Detection of estradiol with a digital immunoassay using an anti-immunocomplex antibody and single-molecule observation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been widely applied to molecular sensors due to their optical properties. We previously reported a molecular detection by observing the scattered light of AuNPs at a single nanoparticle level using dark field microscopy (DFM). Recently, a molecular detection method using digital immunoassay has been reported, taking advantage of the characteristics of DFM. However, the digital immunoassays reported so far have been performed by a conventional sandwich immunoassay, which is difficult to apply to the detection of small molecules. In this study, with the aim of small molecule detection, we developed a digital immunoassay method using an anti-immunocomplex antibody that specifically recognizes immunocomplexes of small molecules with antibodies. The number of AuNPs modified with anti-immunocomplex antibody bound to immunocomplex of estradiol and anti-estradiol antibody was counted at a single nanoparticle level using DFM. We demonstrated for the first time that estradiol molecule can be detected by digital immunoassay using DFM and an anti-immunocomplex antibody with a detection sensitivity of 1 pg/mL.

Colorimetric detection of thrombin based on signal amplification by transcription-reverse transcription concerted reaction using non-crosslinking aggregation of gold nanoparticles.

Gold nanoparticles (AuNPs) have been used as colorimetric biosensors by utilizing the difference in color between the dispersed (red) and aggregated (blue) states. We previously developed a biosensor that converts sandwich-type thrombin recognition to RNA amplification and color difference of AuNPs. But the sensitivity was insufficient because of the linear signal amplification mechanism. In this study, we designed an exponential signal amplification biosensor based on transcription-reverse transcription concerted (TRC) reaction.

Transcription-Based Amplified Colorimetric Thrombin Sensor Using Non-Crosslinking Aggregation of DNA-Modified Gold Nanoparticles.

Gold nanoparticles (AuNPs) have been employed as colorimetric biosensors due to the color difference between their dispersed (red) and aggregated (blue) states. Although signal amplification reactions triggered by structural changes of the ligands on AuNPs have been widely used to improve measurement sensitivity, the use of ligands is limited. In this study, we designed a AuNP-based signal-amplifying sandwich biosensor, which does not require a conformational change in the ligands. Thrombin was used as a model target, which is recognized by two different probes. In the presence of the target, an extension reaction occurs as a result of hybridization of the two probes. Then RNA synthesis is started by RNA polymerase activation due to RNA promoter duplex formation. The amplified RNA drives aggregation or dispersion of the AuNPs, and a difference of the color if the AuNP solution is observed. As this detection system does not require a conformational change in the ligand, it can be generically applied to a wide range ligands.

Molecular detection using aptamer-modified gold nanoparticles with an immobilized DNA brush for the prevention of non-specific aggregation.

Gold nanoparticles (AuNPs) are often used for biosensing. In particular, aptamer-modified AuNPs are often used for colorimetric molecular detection, where target molecule-induced AuNP aggregates can be recognized by a color change from red to blue. However, non-specific aggregation could be induced by various compounds, leading to false-positive results. In this work we employed high-density ssDNA modification on the AuNP surface to prevent non-specific aggregation. The covalently immobilized DNA brush was used as an anchor for an aptamer specific for the target molecule. Herein, as a proof-of-concept study, we demonstrated detection of estradiol (E2), one of the endocrine-disrupting estrogen molecules as a model target, in the presence of antibiotic kanamycin (KN) as a model of co-contaminating compounds that induce non-specific aggregation of AuNPs. We also developed a smartphone dark field microscope (DFM) to visualize AuNP aggregation. Our previous study demonstrated that the observation of light scattering by AuNP aggregates with DFM can be applied for versatile molecular detection. In this work, we could successfully detect E2 with the smartphone DFM, and the results were verified by the results from a conventional benchtop DFM. This study would contribute to the future field applicability of AuNP-based sensors.

A DNA duplex-based, tailor-made fluorescent sensor for porphyrin derivatives.

We synthesized a DNA-based fluorescent sensor, tailored to the recognition and signaling for hydrophobic guest molecules. The sensor molecule is made of three functional moieties, per-O-methylated beta-cyclodextrin (per-Me-beta-CD), DNA, and pyrene as guest-binding, dimerizing, and signaling ones, respectively. Addition of a porphyrin derivative as a hydrophobic guest molecule induced emission switching of the sensor molecule from monomer to excimer due to the DNA hybridization promoted by the host-guest binding.

A general and versatile molecular design for host molecules working in water: a duplex-based potassium sensor consisting of three functional regions.

A general and versatile molecular design for host molecules was proposed based on the structural motif of antibodies. A water-soluble potassium sensor was developed by this molecular design, which consists of three functional regions, benzo-15-crown-5 ether, DNA, and pyrene as guest-binding, dimerising, and signaling sites, respectively. The molecular sensor emitted the monomer fluorescence of the pyrene fluorophore predominantly when existing as a random structure, while it formed a duplex upon recognition of K+ to bring about monomer-excimer emission switching.