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 ...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.展开更多
基金supported by the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(Nos.2019R1A2C2002556 and 2020R1C1C1009668)supported by Nano-Material Technology Development Program through the NRF funded by the Ministry of Science,ICT and Future Planning(No.2009-0082580).
文摘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.
