MicroRNAs are useful in early diagnosis and prognosis of cancer. Therefore, it is highly desirable to develop miniaturized biosensors with high sensitivity to microRNAs. In a recently published article in the magazine Analytica Chimica Actathe researchers constructed a new microfluidic dual-SERS biosensor to detect microRNAs, integrating multifunctional nano-immobilized nanoparticles into the system.
Study: A microfluidic-based SERS biosensor with multifunctional nano-surface immobilized nanoparticles for sensitive detection of MicroRNA. Image Credit: Love Employee/Shutterstock.com
The designed nanosurface was composed of porous anodic aluminum oxide (AAO) packed with gold nanoparticles (AuNPs) which served as a good surface enhanced Raman scattering (SERS) substrate. Silver-coated, p-mercaptobenzoic acid surface-decorated ultra-small gold core shell NPs ([email protected]) were used as SERS nanotags. Furthermore, a single standard DNA (ssDNA) was used to capture microRNA and immobilize the nanotags.
The accuracy of the constructed biosensor was improved by dividing the AAO membrane into AAO/Au array and AAO/[email protected] array, the former acting as the primary detector and reactor, and the latter as the 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, immune cell/system development, human tumor cell expression and apoptosis.
The expression of microRNA provides essential information for the early detection of cancer. Nevertheless, the sensitive detection of microRNA is challenging due to its small size, sequence homology between family members and low abundance in samples. Therefore, developing a microRNA detection technique with high specificity, sensitivity and stability is particularly important.
Northern blotting, polymerase chain reaction (PCR), and microarray methods are conventional technologies used to detect microRNA. However, their detection capability is limited to tissues and is unsuitable for body fluids. The most recent approaches for microRNA detection include colorimetry, fluorescence, electrochemiluminescence and SERS.
SERS is a robust analytical method often used in the detection of biomarkers. Its low background noise, anti-interference and high sensitivity make it a suitable technique for complicated environments. The main challenge in developing a highly efficient SERS approach for detecting biomolecular targets is to prepare SERS-active substrate with good reproducibility and multi-level electromagnetic hot spots.
AAOs are highly ordered and easily controlled nanostructures with tunable geometry that are ideal nanotemplates to prepare SERS substrate. The three-dimensional (3D) structure of AAO and the highly ordered porous nanostructure form a SERS-active substrate with multi-level electromagnetic hot spots and good reproducibility.
Duplex-specific nuclease (DSN)-assisted target recycle amplification is a useful strategy for sensitive detection of microRNA due to its ability to cleave double-stranded (ds) DNA or DNA/RNA heteroduplexes. Previously used microRNA detection strategies via signal amplification had high specificity, a low limit of detection and a wide linear range.
Microfluidic-based SERS biosensor for microRNA . detection
In the present study, AuNP-coated highly ordered porous AAO (AAO/Au) was used as a DSN-assisted target recycling amplification reactor that served as a primary detector for microRNA-sensitive detection.
The AAO array chamber, coated with [email protected] NPs, resulted in AAO/[email protected] nanoplatforms, which were used to detect the SERS nanotags in their dissociated form, were released from the reactor, indirectly confirming the microRNA concentration. Here, AAO/[email protected] nanosurface served as a collector and secondary detector.
The presence of microRNA near the nano surface triggered a hybridization reaction that allowed the ssDNA to capture the microRNA, forming DNA/microRNA heteroduplexes. Then, the DSN-assisted target recycling process was started to cleave the freshly formed DNA/microRNA heteroduplexes into DNA fragments and ss microRNA.
Initially, the SERS nanotags dissociating from the nanosurface resulted in a reduced SERS signal. After capturing the spliced microRNA, the next cycle of SERS nanotags delivery was initiated, amplifying the detection signal, which correlated with the concentration of microRNA. The 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 to 10 nanomole.
The microfluidic dual-SERS detection strategy has a single detection mode that reduces the possibility of false positive or negative and allows the simultaneous detection of multiple microRNAs by integrating different probes.
In summary, a microfluidic-based biosensor with a dual-SERS detection mode consisting of a DSN-assisted target recycle amplification strategy and functionalized AAO substrate was built to detect microRNA in samples. In the construction of this biosensor, the functionalized AAO substrate was divided into two zones.
One zone with an AuNP-loaded AAO array containing AuMBAs@Ag SERS nanotags were used for the DSN-assisted target recycling amplification process. The other zone with [email protected] NP-decorated AAO array was used to collect and detect the detached SERS nanotags, thereby indirectly achieving microRNA detection in the sample. Monitoring SERS signal in two different functional zones correlated with microRNA concentration.
Ma, W., Liu, L., Zhang, X., Liu, X., Xu, Y., Li, S., Zeng, M. A microfluidic-based SERS biosensor with multifunctional nano-surface immobilized nanoparticles for sensitive detection of microRNA. Analytica Chimica Acta† https://doi.org/10.1016/j.aca.2022.340139
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