Rapid and highly sensitive detection of miRNAs in serum of colorectal cancer patients by surface-enhanced Raman spectroscopy microfluidic chip based on catalytic hairpin assembly

Colorectal cancer (CRC) is among the most prevalent forms of cancer globally, with a high mortality rate. In response to the urgent need for improved diagnostics, this study presents a novel approach that employs catalytic hairpin assembly (CHA) as a signal amplification strategy, in conjunction with the rapid enrichment and separation capabilities of magnetic beads (MBs), to construct a surface-enhanced Raman spectroscopy (SERS) microfluidic chip. This chip is specifically designed for the detection of two CRC-related microRNAs, miR-122-3p and miR-150-5p. The SERS microfluidic chip is fabricated by synthesizing gold nanocages (Au nanocage, AuNCs) as a liquid enhancement substrate. Two Raman signal molecules, 4-aminothiophenol (4-ATP) and 5,5 '-dithiobis(2-nitrobenzoic acid) (DTNB), along with hairpin DNA1 (HP1), are used to modify the surface of AuNCs, creating two types of SERS probes (AuNCs@4-ATP@HP1-1 and AuNCs@DTNB@HP1-2). Additionally, hairpin DNA2 (HP2) is immobilized on the surface of MBs to prepare two capture probes (MBs@HP2-1 and MBs@HP2-2). The detection process involves introducing the sample solution, SERS probes, and capture probes into three separate inlets of the microfluidic chip. The target miRNA in the sample triggers a CHA reaction, leading to the formation of a large number of HP1-HP2 complexes (AuNCs@4-ATP@HP1-1-MBs@HP(2-1 )and AuNCs@DTNB@HP1-2-MBs@HP2-2). These complexes are then enriched in a rectangular collection chamber under the influence of a miniature magnet, significantly amplifying the SERS signal. The presence and concentration of miR-122-3p and miR-150-5p are determined by the characteristic peak positions and intensities of 4-ATP and DTNB at 1083 and 1330 cm(-1), respectively. The SERS microfluidic chip offers a linear detection range for miR-122-3p and miR-150-5p from 100 amol/L to 1 nmol/L, with limits of detection (LOD) as low as 19.74 amol/L and 16.35 amol/L, respectively. This method is characterized by its simplicity, speed, and ease of operation, with the entire detection process being completed within 5 minutes. It also offers a wide linear range and low detection limits. Moreover, the SERS microfluidic chip exhibits high stability, strong specificity, and excellent repeatability. Using the SERS microfluidic chip, the expression differences of miR-122-3p and miR-150-5p in the serum of healthy individuals and CRC patients were accurately detected. The results indicated that the expression levels of miR-122-3p and miR-150-5p in the serum of CRC patients are significantly higher than those in healthy individuals (*p<0.05). The SERS microfluidic technology provides a reliable alternative approach for the detection of miRNA in clinical samples, which is instrumental for the early diagnosis and prognostic monitoring of CRC.