grade CMOS cameras do not provide the same level of image quality as CCD detectors (Lustica, 2011), the technology has reached a point where image quality is sufficient to perform image processing tasks at the single nanoparticle level. The other main advantages that these CMOS cameras provide are significantly lower costs of production, the capability for fast read-out speeds and their wide availability in commercially available cameras and smartphones (Waltham, 2013). The last point is especially significant as this would allow for smartphones to be employed in biosensing, a step towards a point-of-care device.
In this study, we demonstrate the ability of a commercially available consumer-grade CMOS camera, when coupled with a dark-field microscope, to analyse the LSPR of single plasmonic nanoparticles. Dark-field images are analysed using an in-house developed MATLAB algorithm that can process thousands of nanoparticles within seconds, a signficant improvement on the longer times of previous spectroscopic techniques. Furthermore, through the calibration of the CMOS camera, we demonstrate that the λmax of up to 5000 nanoparticles can be obtained within seconds, without the use of a spectrometer. In order to demonstrate the ability of this technique to be employed in the detection of an analyte, interleukin-6 (IL-6) was chosen as the model analyte. IL-6 is known to be associated in the inflammatory and auto-immune processes of many diseases, such as Alzheimer's disease (Wang et al., 2015). Using a core-satellite binding assay, we demonstrate the potential of this technique to be employed in biosensing.
Section snippets
Chemicals and materials
Sodium citrate tribasic dihydrate, gold(III) chloride trihydrate (HAuCl4·3H2O), (3-aminopropyl)triethoxysilane (APTES), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) and streptavidin were purchased from Sigma-Aldrich (Sydney, Australia). Small glass coverslips were purchased from Thermo Fisher Scientific (Australia) (22 × 22 mm, 0.16 – 0.19 mm thickness). Large coverslips for use in the flow cell were purchased from Thermo Scientific (USA) (Gold Seal™, 35 ×
Results and discussion
The light scattered by plasmonic nanoparticles is observed as a colored point-spread-function (PSF), of which the color is dependent on the size, shape, material and the local environment of the nanoparticle. Using an inexpensive CMOS-sensor based digital camera, which is readily available in many consumer products (e.g. smartphones), a new colorimetric technique was developed to allow for the rapid and high-throughput analysis of single plasmonic nanoparticles, as shown in Scheme 1. Using this
Conclusion
In summary, we have developed a new digital analysis technique that enables readily-available CMOS digital cameras, to perform rapid and high-throughput analyses of single plasmonic nanoparticles. This technique is based on observing the change in color of scattered light from plasmonic nanoparticles as seen under a dark-field microscope. By calibrating hue, measured by a digital camera with a CMOS sensor, against wavelength, and by using an in-house developed MATLAB program, up to 5000
Acknowledgements
The authors acknowledge the generous financial support from the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036), an ARC Australian Laureate Fellowship (FL150100060 to J.J.G.) and UNSW Vice-Chancellor's Research Fellowships (to S.R.C.V.). This research used the facilities at the Electron Microscope Unit at UNSW. This work was supported through access to the Australian National Fabrication Facility (ANFF) Design House software at the
References (22)
- et al.
Gold nanoparticle-based signal amplification for biosensing
Anal. Biochem.
(2011) - et al.
Immunosensing by colorimetric darkfield microscopy of individual gold nanoparticle-conjugates
Sens. Actuators, B Chem.
(2010) - et al.
Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles
Biosens. Bioelectron.
(2011) - et al.
Role of pro-inflammatory cytokines released from microglia in Alzheimer's disease
Ann. Transl. Med.
(2015) - et al.
Biosensing with plasmonic nanosensors
Nat. Mater.
(2008) - et al.
Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm
Langmuir
(2011) - et al.
Stochastic protein interactions monitored by hundreds of single-molecule plasmonic biosensors
Nano Lett.
(2015) - et al.
Shape-and size-dependent refractive index sensitivity of gold nanoparticles
Langmuir
(2008) - et al.
Wide-field single metal nanoparticle spectroscopy for high throughput localized surface plasmon resonance sensing
Lab Chip
(2011) - et al.
Immunosorbent assay on large arrays of individual particles made by electron beam lithography
ACS Nano
(2013)
Single-molecule sensors: challenges and opportunities for quantitative analysis
Angew. Chem. - Int. Ed.
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These authors contributed equally to this work.