Amplification Strategy of Silver Nanoclusters with a Satellite-Nanostructure for Substrate-Free Assay of Alkaline Phosphatase by ICP-MS.

Alkaline phosphatase (ALP) plays critical roles in signal transmission and cell growth/apoptosis. Its abnormal level in serum/cell is tightly related to diseases, thus, serum and cellular ALP detection is of great significance for disease diagnosis. Herein, a novel approach for ALP assay based on a satellite-nanostructure is developed by conjugating lanthanide upconversion nanoparticles (UCNPs) with silver nanoclusters (AgNCs) through DNA bridging. UCNPs serve as the cores to conjugate with DNA fragments, followed by assembly of AgNCs as the satellites on UCNPs surface through the AgNCs-cytosine affinity, to produce the satellite-nanostructure of UCNPs@DNA-AgNCs. The presence of ALP converts phosphate groups into hydroxyl groups at DNA helix, weakening the coordination of DNA with UCNPs. As a result, the satellite AgNC labeling on DNA fragments strips off the UCNP surface. Silver is quantified by measuring isotope 107Ag with ICP-MS, which further derives the content of ALP by correlation to the number of AgNCs. A linear calibration range is obtained in 0.005-120 U/L with a detection limit of 1.8 mU/L. The distinct advantage of this strategy, on one hand, is the substrate-free feature that eliminates the intermediate process of substrate reaction, where the substrate activity decrease and its instability may significantly deteriorate the sensitivity. On the other hand, ALP triggers the production of a large number of AgNCs resulting in substantial amplification on ICP-MS signal to give a favorable sensitivity. This is the first attempt for ALP detection by inductively coupled plasma mass spectrometry.

Placeholder Strategy with Upconversion Nanoparticles-Eriochrome Black T Conjugate for a Colorimetric Assay of an Anthrax Biomarker.

The timely warning of the germination of bacterial spores and their prevention are highly important to minimize their potential detrimental effects and for disease control. Thus, a sensitive and selective assay of biomarkers is most desirable. In this work, a nanoprobe is constructed by conjugating lanthanide upconversion nanoparticles (UCNPs) with sodium tripolyphosphate (TPP) and eriochrome black T (EBT). The nanoprobe, UCNPs-TPP/EBT, serves as a platform for the detection of the anthrax biomarker, dipicolinic acid (DPA). In principle, DPA displaces EBT from the UCNPs-TPP/EBT nanoconjugate, resulting in a color change from magenta to blue because of the release of free EBT into the aqueous solution. The binding sites on UCNPs are partly preblocked with TPP as the placeholder molecule, leaving a desired number of binding sites for EBT conjugation. On the basis of this dye displacement reaction, a novel colorimetric assay protocol for DPA is developed, deriving a linear calibration range from 2 to 200 µM with a detection limit of 0.9 µM, which is well below the infectious dose of the spores (60 µM). The assay platform exhibits excellent anti-interference capability when treating a real biological sample matrix. The present method is validated by the analysis of DPA in human serum, and its practical application is further demonstrated by monitoring the DPA release upon spore germination.

Highly Sensitive Detection of MicroRNA-21 with ICPMS via Hybridization Accumulation of Upconversion Nanoparticles.

A highly sensitive platform is developed for the determination of microRNA-21 (miRNA-21) with inductively coupled plasma mass spectrometry (ICPMS). It includes the following operations: Hairpin structures DNA H1 and H2 are designed, and DNA H1 is bound to ultrasmall lanthanide upconversion nanoparticles (UCNPs) to produce UCNPs@DNA conjugate probes. Target miRNA triggers a chain reaction for alternating hybridization between DNA H1 (bound on UCNPs@DNA probe) and DNA H2. This leads to UCNPs accumulation and serves as an efficient amplification strategy for UCNPs. The concentration of miRNA-21 is closely correlated to the number of UCNPs; thus, the detection of 89Y by ICPMS provides a promising approach for miRNA quantification. This protocol exhibits high sensitivity to miRNA-21 within 0.1-500 fM, along with a detection limit of 41 aM, which is among the hitherto reported most sensitive procedures. It is worth mentioning that rare earth elements are scarcely present in living systems, which minimizes the background for ICPMS detection and excludes potential interferences from the coexisting species, which is most suited for biological assay.