Ultrasensitive Hypoxia Sensing at the Single-Molecule Level via Super-Resolution Quantum Dot-Linked Immunosandwich Assay

Abstract

Activated hypoxia-inducible factor-1alpha (HIF-1α) plays an important role in the adaptive response of tumor cells to oxygen changes through the transcriptional activation of genes that regulate important biological processes required for tumor survival and progression. In this study, we developed an ultrasensitive hypoxia sensor based on read-out with quantum dots on a gold nanodisc (quantum dot-linked immunosandwich assay, QLISA) with excellent selectivity for HIF-1α. The immunoassay platform was established by comparing the immune response results using Qdot525 as a detection nanoprobe instead of a fluorescent dye (Alexa488) (fluorescent-linked immunosandwich assay, FLISA). In addition, using three-dimensional total internal reflection fluorescence microscopy, the platform was optically sectioned along the z-axis at 10 nm intervals to compare the height difference between the nanodisc and the nanoprobe following the QLISA and FLISA procedures and to localize the target location. Here, the super-resolution QLISA (srQLISA)-based hypoxia sensor exhibited high accuracy and precision for the detection of HIF-1α-extracted samples in cancer spheroids compared with the super-resolution FLISA (srFLISA) method. The developed nanobiosensor method demonstrated a wide dynamic linear detection range of 32.2 zM− 8.0 pM with a limit of detection of 16 zM under optimal experimental conditions for HIF-1α, an approximate 10 6 -fold enhanced detection sensitivity compared with the conventional enzyme-linked immunosorbent assay method based on absorbance. The detection of HIF-1α using the newly developed srQLISA sensor allows for independently predicting tumor progression and early cancer onset increases in the microvasculature density of tumor lesions.

Super-resolution Multispectral Imaging Nanoimmunosensor for Simultaneous Detection of Diverse Early Cancer Biomarkers

“…However, the amide I and II peaks, which are characteristic wavenumbers for the α–helix structure of a protein, are observed at 1656 and 1545 cm –1 , respectively (Figure S2c). , These results demonstrate the stable binding of DSP and protein A/G to the gold surface.…”

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CdTe/Silica Nanospheres in a Lateral Flow Immunoassay for Prostate-Specific Antigen

“…UQSN-115 is an optimal label for LFIA because nanospheres of this size are able to remodel the surface sufficiently during synthesis to encapsulate the inner CdTe QDs. This sufficient self-encapsulation ensures the protection of the internal QDs and reduces the loss of fluorescence during the coupling process. , …”

Wavelength-Dependent Metal-Enhanced Fluorescence Biosensors via Resonance Energy Transfer Modulation

“…These AuNPs enhanced the fluorescence emission of three fluorescent dyes (Alexa Fluor 488, Alexa Fluor 546, and PE Cy7) approximately 150-fold on a randomly arranged MEF immunosensor to achieve a detection limit of 28.67 pM (4.3 ng/mL) for immunoglobulin detection [ 59 ]. Kang’s group arranged 300 nm gold nanodisks using electron-beam lithography and used them as sensors for the high-sensitivity detection of biomolecules [ 60 ]. They designed the analyte to be sandwiched between a capture antibody immobilized on a gold nanodisk and a detection antibody conjugated with QDs.…”