Tagging the rolling circle products with nanocrystal clusters for cascade signal increase in the detection of miRNA

Abstract

A new method to label and detect the long ssDNA products from rolling circle (RC) amplification was reported. ZnS nanocrystal clusters (NCCs) were used to tag the RC products. The NCCs release millions of cations to trigger millions of metal-responsive dyes for cascade signal amplification. Selective and sensitive quantification of miRNA was demonstrated; and the NCCs delivered much lower detection limits compared to using peroxidase or quantum dots as the labels.

Expanding the Chemical Repertoire of DNA Nanomaterials Generated by Rolling Circle Amplification

“…In contrast, substitution of dUTP with the larger alkyne Oct-dUTP 4 led to a 76% reduction in total DNA yield and a significantly reduced amount of DNA present in the DNFs ( Figure 3B). A similar trend was observed with the azide triphosphates N3-dUTP 1 and the bulkier AHP-dUTP 5 ( Figure 3C and D), albeit to a smaller degree (16% and 42% reduction in total DNA respectively), suggesting that phi29 tolerates smaller nucleobase modifications better than larger ones, which is in agreement with other reports (31)(32)(33)(34)(35).…”

“…To achieve a high level of functionality in the DNFs we needed to identify dN*TPs that could be incorporated to high degree without altering the DNF structure. Whilst dN*TPs have been used to add functionality to RCA products, phi29 does not incorporate bulky dN*TPs without a reduction in polymerase efficiency (31)(32)(33)(34)(35), and the standard approach in RCA is to introduce dN*TPs at low concentration in the reaction mixture (typically 40 µM) in the presence of a much higher concentration of the four canonical triphosphates (typically 2 mM of each triphosphate) (36). This obviously gives very low labelling density.…”

Nanotechnology‐Based Strategies for the Detection and Quantification of MicroRNA

“…This signal amplification mechanism allowed detection of as little as 10 amol miR‐21 (100 f M in 100 μL sample solution during hybridization). In the second approach, ZnS NCs are recruited to the surface of magnetic beads after rolling circle amplification (RCA) of a circular template generated as a consequence of miRNA hybridization with a padlock probe 62b. More specifically, the long ssDNA produced by this enzymatic reaction carry multiple biotin tags due to incorporation of biotin–dATP and can be used to capture strepavidinated ZnS NCs.…”

Portable aptamer biosensor of platelet-derived growth factor-BB using a personal glucose meter with triply amplified

“…Due to the unique attributes of rapid kinetics at room temperature (orders of magnitude faster than in the bulk), the tuning of reactivity via control of nanocrystal size, shape, surface faceting and the avoidance of strong acids or corrosive oxidants, CX-based amplification has been employed as a convenient tool for sensitive detection of a variety of targets, such as DNA (Zhang et al, 2015a(Zhang et al, , 2015b(Zhang et al, , 2015c, microRNA (Li et al, 2009a), femtomolar proteins (Yao et al, 2012a(Yao et al, , 2012b, telomerase , glutathione (Shi et al, 2013) and so on. Zhong's group has reported a series of work on cationexchange-based fluorescence amplification (Li et al, 2009a(Li et al, , 2009bYao et al, 2011Yao et al, , , 2012aYao et al, , 2012bYao et al, , 2013. By exchanging nonfluorescent CdSe or ZnS NC clusters with Ag + , Ag 2 Se NCs together with large amounts of the released Cd 2+ or Zn 2+ cations were formed.…”