Plasmonic hotspots of dynamically assembled nanoparticles in nanocapillaries: Towards a micro ribonucleic acid profiling platform

Abstract

Plasmonic hot spots, generated by controlled 20-nm Au nanoparticle (NP) assembly, are shown to suppress fluorescent quenching effects of metal NPs, such that hair-pin FRET (Fluorescence resonance energy transfer) probes can achieve label-free ultrasensitive quantification. The micron-sized assembly is a result of intense induced NP dipoles by focused electric fields through conic nanocapillaries. The efficient NP aggregate antenna and the voltage-tunable NP spacing for optimizing hot spot intensity endow ultra-sensitivity and large dynamic range (fM to pM). The large shear forces during assembly allow high selectivity (2-mismatch discrimination) and rapid detection (15 min) for a DNA mimic of microRNA. V C 2013 AIP Publishing LLC.[http://dx.doi.org/10.1063/1.4832095] Irregular expressions of a panel of microRNAs (miRNA) in blood and other physiological fluids may allow early diagnosis of many diseases, including cancer and cardiovascular diseases. 1 However, quantifying all relevant miRNAs (out of 1000), with similar sequences over 22 bases 2 and large variations in expression level (as much as 100 fold) at small copy numbers, requires a new molecular diagnostic platform with high-sensitivity, high-selectivity, and large dynamic range. Current techniques for miRNA profiling, such as Northern blotting, 3 microarray-based hybridization, 4 and real-time quantitative polymerase chain reaction 5 are expensive and complex. A simple and rapid miRNA array would allow broad distribution of molecular diagnostic devices for cancer and chronic diseases, eventually into homes for frequent prescreening of many diseases.At their low concentrations in untreated samples, optical sensing of miRNA is most promising. Plasmonically excited Raman scattering (SERS) and fluorescence sensors from metallic nanoparticles (NPs) or surfaces have enhanced the sensitivity of optical molecular sensors by orders of magnitude. 6-9 However, probe-less SERS sensing or fluorescent sensing of unlabeled targets are insufficiently specific for miRNA targets in heterogeneous samples. Plasmonic detection is also very compatible with FRET probes whose donor dye offers small light sources to excite fluorescently labelled targets upon hybridization. 7,10 A particular family of FRET reporters does offer label-free sensing: hairpin oligo probes whose end-tagged fluorophores are quenched by the Au NP to which they are functionalized. 11 The fluorescent signal is only detected when the hairpin is broken by the hybridizing target nucleic acid or protein (for an aptamer probe), and the more rigid paired segment separates the end fluorophore from the quenching surface to produce a fluorescent signal. It is often hoped that plasmonics on the metal surface will enhance the intensity to overcome the quenching effect, if the linearized hairpin is within the NP plasmonic penetration length. However, since fluorescent quenching decays slowly (linearly) with fluorophore-metal spacing 10 whereas the plasmonic intensity decays exponentially from a flat surface, careful ex...

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“…6(d)-6(f)) permits us to monitor the NP assembly dynamics, location, size, and spacing. 136 Computer simulations show that the NP assembly location coincides with the counter-ion concentration maximum under enrichment conditions (Fig. 6(g)).…”

“…[147][148][149][150][151][152] For example, we can pack 25 nm to 100 nm oligo-functionalized Au, silica and polystyrene NPs into iontrack etched polyethylene terephthalate (PET) polymer conic nanopores, 134,135 and into conic silica nanocapillaries pulled by laser assisted drawing. 130,136,137 We have studied the NP assembly mechanism with two-dimensional nano-wedge models fabricated by focused ion beam milling. 153 In a conic nanopore, NPs larger than the conic tip diameter ($100 nm) can be assembled by electrophoresis either on its tip or through its base.…”

“…There are several low-cost nanopore fabrication technologies for making single and multiple solid-state nanopore membranes (see, for example, laser-assisted pulling and ion-track fabrication technologies that we have employed [134][135][136][137][138] ). Among these, track etching of polyethylene terephthalate (PET) polymer membranes and chemical etching can produce solid-state conic nanopores with very small tip diameters ranging from 5 to 10 nm.…”

“…Validation was done with hairpin probes for miR146a with fluorescent reporters and quenchers enhanced by NP plasmonic hotspots. 136,161 Gold NPs were functionalized with a thiolated fam-labeled hairpin oligo-probe (HOP) (Fig. 6(a)).…”

A bead-based fluorescence immunosensing technique enabled by the integration of Förster resonance energy transfer and optoelectrokinetic concentration

“…7 Similar bio-analysis approaches have been reported in hybridization assays for oligonucleotides, 8,9 quantum dot (QD)-based molecular beacons for aptamer binding assays, 10,11 and immunoassays. 12,13 Among them, Liu et al 14 reported a novel detection of gold nanoparticles (AuNPs) functionalized with fluorescently labelled dsDNA by forming plasmonic hotspots through glass nanocones. A minimum concentration of 1 fM was achieved, and the intensity reached a maximum after minutes of packing time.…”