Orthogonal Tip-to-Tip Nanocapillary Alignment Allows for Easy Detection of Fluorescent Emitters in Femtomolar Concentrations

Abstract

Here we present the realization of a novel fluorescence detection method based on the electromigration of fluorescent molecules within a nanocapillary combined with the laser excitation through a platinum (Pt)-coated nanocapillary. By using the Pt nanocapillary assisted focusing of a laser beam, we completely remove the background scattering on the tip of the electrophoretic nanocapillary. In this excitation geometry, we demonstrate a 1000-fold sensitivity enhancement (1.0 nM to 1.0 pM) compared to the detection in microcapillaries with epifluorescence illumination and fluorescence spectrophotometry. Due to a significant electroosmotic flow, we observe a decelerating migration of DNA molecules close to the tip of the electrophoretic nanocapillary. The reduced DNA translocation velocity causes a two-step stacking process of molecules in the tip of the nanocapillary and can be used as a way to locally concentrate molecules. The sensitivity of our method is further improved by a continuous electrokinetic injection of DNA molecules followed by sample zone stacking on the tip of the nanocapillary. Concentrations ranging from 0.1 pM to 1.0 fM can be directly observed on the orifice of the electrophoretic nanocapillary. This is a 1000-fold improvement compared to traditional capillary electrophoresis with laser-induced fluorescence.

Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids

“…This value was approximately 1 order of magnitude smaller than the K d value obtained from bulk SPR measurements (Figure c). The smaller dissociation constant could be attributed to factors such as (i) analyte molecules might slow down when they arrive near the tip of the nanopipette and (ii) the significant reduction in DNA migration caused by electro-osmotic flow (EOF) at the pipette tip, resulting in the accumulation of analytes in the vicinity of the nanopore. , …”

“…The smaller dissociation constant could be attributed to factors such as (i) analyte molecules might slow down when they arrive near the tip of the nanopipette 48 and (ii) the significant reduction in DNA migration caused by electro-osmotic flow (EOF) at the pipette tip, resulting in the accumulation of analytes in the vicinity of the nanopore. 49,50 The detection method exhibited high efficiency over a broad dynamic range spanning 5 orders of magnitude (100 fM to 10 nM, Figure 4b). Due to the protein binding signals originating from the ternary sandwich structure formed by the target, false positives were relatively infrequent in the absence of the A29 target protein.…”