An enzymatic reaction-based phenylboronic acid-modified microporous array chip for chiral differentiation and high-throughput detection of D-amino acids

D-amino acids (D-AAs), which are potential biomarkers, are found at considerably higher levels in the saliva of individuals with early gastric cancer (GC), making the development of a rapid and sensitive assay imperative. In this paper, a Raman-active boronate modified surface-enhanced Raman scattering (SERS) microporous array chip based on enzymatic reaction was constructed for reliable, sensitive and quantitative monitoring of D-Proline (D-Pro) and D-Alanine (D-Ala) in saliva. Initially, 3-mercaptophenylboronic acid (3-MPBA) was bonded to Au-coated Si nanocrown arrays (Au/SiNCA) via Au-S bonding. Following this, H₂O₂ obtained from D-Amino acid oxidase (DAAO)-specific catalyzed D-AAs further reduced 3-MPBA to 3-hydroxythiophenol (3-HTP) with a new Raman peak at 882 cm^-1. Meanwhile, the original characteristic peak at 998 cm^-1 remained unchanged. Therefore, the I₈₈₂/I₉₉₈ ratio increased as the D-AAs content in the sample to be tested rose, allowing D-AAs to be quantitatively detected. Proudly, the Au/SiNCA with large-area periodic crown structure prepared in this paper could provide numerous, uniform “hot spots”, and the microporous array chip with 16 detection units was employed as the platform for SERS analysis, realizing high-throughput, high sensitivity, high specificity and high reliability quantitative detection of D-AAs (D-Pro and D-Ala). The limits of detection (LOD) were down to 10.1 μM and 13.7 μM throughout the linear range of 20-500 μM. The good results of the saliva detection suggested that this SERS sensor could rapidly differentiate between early-stage GC patients and healthy individuals.