MicroRNAs are useful in the diagnosis and early prognosis of cancer. Therefore, the development of miniaturized biosensors with high sensitivity to microRNAs is highly desirable. In a recent article in the journal Analytica Chimica Acta, researchers built a new dual-SERS microfluidic biosensor to detect microRNAs, integrating multifunctional nanosurface immobilized nanoparticles into the system.
Study: a microfluidic-based SERS biosensor with nanoparticles immobilized on multifunctional nanosurface for sensitive detection of MicroRNA. Image Credit: Love Employee / Shutterstock.com
The designed nanosurface was composed of porous anodic aluminum oxide (AAO) filled with gold nanoparticles (AuNPs) that served as a good surface-enhanced Raman dispersion substrate (SERS). Ultra-thin gold shell NPs, coated with silver and p-mercaptobenzoic acid ([email protected]) were used as SERS nanotags. In addition, a single standard DNA (ssDNA) was used to capture microRNAs and immobilize nanotags.
The accuracy of the constructed biosensor was improved by subdividing the AAO membrane into matrix AAO / Au and AAO /[email protected] matrix, where the former acted as a primary detector and reactor, and the latter served as a secondary detector and collector. Dual SERS mode on primary and secondary detectors prevented false positives or false negatives during microRNA detection.
MicroRNA detection methods
MicroRNAs are a class of endogenous ssRNAs with 19 to 23 base pairs. These microRNAs modulate post-transcriptional gene expression in living systems. MicroRNAs are vital for various biological processes such as repression, cell / immune system development, human tumor cell expression, and apoptosis.
MicroRNA expression provides vital information for early detection of cancer. However, sensitive detection of microRNAs is a challenge due to their small size, sequence homology among family members, and low abundance of samples. Therefore, the development of a microRNA detection technique with high specificity, sensitivity, and stability is especially important.
Northern blotting, polymerase chain reaction (PCR) and microarray methods are conventional technologies used to detect microRNAs. However, its ability to detect is limited to tissues and is not suitable for body fluids. The most recent approaches for microRNA detection include colorimetry, fluorescence, electro-chemiluminescence, and SERS.
SERS is a robust analytical method that is often applied to the detection of biomarkers. Its low background noise, anti-interference and high sensitivity make it a suitable technique for complicated environments. The main challenge to develop a highly efficient SERS approach to detect biomolecular targets is to prepare an SERS active substrate with good reproducibility and multi-level electromagnetic hotspots.
AAOs are highly ordered and easily controllable nanostructures with adjustable geometry that are nanoparticles ideal for preparing SERS substrate. The three-dimensional (3D) structure of the AAO and its highly ordered porous nanostructure form a SERS active substrate with electromagnetic hot spots of various levels and good reproducibility.
Targeted specific nuclease-assisted (DSN) recycling target amplification is a convenient strategy for sensitive detection of microRNAs due to their ability to cleave double-stranded DNA (ds) or heteroduplex DNA / RNA . The microRNA detection strategies used previously by signal amplification had high specificity, a low detection limit, and a wide linear range.
Microfluidic-based SERS biosensor for microRNA detection
In the present study, porous AAO (AAO / Au) coated with AuNP and highly ordered was used as a DSN-assisted target recycling amplifier reactor that served as the primary detector for microRNA-sensitive detection.
The AAO matrix chamber, coated with [email protected] NPs, resulted in AAO /[email protected] nanoplatforms, which were used to detect SERS nanotags in their dissociated, reactor-released forms, thus indirectly confirming the concentration of microRNAs. Here, AAO /[email protected] nanosurface served as a secondary collector and detector.
The presence of microRNA near the nanosurface triggered a hybridization reaction that facilitated the ssDNA to capture the microRNA, forming a DNA / microRNA heteroduplex. The DSN-assisted target recycling process was then initiated to cleave newly formed DNA / microRNA heteroduplexes into ss DNA and microRNA fragments.
Initially, SERS nanotags that dissociated from the nanosurface resulted in a decrease in the SERS signal. After capturing the cleaved microRNA, the next SERS nanotag release cycle was initiated, thus amplifying the detection signal, which correlated with the microRNA concentration. MicroRNA detection was achieved with a 30 microliter sample and a 10 microliter enzyme to obtain a wide linear range of concentrations between 10 femtomoles and 10 nanomoles.
The dual-SERS microfluidic detection strategy has a single detection mode that reduces the possibility of false positives or negatives and allows the simultaneous detection of multiple microRNAs by integrating different probes.
Conclusion
In summary, a microfluidic-based biosensor was constructed with a dual SERS detection mode consisting of a DSN-assisted target recycling amplification strategy and a functionalized AAO substrate to detect microRNAs in the samples. In the construction of this biosensor, the functionalized AAO substrate was divided into two zones.
An area with an AuNP-loaded AAO matrix with AuMBA @ Ag SERS nanotags was used for the DSN-assisted goal recycling amplification process. The other area with [email protected] An NP-decorated AAO matrix was used to collect and detect dissociated SERS nanotags, thus indirectly achieving microRNA detection in the sample. Monitoring of the SERS signal in two different functional areas correlated with microRNA concentration.
Reference
Ma, W., Liu, L., Zhang, X., Liu, X., Xu, Y., Li, S., Zeng, M. A microfluidic-based SERS biosensor with nanoparticles immobilized on multifunctional nanosurface for detection sensitive to microRNA. Analytica Chimica Acta. https://doi.org/10.1016/j.aca.2022.340139
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