RESUMO
Assays utilizing fluorophores are common throughout life science research and diagnostics, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve higher signal-to-noise. We describe how the synergistic coupling of plasmonic and photonic modes can significantly boost the emission from fluorophores. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) with the absorption and emission spectrum of the fluorescent dye, a 52-fold improvement in signal intensity is observed, enabling individual PFs to be observed and digitally counted, where one PF tag represents one detected target molecule. The amplification can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency, and increased rate of spontaneous emission. The applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease is demonstrated. A limit of detection of 10 fg mL-1 and 100 fg mL-1 in buffer and human plasma respectively, is achieved, representing a capability nearly three orders of magnitude lower than standard immunoassays.
Assuntos
Nanopartículas , Humanos , FluorescênciaRESUMO
Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Ácidos Nucleicos Imobilizados/química , SARS-CoV-2/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Humanos , Limite de Detecção , Microscopia/métodos , Óptica e Fotônica/instrumentação , Óptica e Fotônica/métodos , SARS-CoV-2/química , Saliva/virologia , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between light and biological matter. A common objective is to concentrate the electromagnetic energy associated with light into nanometer-scale volumes that, in many cases, can extend below the conventional Abbé diffraction limit. Dating back to the first application of surface plasmon resonance (SPR) for label-free detection of biomolecular interactions, resonant optical structures, including waveguides, ring resonators, and photonic crystals, have proven to be effective conduits for a wide range of optical enhancement effects that include enhanced excitation of photon emitters (such as quantum dots, organic dyes, and fluorescent proteins), enhanced extraction from photon emitters, enhanced optical absorption, and enhanced optical scattering (such as from Raman-scatterers and nanoparticles). The application of photonic metamaterials as a means for enhancing contrast in microscopy is a recent technological development. Through their ability to generate surface-localized and resonantly enhanced electromagnetic fields, photonic metamaterials are an effective surface for magnifying absorption, photon emission, and scattering associated with biological materials while an imaging system records spatial and temporal patterns. By replacing the conventional glass microscope slide with a photonic metamaterial, new forms of contrast and enhanced signal-to-noise are obtained for applications that include cancer diagnostics, infectious disease diagnostics, cell membrane imaging, biomolecular interaction analysis, and drug discovery. This paper will review the current state of the art in which photonic metamaterial surfaces are utilized in the context of microscopy.
Assuntos
Técnicas Biossensoriais , Microscopia , Óptica e Fotônica , Fótons , Ressonância de Plasmônio de SuperfícieRESUMO
Silver nanoislands are key platforms for plasmonic photocatalysis, SERS sensing and optical metamaterials due to their localized surface plasmon resonances. The low intrinsic loss in Ag enables high local electromagnetic field enhancements. Solution-based fabrication techniques, while cheap, result in highly non-reproducible plasmonic substrates with wide sample-to-sample variability in geometry, optical resonances and Q-factors. Herein, we present a non-lithographic method of forming silver nanoislands based on sputter deposition of Ag films followed by elevated temperature annealing to induce spontaneous dewetting. The resulting plasmonic substrates show reproducible, well-defined LSPR resonances with high ensemble Q-factors whose optical properties could be modeled using spectroscopic ellipsometry to yield n and k values across the visible range. UV-Vis-NIR, and XRD analyses define the optical and crystallographic characteristics of the Ag nanoisland samples. FESEM was utilized to discern the geometry and architecture of the Ag nanoisland as well as their uniformity and monodispersity. Our vacuum deposited Ag nanoislands demonstrated excellent photocatalytic activity for the transformation of 4-nitrobenzenethiol (4-NBT) and 4-aminothiophenol (PATP) into p,p'-dimercaptoazobenzene (DMAB).
RESUMO
While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence.