Competitive recognition strategy based on a gold nano-hexagons microarray chip for simultaneous high-sensitivity detection of diabetic retinopathy-associated miR-21 and miR-152

Abstract

Early detection of biomarkers has a crucial role in preventing blindness caused by diabetic retinopathy (DR). In this work, we developed a rapid, highly sensitive method for the simultaneous detection of DR-related biomarkers (miR-21 and miR-152) based on an Au nano-hexagon (AuNHs) integrated microarray chip combined with a competitive recognition strategy. The nucleic acid aptamer-modified AuNHs array served as capture substrate, while Au nano-trisoctahedrons (AuNTs) with complementary strands of nucleic acid aptamers mounted on their surface and Raman signaling molecules served as SERS probes (AuNTs@4-MBA@H₁ and AuNTs@DTNB@H₂). SERS probes carrying Raman signaling molecules (4-MBA and DTNB) were displaced by the target miRNAs in the experiments because they had a higher binding affinity to nucleic acid aptamers than their structural analogs (H₁ and H₂). This approach led to a notable decrease in the Raman signal. The competitive recognition strategy was straightforward and useful, and required only a one-step dropwise reaction to detect both miRNAs simultaneously and effectively. The microarray chip designed accordingly had the advantages of sensitive, rapid and high-throughput detection, and the detection time was only 8 min. The limits of detection (LOD) for miR-21 and miR-152 were as low as 2.76 × 10⁻¹⁴ M and 1.94 × 10⁻¹⁴ M, respectively. The utility of the method in complex systems was verified by detecting miRNAs in the serum of DR rats, and the accuracy of the method was verified against the detection results of a RT-qPCR method. This method provides a new idea and manner for the detection of miRNAs in serum, which is crucial for the early diagnosis of DR.