Our simple and scalable solution-phase growth methodology affords uniform, plasmonic, nanostructured gold films on glass slides, capable of intensifying the emission of near-infrared fluorophores. Fluorescence enhancement is found to be dependent on plasmon resonance, gold film structure, and the fluorophores used to probe the fluorescence enhancement phenomenon. Employment of μArray/Au assays for quantification of protein biomarkers results in significantly improved detection limits and broader dynamic ranges than traditional protein microarrays and ELISA. Moreover, multiplexed protein microarrays performed on μArray/Au benefit from enhanced feature intensity and low-autofluorescence background, compared with commercially available glass and nitrocellulose substrates, thus providing a broader dynamic range with increased sensitivity for protein microarrays.
Excitation field enhancement, resulting in an increased optical transition rate, is believed to be a contributing factor to our observations of NIR-FE20,21
. Previous results suggest that excitation field enhancement is strongest within the gaps of the gold island structure of Au/Au films17
. In this study, we found that optimal NIR-FE is provided by Au/Au films containing dense gold islands with an average inter-island gap distance of ~36 nm. On the other hand, thick Au/Au films containing a semi-continuous layer of gold (few gaps present) yielded little fluorescence enhancement relative to a bare glass substrate, whereas completely continuous gold films (no gaps present) resulted in quenching of fluorescence. We therefore attribute the nanoscale gaps in our Au/Au films, and the resulting enhanced excitation electric fields, as one of the causes of fluorescence enhancement on our μArray/Au platform20
Proximity of a fluorophore to a metal structure could quench fluorescence emission due to a greater enhancement of the non-radiative decay rate knr
than the radiative decay rate krad
). Fluorophore coupling to the scattering component of plasmonic extinction is responsible for increased radiative decay rates resulting in fluorescence enhancement, whereas the absorption component is responsible for enhancing the rate of non-radiative decay and thus fluorescence quenching15
. The magnitude of absorption and scattering components of the optical spectra of metal nanoparticles depends on both the size and shape of the metal structures, as described by Mie theory13,26
. Significantly increased non-radiative decay rates could be the cause of fluorescence quenching, rather than enhancement, on continuous films.
The scattering (re-radiating) efficiency of our Au/Au film for fluorescence enhancement is large in the near-infrared where the plasmonic peaks of the Au/Au films overlap with the fluorescence emission wavelengths of the IR800 and Cy5 fluorophores. The plasmonic resonance peaks of our Au/Au films reside in this NIR region due to the wavelength-dependent dielectric constant of gold, suitable sizes of the gold islands, and possibly the elongated shapes of the islands13,27
in the Au/Au films. We attribute NIR-FE of IR800 and Cy5 labels in our μArray/Au to the optimal gold island size (on the order of 104
) and the coupling of the dipolar components of the plasmonic modes of these nanoislands to the emission of fluorescence. The suitable Au islands sizes and plasmonic coupling in these nanostructured Au/Au films may have led to an increase in krad
that outweighed knr
enhancement of the near-infrared fluorophores used herein for microarray protein assays, contributing to an increased apparent quantum yield and improved signal-to-noise ratios for protein detection. Further research into the effect of our nanostructured Au/Au film on radiative and non-radiative decay rates of IR800 and Cy5 is underway.
