Label free imaging of the chemical environment of biological specimens would readily bridge the supramolecular and the cellular scales, if a chemical fingerprint technique such as Raman scattering can be coupled with super resolution imaging. We demonstrate the possibility of label-free super-resolution Raman imaging, by applying stochastic reconstruction to temporal fluctuations of the surface enhanced Raman scattering (SERS) signal which originate from biomolecular layers on large-area plasmonic surfaces with a high and uniform hot-spot density (>1011/cm2, 20 to 35 nm spacing). A resolution of 20 nm is demonstrated in reconstructed images of self-assembled peptide network and fibrilated lamellipodia of cardiomyocytes. Blink rate density is observed to be proportional to the excitation intensity and at high excitation densities (>10 kW/cm2) blinking is accompanied by molecular breakdown. However, at low powers, simultaneous Raman measurements show that SERS can provide sufficient blink rates required for image reconstruction without completely damaging the chemical structure.
Although label-free biosensors comprised of optical microcavities inherently possess the capability of resolving molecular interactions at individual level, this extreme sensitivity restricts their convenience for large scale applications by inducing vulnerability towards non-specific interactions that readily occur within complex media. Therefore, the use of optical microresonators for biosensing is mostly limited within strictly defined laboratory conditions, instead of field applications as early detection of cancer markers in blood, or identification of contamination in food. Here, we propose a novel surface modification strategy suitable for but not limited to optical microresonator based biosensors, enabling highly selective biosensing with considerable sensitivity as well. Using a robust, silanebased surface coating which is simultaneously protein resistant and bioconjugable, we demonstrate that it becomes possible to perform biosensing within complex media, without compromising the sensitivity or reliability of the measurement. Functionalized microtoroids are successfully shown to resist nonspecific interactions, while simultaneously being used as sensitive biological sensors. This strategy could pave the way for important applications in terms of extending the use of state-of-theart biosensors for solving problems similar to the aforementioned.Label-free detection of chemical and biological species is an indispensable analytical method providing fast and reliable qualitative and quantitative information regarding the composition of analyte, and comprehension of interaction among biomolecules 1-3 . Among various label free biodetection techniques such as surface plasmon resonance 4 and quartz crystal microbalance 5 , optical microresonators of whispering gallery mode (WGM) type, where light is confined in a circular path inside the periphery of the resonator by means of continuous total internal reflection 6 , particularly possess outstanding potentials by virtue of their extreme sensitivity towards the alterations of the refractive index of the media they reside within 7 . Light can be evanescently coupled to these optical microcavities using tapered optical fibres as resonant modes 8 , which can be tracked by continuously scanning the wavelength of a tuneable laser around the resonant wavelength and observed as Lorentzian shaped dips in the transmission spectrum of the tapered fibre 9,10 . The linewidth of the resonant mode is inversely proportional to the quality factor (Q) of the microresonator, representing its ability of storing optical power 11 . Especially surface tension induced microcavities, which surface roughness, one of the main sources of optical losses due to scattering from the surface, is diminished by a thermal treatment causing the melting and reflowing of the material constituting the microresonator 12 , such as microspheres 13 and microtoroids 14 , have extremely narrow resonance linewidths. This enables detection of even single adsorption events causing shifts in the resonance wave...
Mictoroids, as optical biosensors, can provide beneficial biosensing platforms to understand DNA alterations. These alterations could have significant clinical importance, such as the case of Pseudomonas aeruginosa, which is a commonly found pathogen in Cystic Fibrosis (CF) patientscausing poor prognosis by undergoing mutations during disease steps, gaining virulence and drug resistance. To provide a preliminary diagnosis platform for early-stage bacterial mutations, biosensing with a selective microtoroid surface was suggested. For this purpose, microtoroids with high quality factors were fabricated. The microtoroid surfaces were coated with (3-aminopropyl) triethoxysilane (APTES)/trimethylmethoxysilane (TMMS) mixed silane solution followed by EDC/NHS chemistry for covalent conjugation of DNA probes. Ethanolamine capping was applied to avoid unspecific interactions. The confocal studies confirmed homogeneous functionalization of the microtoroid surface. The DNA hybridization was demonstrated to be affected from the probe length. The optical biosensors showed a significant response (∼22 pm) to the complementary strand of the mutated type P. aeruginosa DNA, while showing substantially low and late response (∼5 pm) to the point mismatch strand. The limit of detection (LOD) for the complementary strand was calculated as 2.32 nM. No significant response was obtained for the noncomplementary strand. The results showed the microtoroids possessed selective surfaces in terms of distinguishing DNA alterations.
Roll-to-roll UV nanoimprint lithography has superior advantages for high-throughput manufacturing of micro- or nano-structures on flexible polymer foils with various geometries and configurations.
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