An innovative “unlocked mechanism” by a double key avenue for one-pot detection of microRNA-21 and microRNA-141

Abstract

The accurate and quantitative detection of microRNAs (miRNAs) as next-generation, reliable biomarkers will provide vital information for cancer research and treatment. However, their unique, intrinsic features pose quite a challenge for miRNA profiling, especially for multiplexed detection. Thus, there is a strong and an ever-growing need to develop an accurate, simple, sensitive and specific miRNA sensing method.Methods: In this study, a simple and novel sensor is presented that uses a flow cytometry (FCM) method based on the double key “unlocked mechanism” and a fluorescence enrichment signal amplification strategy. The “unlocked mechanism” was cleverly designed via using hairpin DNA probes (HDs) labeled by fluorescent particles (FS) as the lock to block part of them, which can specifically hybridize with the probe on polystyrene microparticles (PS). The target miRNA and duplex-specific nuclease (DSN) forming the double key can specifically open the HDs and cleave a single-stranded DNA (ssDNA) into DNA/RNA dimers circularly in order to unlock the special part of the HDs to be specially enriched further on the PS.Results: The designed sensor with a hairpin structure and DSN special performance was found to have a high specificity. The circularly unlocking fluorescent probes and fluorescent signal enrichment can be beneficial for achieving a high sensitivity with a detection limit of 3.39 fM for miRNA-21. Meanwhile, the performance of multiplexing was estimated by simultaneous detection of miR-21 and miR-141, and the method also allowed for miR-21 detection in breast cancer blood samples.Conclusion: The designed sensor based on an “unlocked mechanism” and a signal enrichment strategy resulted in a one-pot, highly specific and sensitive detection of multiplex miRNAs. The whole detection without the need for a complex purification process is based on a FCM and is expected to have a great value in cancer diagnosis and biomedical research.

The Application of Nucleic Acid Probe–Based Fluorescent Sensing and Imaging in Cancer Diagnosis and Therapy

“…In the presence of miRNA targets, both miRNA targets and duplex-specific nuclease (DSN) act as the double key to specifically unlock HDs and increase the enrichment of HDs on PS microparticles. Then, the unlocked fluorescent probes lead to the enrichment of the fluorescent signal ( Peng et al, 2019b ). The FCM-based DNA probe sensor is allowed to measure miRNA-21 and miRNA-141 in breast cancer blood samples with higher sensitivity ( Peng et al, 2019a ).…”

“…However, most of AgNC-MBs have limited versatility; the reason is that fluorescence properties of DNA-AgNCs will be severely damaged when the AgNC-stabilizing sequence is embedded into the MB sequence. Based on toehold-mediated DNA strand displacement, a new type of AgNC-MB is constructed by combining with total internal reflection fluorescence (TIRF)–based single-molecule fluorescence imaging ( Peng et al, 2019b ). The AgNC-MB platform can simultaneously measure two breast cancer–related miRNAs (miRNA-21 and let-7a) and distinguish the mutant-type targets at low abundance ( Peng et al, 2019a ).…”

A Universal Strategy for Enhancing the Circulating miRNAs’ Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock

“…Numerous studies have achieved effective nucleic acid detection using padlock-assisted RCA because of its advantages of efficient isothermal amplification, easy editing, and flexible signal output. However, for nucleic acid molecules with low abundance and high intermolecular homology, such as circulating miRNAs, , the conventional padlock still has some shortcomings. For instance, the target recognition process only depends on base complementary pairing and the fidelity of the utilized ligase to ensure specificity, which is insufficient for distinguishing single-base mismatch .…”

Research on personalized recommendation algorithm for micromechanical sensors based on cloud model

“…Micromechanical sensors are a new generation of sensor devices based on semiconductor process technology with miniaturization, intelligence, low power consumption, and easy integration, and they are one of the most representative achievements in the field of microelectronics []- [3]. There are seven broad types of micromechanical sensors, namely micromechanical gyroscopes, micro gas sensors, micromechanical temperature sensors, micromechanical pressure sensors, microacceleration sensors, microflow sensors, and other micromechanical sensors []- [7]. These sensors are widely used in the fields of chemistry, medicine, and manufacturing to optimize data transmission and technical control effects through material analysis and equipment processing and have great application prospects []- [12].…”