Digital Duplex Homogeneous Immunoassay by Counting Immunocomplex Labeled with Quantum Dots.

Digital multiplexed homogeneous immunoassay is supposed to have the advantages of high sensitivity, high analytical throughput, small sampling errors, and low consumption. We present a spectral imaging-based multiplex, homogenous immunoassay by counting sandwich-structured immunocomplexes in the form of quantum dot (QD) aggregates. As a proof of concept, the method was utilized to detect two tumor biomarkers: carcino-embryonic antigen (CEA) and α-fetoprotein (AFP). The immunocomplex induced by CEA contained QD 655 and QD 585 and were recognized by the spectral pattern of dual-color QD aggregates under a transmission-grating-based spectral imaging microscope. Immunocomplexes induced by AFP were labeled with the QD 585 aggregate and were identified by the spectral blue-shift pattern of same-color QD aggregates. Limits of detection for AFP and CEA were calculated to be 0.02 and 0.10 pM at a signal-to-noise ratio of 3, respectively. Further successful quantification of the model proteins in human plasma demonstrated the accuracy and reliability of our approach.

A homogeneous digital biosensor for circulating tumor DNA by the enumeration of a dual-color quantum dot complex.

Monitoring ctDNA in blood is important for cancer management. Here, a one-step single particle counting approach was developed for directly quantifying ctDNA in plasma. Hairpin DNA containing a triple helix stem was immobilized onto QD 585 as a probe. The hairpin was opened by the target, and therefore hybridized with assistant DNA on QD 655, resulting in an aggregate of QD 585 and QD 655. The two-color QD aggregate was regarded as the target. Observed under a single particle transmission grating-based spectral microscope, the two-color QD aggregate was distinguished by a unique spectral pattern of two first-order streaks, and it was counted. The difference in the responses of the probes to perfect-match DNA, single-base mismatch DNA, and non-match DNA indicated that the probe had sufficient single-base discrimination capabilities. The success in plasma recovery tests demonstrated the feasibility of carrying out the direct detection of ctDNA in plasma.

Single Gold Nanoparticle-Based Colorimetric Detection of Picomolar Mercury Ion with Dark-Field Microscopy.

Mercury severely damages the environment and human health, particularly when it accumulates in the food chain. Methods for the colorimetric detection of Hg(2+) have increasingly been developed over the past decade because of the progress in nanotechnology. However, the limits of detection (LODs) of these methods are mostly either comparable to or higher than the allowable maximum level (10 nM) in drinking water set by the US Environmental Protection Agency. In this study, we report a single Au nanoparticle (AuNP)-based colorimetric assay for Hg(2+) detection in solution. AuNPs modified with oligonucleotides were fixed on the slide. The fixed AuNPs bound to free AuNPs in the solution in the presence of Hg(2+) because of oligonucleotide hybridization. This process was accompanied by a color change from green to yellow as observed under an optical microscope. The ratio of changed color spots corresponded with Hg(2+) concentration. The LOD was determined as 1.4 pM, which may help guard against mercury accumulation. The proposed approach was applied to environmental samples with recoveries of 98.3 ± 7.7% and 110.0 ± 8.8% for Yuquan River and industrial wastewater, respectively.

A Single-Molecule Homogeneous Immunoassay by Counting Spatially "Overlapping" Two-Color Quantum Dots with Wide-Field Fluorescence Microscopy.

We developed a single-molecule homogeneous immunoassay by counting spatially "overlapping" two-color quantum dots (QD) under a wide-field fluorescence microscope. QD 655 with red fluorescence and QD 565 with green fluorescence were modified with capture and detection antibodies, respectively. A capture antibody-modified QD 655 and a detection antibody-modified QD 565 were conjugated by a corresponding antigen molecule to form a "sandwich" immunocomplex. The conjugated QD 655 could not be distinguished from the conjugated QD 565 by fluorescent microscopy because the distance between them was smaller than the resolution of an optical microscope (approximately 200 nm). The immunocomplex color became yellow because of the spatial "overlap" of the red and green fluorescence. The number of the yellow spots was equal to the number of immunocomplex molecules, while the concentration of the antigen was related to the ratio of the yellow dots to the red dots. The successful quantification of two model proteins in the human plasma, namely, alpha-fetoprotein and carcinoembryonic antigen, demonstrated the accuracy and reliability of our approach.