Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy.

Conjugates of semiconductor quantum dots (QDs) and antibodies have emerged as a promising bioprobes due to their great combination of QD's efficient fluorescence and the high specificity of antigen-antibody reactions. For further developments in this field, it is essential to understand the molecular conformation of the QD-antibody conjugates at the single-molecule scale. Here, we report on the direct imaging of QD-antibody conjugates at the single-molecule scale by using high-speed atomic force microscopy (HS-AFM). Owing to the high spatiotemporal resolution of HS-AFM, we observed the dynamic splitting of individual antibodies during the conjugation process. QD-antibody conjugates were also clearly visualized at the single-molecule scale details. Several important features were even discovered through dynamic observation of the QD-antibody conjugates. We observed an intermediate state of conjugation, where the antibodies attached and detached to QDs repeatedly. We also revealed that the attached antibodies were not steady but drastically fluctuated in their recognition areas due to the Brownian motion. We also demonstrated that HS-AFM observation is useful for the quantitative analysis of fabricated conjugates.

Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay.

Precise Fractionation of CdSe/ZnS Quantum Dot-Organic-Dye Conjugates Using a Gel Filtration Column.

Conjugates of semiconductor quantum dots (QDs) and organic dyes have been receiving attention as fluorescence biological sensing materials. In designing such sensors, a most important parameter is the number of organic-dye molecules that conjugate to a QD. If a precise separation method was developed, it might be possible to control conjugation without knowing the exact number of conjugated dye molecules per QD. In this study, the difference in linear velocities in a gel filtration column between CdSe/ZnS QDs and 5-(and 6)-carboxynaphthofluorescein succinimidyl ester is used. The velocities differ because the hydrophilicity of CdSe/ZnS QDs is much higher than that of the organic dye; hence, CdSe/ZnS-organic-dye conjugation can be controlled by changing the fraction number. Furthermore, the concentrations of CdSe/ZnS QDs and organic dye in fractionated solutions can be determined by measuring fluorescence spectra, and we demonstrate a fluorescence-type pH sensor based on the conjugate, which has a pH-sensitivity range from 7.5 - 9.5.