The observed variation in enhancement factor between Cy5 and IR800 on a given Au/Au film is likely due to several contributing factors. First, because the observed quantum yield scales with the ratio of radiative decay rate to total decay rate, the radiative decay rate could be enhanced relatively more than the non-radiative decay rate for inherently low quantum yield emitters12
(η~7% for IR800 versus ~20% for Cy5). Also, the different excitation and emission energies of the fluorophores may interact differently with absorbance and scattering components of an Au/Au film with fixed plasmon resonance. It is presumed that only the scattering component of metal nanoparticles contributes to far field radiation10
, which is greater at longer wavelengths, such as those characteristic of IR800. Finally, the incident field enhancement efficiency of metal nanostructures is dependent on both nanoparticle spacing as well as wavelength. Peak field enhancements are obtained at longer wavelengths when nanoparticle separation distances are small28
Combined with the benefit of low biological autofluorescence in the near-infrared excitation/emission region and increased surface area, the improved signal-to-noise ratios of μArray/Au assays provide vastly improved sensitivity for quantification of disease biomarkers in biological media. Unlike chemical amplification methods, metal-enhancement of NIR fluorescence emission increases signal without creating undesirable background or extra noise. μArray/Au assays afford increased signal-to-noise ratios by ~15-fold for Cy5-labelled sandwich assays and ~100-fold for IR800-labelled sandwich assays relative to identical protein microarray sandwich assays on glass slides. The fivefold increase in fluorescence enhancement for Cy5 and IR800 employed in immunoassay experiments compared with drop-dried samples is probably caused by alleviation of non-radiative energy transfer to the metallic film at greater separation distances, in agreement with previous literature29
. For detection and quantification of CEA spiked into 100% serum, such gains in signal-to-noise resulted in improved detection limits to 5 fM with a dynamic range over six orders of magnitude.
Detection of CEA in the serum of xenograft mouse models bearing LS174T tumours was accomplished by use of μArray/Au assays. In comparison to a calibration curve, serum CEA concentrations in the range of ~30 fM to 100 fM were detectable when tumour volumes were well below 100 mm3. We note that with detection limits in the range of 1–5 pM, commercially available CEA ELISA kits fail to provide adequate sensitivity to detect or quantify CEA in the majority of the ex vivo serum samples analysed in the present study. Moreover, protein diagnostics based on highly sensitive μArray/Au assays require far less sample volume than ELISA and may be multiplexed to analyse a variety of protein biomarkers at once.
To demonstrate the multiplexing capabilities of μArray/Au substrates, a panel of human autoantigens was printed onto μArray/Au substrates, as well as commercially available nitrocellulose membrane substrates and glass slides. The autoantigens printed represent both well-characterized and poorly characterized targets of human autoantibodies implicated in a range of autoimmune diseases including systemic lupus erythematosus (SLE), Sjögren syndrome, mixed connective tissue disease, systemic sclerosis, celiac disease, Good pasture’s syndrome, and others4,30–33
. Incubation of a mixture of human sera (Methods) containing known reactivity towards several autoantigen targets led to the observation of a broad range of reactivities, represented as microarray feature intensities, with which the various assay substrates could be compared and contrasted.
Nitrocellulose-based substrates were often used for protein microarrays, because they provide a high-surface area, 11 μm thick, three-dimensional polymer layer for capture reagent immobilization, with a protein binding capacity34
of ~4,000 ng mm−3
. In contrast, planar surfaces, such as μArray/Au and glass, provide binding capacities in the range of 10–100 ng mm−2
. Therefore, protein micro-arrays, such as autoantigen arrays, on nitrocellulose membranes yield high feature intensities through increased capture efficiency (vide supra). On the other hand, nitrocellulose films are known to exhibit high autofluorescence in the visible, limiting their dynamic range and utility. Autofluorescence of nitrocellulose is mitigated to an extent by employing fluorescence excitation and emission energies in the near-infrared (for example, Cy5 and IR800 dyes); however, we have observed that sufficient background intensity remains, even at emission wavelengths from 700–800 nm emission (; Supplementary Fig. S11
), to prevent identification of dilute or low-reactivity autoantibodies. The dynamic range of this IR800-labelled autoantigen array on nitrocellulose was half-an-order of magnitude, whereas the background intensity and maximum observed feature intensity of an autoantigen array labelled by Cy5 differed only by a factor of 2.
