Universal Surface-Enhanced Raman Scattering Amplification Detector for Ultrasensitive Detection of Multiple Target Analytes

Up to now, the successful fabrication of efficient hot-spot substrates for surface-enhanced Raman scattering (SERS) remains an unsolved problem. To address this issue, we describe herein a universal aptamer-based SERS biodetection approach that uses a single-stranded DNA as a universal trigger (UT) to induce SERS-active hot-spot formation, allowing, in turn, detection of a broad range of targets. More specifically, interaction between the aptamer probe and its target perturbs a triple-helix aptamer/UT structure in a manner that activates a hybridization chain reaction (HCR) among three short DNA building blocks that self-assemble into a long DNA polymer. The SERS-active hot-spots are formed by conjugating 4-aminobenzenethiol (4-ABT)-encoded gold nanoparticles with the DNA polymer through a specific Au–S bond. As proof-of-principle, we used this approach to quantify multiple target analytes, including thrombin, adenosine, and CEM cancer cells, achieving lowest limit of detection values of 18 pM, 1.5 nM, and 10 cells/mL, respectively. As a universal SERS detector, this prototype can be applied to many other target analytes through the use of suitable DNA-functional partners, thus inspiring new designs and applications of SERS for bioanalysis

Ultrasensitive Detection of Single Nucleotide Polymorphism in Human Mitochondrial DNA Utilizing Ion-Mediated Cascade Surface-Enhanced Raman Spectroscopy Amplification

Although surface-enhanced Raman spectroscopy (SERS) has been featured by high sensitivity, additional signal enhancement is still necessary for trace amount of biomolecules detection. In this paper, a SERS amplified approach, featuring “ions-mediated cascade amplification (IMCA)”, was proposed by utilizing the dissolved silver ions (Ag⁺) from silver nanoparticles (AgNPs). We found that using Ag⁺ as linkage agent can effectively control the gaps between neighboring 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNPs@4-ABT) to form “hot spots” and thus produce SERS signal output, in which the SERS intensity was proportional to the concentration of Ag⁺. Inspired by this finding, the IMCA was utilized for ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA (16189T → C). Combining with the DNA ligase reaction, each target DNA binding event could successfully cause one AgNP introduction. By detecting the dissolved Ag⁺ from AgNPs using IMCA, low to 3.0 × 10^–5 fm/μL targeted DNA can be detected, which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic β-cell lines from diabetes patients. This IMCA approach is expected to be a universal strategy for ultrasensitive detection of analytes and supply valuable information for biomedical research and clinical early diagnosis

Fabricating a Reversible and Regenerable Raman-Active Substrate with a Biomolecule-Controlled DNA Nanomachine

A DNA configuration switch is designed to fabricate a reversible and regenerable Raman-active substrate. The substrate is composed of a Au film and a hairpin-shaped DNA strand (hot-spot-generation probes, HSGPs) labeled with dye-functionalized silver nanoparticles (AgNPs). Another ssDNA that recognizes a specific trigger is used as an antenna. The HSGPs are immobilized on the Au film to draw the dye-functionalized AgNPs close to the Au surface and create an intense electromagnetic field. Hybridization of HSGP with the two arm segments of the antenna forms a triplex-stem structure to separate the dye-functionalized AgNPs from the Au surface, quenching the Raman signal. Interaction with its trigger releases the antenna from the triplex-stem structure, and the hairpin structure of the HSGP is restored, creating an effective “off–on” Raman signal switch. Nucleic acid sequences associated with the HIV-1 U5 long terminal repeat sequences and ATP are used as the triggers. The substrate shows excellent reversibility, reproducibility, and controllability of surface-enhanced Raman scattering (SERS) effects, which are significant requirements for practical SERS sensor applications

Molecular Recognition-Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes

Exosomes are membrane-enclosed extracellular vesicles derived from cells, carrying biomolecules that include proteins and nucleic acids for intercellular communication. Owning to their advantages of size, structure, stability, and biocompatibility, exosomes have been used widely as natural nanocarriers for intracellular delivery of theranostic agents. Meanwhile, surface modifications needed to endow exosomes with additional functionalities remain challenging by their small size and the complexity of their membrane surfaces. Current methods have used genetic engineering and chemical conjugation, but these strategies require complex manipulations and have only limited applications. Herein, we present an aptamer-based DNA nanoassemblies on exosome surfaces. This in situ assembly method is based on molecular recognition between DNA aptamers and their exosome surface markers, as well as DNA hybridization chain reaction initiated by an aptamer-chimeric trigger. It further demonstrated selective assembly on target cell-derived exosomes, but not exosomes derived from nontarget cells. The present work shows that DNA nanostructures can successfully be assembled on a nanosized organelle. This approach is useful for exosome modification and functionalization, which is expected to have broad biomedical and bioanalytical applications

Aptasensor with Expanded Nucleotide Using DNA Nanotetrahedra for Electrochemical Detection of Cancerous Exosomes

Exosomes are extracellular vesicles (50–100 nm) circulating in biofluids as intercellular signal transmitters. Although the potential of cancerous exosomes as tumor biomarkers is promising, sensitive and rapid detection of exosomes remains challenging. Herein, we combined the strengths of advanced aptamer technology, DNA-based nanostructure, and portable electrochemical devices to develop a nanotetrahedron (NTH)-assisted aptasensor for direct capture and detection of hepatocellular exosomes. The oriented immobilization of aptamers significantly improved the accessibility of an artificial nucleobase-containing aptamer to suspended exosomes, and the NTH-assisted aptasensor could detect exosomes with 100-fold higher sensitivity when compared to the single-stranded aptamer-functionalized aptasensor. The present study provides a proof-of-concept for sensitive and efficient quantification of tumor-derived exosomes. We thus expect the NTH-assisted electrochemical aptasensor to become a powerful tool for comprehensive exosome studies