Ultrasensitive Electroanalysis of Low-Level Free MicroRNAs in Blood by Maximum Signal Amplification of Catalytic Silver Deposition Using Alkaline Phosphatase-Incorporated Gold Nanoclusters

Abstract

An ultrasensitive sandwich-type analysis method has been initially developed for probing low-level free microRNAs (miRNAs) in blood by a maximal signal amplification protocol of catalytic silver deposition. Gold nanoclusters (AuNCs) were first synthesized and in-site incorporated into alkaline phosphatase (ALP) to form the ALP-AuNCs. Unexpectedly, the so incorporated AuNCs could dramatically enhance the catalysis activities of ALP-AuNCs versus native ALP. A sandwiched hybridization protocol was then proposed using ALP-AuNCs as the catalytic labels of the DNA detection probes for targeting miRNAs that were magnetically caught from blood samples by DNA capture probes, followed by the catalytic ligation of two DNA probes complementary to the targets. Herein, the ALP-AuNC labels could act as the bicatalysts separately in the ALP-catalyzed substrate dephosphorylation reaction and the AuNCs-accelerated silver deposition reaction. The signal amplification of ALP-AuNCs-catalyzed silver deposition was thereby maximized to be measured by the electrochemical outputs. The developed electroanalysis strategy could allow for the ultrasensitive detection of free miRNAs in blood with the detection limit as low as 21.5 aM, including the accurate identification of single-base mutant levels in miRNAs. Such a sandwich-type analysis method may circumvent the bottlenecks of the current detection techniques in probing short-chain miRNAs. It would be tailored as an ultrasensitive detection candidate for low-level free miRNAs in blood toward the diagnosis of cancer and the warning or monitoring of cancer metastasis in the clinical laboratory.

Bifunctional magnetic nanobeads for sensitive detection of avian influenza A (H7N9) virus based on immunomagnetic separation and enzyme-induced metallization

“…4C, the stripping peak current increased rapidly with the deposition time until 30 min. This phenomenon may be associated with the decrease of nucleation sites on the deposition electrode and the loss of the enzyme activity with the increase in the deposition time (Qu et al, 2008;Si et al, 2014). So 30 min was chosen as the deposition time in the immunosensor.…”

Electrochemical detection of carcinoembryonic antigen based on silver nanocluster/horseradish peroxidase nanocomposite as signal probe

“…Recent advances have enabled the synthesis of metal nanoclusters utilizing different stabilizing ligands, such as DNA molecules, peptide and polymers [17,18]. The metal nanoclusters exhibit strong photoluminescence, good biocompatibility and photostability [19]. These interesting properties are desirable for applications in biosensing.…”

Enzymatic Hydrogelation-Induced Fluorescence Turn-Off for Sensing Alkaline Phosphatase in Vitro and in Living Cells

“…5−8 To date, a large number of spectroscopic methods (e.g., colorimetric, fluorometric, and surface-enhanced Raman scattering (SERS)) and electrochemical approaches have been reported for detection of ALP activity. 9−14 Due to their operational simplicity and high spatial temporal sensitivity, fluorometric methods using organic dyes, 15,16 fluorescent polymers, 9,17 inorganic quantum dots (QDs), or noble metal nanoclusters 18−20 as indicators for ALP detection have been extensively explored.…”