Wavelength-dependent three-dimensional single-molecule superlocalization imaging for yoctomole detection of thyroid-stimulating hormone on a quantum dot nanobiosensor

A wavelength-dependent three-dimensional(3D)superlocalization imaging method on gold nanoislands(GNIs)chip was developed as a supersensitive single-molecule thyroid-stimulating hormone(TSH)nanobiosensor.Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots(QDs)nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection(TIR)of incident light using a dual-view device.The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10nm intervals before/after immunoreaction to identify the optimal conditions for detection.The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function.The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy(3D TIRF-SLM)imaging technique showed an excellent detection limit of 90 yoctomoles(~54 molecules)and a wide linear dynamic range of 1.14 zmol/L-100 pmol/L for TSH.The detection sensitivity was about 4.4×10^(9)times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum.The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-tracebiomolecules.

Super-resolution morphological dissemination of intercalating dye in single DNA molecules via binding activated localization microscopy

Fluorescent dye （YOYO-I） intercalated with single DNA molecules were investigated via bindingactivated localization microscopy （BALM） at sub-diffraction limit resolutions. Various dye-to-DNA base pair （bp） ratios were imaged using the blinking property of YOYO-1 dye under optimum BALM switching buffer conditions. Individual DNA molecules exhibited regular/irregular intercalating phenomena with respect to dye-to-DNA bp ratio. The acquired images were reconstructed into super-resolution images by applying a Gaussian fit to the centroid of the point spread function. The YOYO-1 intercalated with λ-DNA possessed a non-homogeneous region due to the different binding modes of YOYO-1 with λ-DNA. Each binding mode was imaged at the sub-diffraction limit super-resolution. The distance between homogenously localized intercalating dyes within the DNA molecules was measured to be 34nm （n= 10; dye：DNAbp= 1：100） without photocleavage in 50mmol/L β-mercaptoethylamine buffer. The results were similar to those of the theoretical values without photocleavage in the base pairs of single DNA molecules below the diffraction limit. The results paved the way for an in-depth microscopic analysis of molecular variation with single λ-DNA molecules. With this method, it should be possible to analyze the exact base pair breakdown during various stages of cell apoptosis.

Base pair distance analysis in single DNA molecule by direct stochastic optical reconstruction microscopy

Precise fluorescence imaging of single l-DNA molecules for base pair distance analysis requires a superresolution technique, as these distances are on the order of diffraction limit. Individual l-DNA molecules intercalated with the fluorescent dye YOYO-1 were investigated at subdiffraction spatial resolution by direct stochastic optical reconstruction microscopy（d STORM）. Various dye-to-DNA base pair ratios were imaged by photoswitching YOYO-1 between the fluorescent state and the dark state using two laser sources. The acquired images were reconstructed into a super-resolution image by applying Gaussian fitting to the centroid of the point spread function. By measuring the distances between localized fluorophores, the base pair distances in single DNA molecules for dye-to-DNA base pair ratios of 1：50,1：100, and 1：500 were calculated to be 17.1 0.8 nm, 34.3 2.2 nm, and 170.3 8.1 nm[17_TD$IF], respectively,which were in agreement with theoretical values. These results demonstrate that intercalating dye in a single DNA molecule can be photoswitched without the use of an activator fluorophore, and that super-localization precision at a spatial resolution of 17 nm was experimentally achieved.