Combination of Mass Signal Amplification and Isotope‐Labeled Alkanethiols for the Multiplexed Detection of miRNAs

Abstract

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO concentrations.

Preparation and evaluation of in situ photocleavable mass tags with facile mass variation for matrix-free laser desorption ionization mass spectrometry

“…23 In previous MALDI-TOF MS analysis of self-assembled monolayers of alkanethiolates on gold surfaces, oligo EG-containing alkanethiolates were mostly observed as sodium and potassium adducts. [3][4][5][6] In addition, for the analysis of multiple analytes simultaneously, multiple mass tags with different masses should be detected in a narrow mass range without complicated peak overlap. As such, amino acid series mass tags with 14 Da mass differences were used for further feasibility studies.…”

“…Gildea et al reported an acid-labile trityl protecting group for oligonucleotide synthesis. 20 They introduced the synthetic route for the NHS-activated 4,4 0 -demethoxytrityl chloride (5). The tritylation of the acid-containing thiol with 5 led to the formation of 6, which was subsequently reacted with amine-containing molecules to afford the PMTs (for detailed experimental protocols to prepare the PMTs, see the ESI †).…”

“…1,2 In particular, the combination of metallic nanoparticle (NP)-based mass tags and matrix-assisted laser desorption/ionization (MALDI) MS has been widely used because pulsed laser irradiation can facilitate the desorption/ionization of small reporter molecules on NPs without the use of organic matrices, which helps amplify the MS signals. [3][4][5][6][7] Photocleavage involving controlled and heterolytic chemical bond breaking through exposure to specic wavelengths of light (particularly those of the laser equipped in MALDI instruments) can afford stable and detectable ionic components in MS analysis. For example, orthonitrobenzyl-based photocleavage is widely employed in photochemistry because of its efficient photocleavage upon exposure to UV light.…”

Programmable Clostridium perfringens Argonaute-Based, One-Pot Assay for the Multiplex Detection of miRNAs

“…It is very challenging to sensitively and specifically detect multiple miRNAs in one pot, not only because miRNAs are in an extremely low concentration and are with short sequence that are susceptible to interference, but also because the multiplexity greatly increases the complexity of detection system. Although various methods such as fluorescence-based approaches have been reported for the detection of miRNAs, these methods suffer from several shortcomings: long detection time and multiple steps (e.g., >3 h), − limited sensitivity, or expensive equipment. , This COP method breaks through the bottleneck of existing one-pot technologies, such as CRISPR-Cas-based approaches − and Ago-mediated methods , (Table S3), and presents many important and outstanding advantages including the ability of multiplex detection, programmable generality, single nucleotide precision, sensitivity, one-step strategy, simplicity, and rapidness. This method holds great potential in the application of molecular medical diagnosis.…”