In contrast, μArray/Au substrates afford higher positive signals owing to fluorescence enhancement as well as tenfold lower autofluorescence backgrounds compared with nitrocellulose. With a much broader fluorescence intensity range (~2 orders of magnitude) than nitrocellulose (~half of an order of magnitude) in this multiplexed autoantigen array, μArray/Au substrates identified not only the highly reactive autoantibodies in the serum mixture, but also other autoantigen features with non-negligible reactivity towards the antoantibody-containing serum (). μArray/Au also outperformed planar glass substrates, with vastly increased positive intensities and higher signal-to-noise ratios owing to NIR-FE.
All assay substrates exhibited positive feature intensities for highly reactive markers of autoimmune conditions such as Lupus (for example, SLE, and subacute cutaneous lupus erythematosus, SCLE), Sjögren Syndrome, systemic sclerosis, poly- and dermatomyositis, and thyroiditis. The sample of mixed autoimmune serum employed here included well-characterized4,24
autoantibodies targeting Ro/SS-A, Jo-1, centromere protein B, thyroglobulin and DNA topoisomerase-1 (scl-70), all of which were detected with high intensity as expected on their conjugate antigen feature. The serum sample also contains autoantibodies towards myeloperoxidase, proteinase 3, histones, and mitrochondrial antigen, which were not included in the autoantigen array. Moreover, it is expected that the serum mixture also includes uncharacterized autoantibodies and other human immunoglobulin Gs that may contribute to intermediate and low levels of array feature intensity. For example, non-negligible intensity was observed on both nitrocellulose and μArray/Au representing reactivity of the serum mixture towards thyroperoxidase, Sm protein B/B′, measles antigen, glomerular basement membrane antigen, complement complex C1q, and components of the U1-snRNP complex (for example, U1-C, U1-A, sm/RNP).
Autoantibodies of PCNA, implicated in SLE, were not observed on any of the substrates tested. However, the μArray/Au substrate revealed slight reactivity, not known a priori, of the incubated autoimmune sera towards autoantigens including double-stranded DNA, PL-12, and PM/Scl-75 (), which, on nitrocellulose, did not present feature intensity of significant difference from the background. These antigens are implicated in autoimmune disorders such as SLE, polymyositis, and polymyositis-systemic sclerosis overlap syndrome, respectively. Our results suggest that in addition to improved detection limits for protein-based diagnostics (for example, early cancer detection), μArray/Au protein assays could significantly enhance our ability to elucidate intermediate and low-level autoantibody reactivity in autoimmune diseases. In general, the higher sensitivity and broader dynamic range afforded by μArray/Au substrates will benefit high-throughput proteomics research, as well as diagnostics, for a wide range of diseases throughout various biological fields.
In conclusion, μArray/Au assays afford a significant improvement in signal-to-noise ratio, resulting in multiplexed microarray protein sandwich assays possessing a broad dynamic range and high sensitivity, with detection limits ~1,000 to 5,000-fold lower than traditional techniques, yet they require no additional assay steps and are compatible with standard protein microarray processing and equipment. The NIR-FE μArray/Au assays rely on physical principles, namely an enhancement in excitation field strength, reduction in excited state lifetime, and overall apparent increase in fluorescence quantum yield, to significantly improve the signal-to-noise ratio over standard protein microarrays.
Our solution phase, bottom-up growth procedure of Au/Au films for fluorescence enhancement applications is scalable, simple and fast. The Au/Au film substrates for NIR-FE applications are stable over time and in biological media, and, moreover, are uniform enough to provide quantitative analysis with a dynamic range of over six orders of magnitude. In addition to affording biomarker quantification at low concentrations, high-throughput screening methods may benefit from the expanded dynamic range afforded by multiplexed μArray/Au assays, where concentrations of analytes, as well as binding constants, may span a significant and unknown range. NIR-FE based on Au/Au films may also find further applications as an in vitro
imaging tool. For example, fluorescent agents bound to the membrane of live cells have been enhanced by Au/Au films35
. Coupled with the simplicity afforded by physical signal enhancement and compatibility with existing microarray tools, μArray/Au assays are expected to find broad use in disease diagnosis and protein biomarker discovery applications.