Glucose Oxidase Coupling with Pistol-Like DNAzyme Based Colorimetric Assay for Sensitive Glucose Detection in Tears and Saliva

Non-invasive monitoring of glucose levels in tears and saliva is crucial for diagnosing and predicting various illnesses, such as diabetic nephropathy. However, the capability of the current glucose detection methods to identify small amounts of glucose with a high sensitivity remains a significant obstacle. This study proposes a simple, visual technique for sensitively detecting glucose levels from tears and saliva using glucose oxidase (GOx) to catalyze glucose and pistol-like DNAzyme (PLDz) to enhance the signal. In particular, the β-D-glucose present in the samples serves as the initial molecule that GOx identifies and catalyzes to generate gluconic acid and hydrogen peroxide (H₂O₂). The H₂O₂ induces the self-cleavage of PLDz, activating the “part b” sequence. This activation initiates catalytic hairpin assembly (CHA) and releases the DNAzyme section in the H1 probe. The DNAzyme acts as a peroxidase analog, facilitating the catalysis of the 3,3′,5,5′-tetramethylbenzidine (TMB)-hydrogen peroxide (H₂O₂) system and resulting in color changes. The proposed method exhibits a broad detection range of six orders of magnitude and a low limit of 0.32 μM for glucose detection. Furthermore, the proposed method was highly effective in detecting glucose in saliva and tears, suggesting that it could potentially diagnose hyperglycemia-related disorders in clinical environments.