A simple and universal enzyme-free approach for the detection of multiple microRNAs using a single nanostructured enhancer of surface plasmon resonance imaging

Abstract

Here we describe a simple approach for the simultaneous detection of multiple microRNAs (miRNAs) using a single nanostructured reagent as surface plasmon resonance imaging (SPRi) enhancer and without using enzymatic reactions, sequence specific enhancers or multiple enhancing steps as normally reported in similar studies. The strategy involves the preparation and optimisation of neutravidin-coated gold nanospheres (nGNSs) functionalised with a previously biotinylated antibody (Ab) against DNA/RNA hybrids. The Ab guarantees the recognition of any miRNA sequence adsorbed on a surface properly functionalised with different DNA probes; at the same time, gold nanoparticles permit to detect this interaction, thus producing enough SPRi signal even at a low ligand concentration. After a careful optimisation of the nanoenhancer and after its characterisation, the final assay allowed the simultaneous detection of four miRNAs with a limit of detection (LOD) of up to 0.5 pM (equal to 275 attomoles in 500 μL) by performing a single enhancing injection. The proposed strategy shows good signal specificity and permits to discriminate wild-type, single- and triple-mutated sequences much better than non-enhanced SPRi. Finally, the method works properly in complex samples (total RNA extracted from blood) as demonstrated by the detection of four miRNAs potentially related to multiple sclerosis used as case study. This proof-of-concept study confirms that the approach provides the possibility to detect a theoretically unlimited number of miRNAs using a simple protocol and an easily prepared enhancing reagent, and may further facilitate the development of affordable multiplexing miRNA screening for clinical purposes.

Inkjet‐Based Technology for Microencapsulation of Gold Nanoparticles within Biocompatible Hydrogels

“…Here the carboxylic acid polyethylene glycol (PEG) improves the stability of AuNPs and enables a better interaction with positively charged chitosan for forming the microcapsules; however, terminal PEG functional groups can of course also be employed for conjugating molecules of interest to the AuNPs. [ 17 ] This protective behavior has already been described for other chitosan‐AuNPs hybrid systems that protected the functionality of sensitive molecules from gastric degradation. [ 24 ] The stability of our inkjet microcapsules was also studied by incubation in simulated gastric fluid that replicates the pH and ion concentration to which microcapsules would be exposed during a potential oral administration route.…”

“…In the biotechnology field, gold nanoparticles (AuNPs) have been used for a number of applications ranging from drug release [ 13,14 ] and photothermal therapy [ 15,16 ] to biosensing. [ 17,18 ] In principle, encapsulating nanoparticles in polymers can be employed to enhance nanoparticle stability, provide sustained release, enhance their bioavailability, or improve other aspects related to their delivery and storage. [ 19 ] One of the main advantages of encapsulating AuNPs in polymers is that the optical properties of the AuNPs can be preserved, [ 20 ] avoiding large unwanted plasmon shifts due to change of solvent and plasmon hybridization.…”

A Novel Amplification-free SARS-CoV-2 Point-of-care Nucleic Acid Detection System based on Hybrid Capture Fluorescence Immunoassay

“…The a nity of the S9.6 for DNA-RNA hybrids, the molar ratio of the monoclonal antibody to the hybrid, and the e ciency of signal ampli cation were of critical importance to determine the detection sensitivity of HC-FIA method. The molar ratio of S9.6 to the RNA-DNA hybrid was documented to be [11][12][13] under certain conditions, that was, a hybrid can bind to more than a dozen antibodies, leading to the signal ampli cation. The chromatographic process might have unavoidable effects on the absolute uorescence values of the T line, as well as the C line, and in turn affected the stability of the results.…”

“…The second-generation of hybrid capture technology (HC2) approved by Food and Drug Administration (FDA) can detect 13 high-risk HPV subtypes simultaneously. The monoclonal antibody was also used to capture the complexes formed by hybridization of small RNA and microRNA with various types of labeled DNA probes for signal conversion and ampli cation [7][8][9][10][11].…”

Surface Plasmon Resonance for Biomarker Detection: Advances in Non-invasive Cancer Diagnosis

“…This colorimetric assay offers a LOD of <1 pM, with the advantage of easy sampling and assay operation and great practical utility. Sguassero et al (2019) report on a simple and universal enzyme-free approach for the detection of multiple miRNAs using a single nanostructured enhancer of SPRi. They immobilize DNA probes complementary to target miRNA to a solid surface.…”