Nucleic Acid and Nanomaterial Synergistic Amplification Enables Dual Targets of Ultrasensitive Fluorescence Quantification to Improve the Efficacy of Clinical Tuberculosis Diagnosis

Interferon-γ (IFN-γ) release assays (IGRAs) are constrained by the limited diagnostic performance of a single indicator and the excessive Mycobacterium tuberculosis (Mtb) antigen stimulation time. This study presents a simultaneous, homogeneous, rapid, and ultrasensitive fluorescence quantification strategy for IFN-γ and IFN-γ-induced protein 10 (IP-10). This method relies on the high-affinity binding of aptamers to IFN-γ and IP-10, the enzyme-free catalytic hairpin assembly reaction, and the heightened sensitivity of CdTe quantum dots to Ag+ and hairpin structure C-Ag+-C and carbon dots to Hg2+ and hairpin structure T-Hg2+-T. Under optimized conditions, the selectivity of IFN-γ and IP-10 was excellent, with a linear range spanning from 1 to 100 ag/mL and low limits of detection of 0.3 and 0.5 ag/mL, respectively. Clinical practicality was confirmed through testing of 57 clinical samples. The dual-indicator combination detection showed 92.8% specificity and 93.1% sensitivity, with an area under the curve of 0.899, representing an improvement over the single-indicator approach. The Mtb antigen stimulation time was reduced to 8 h for 6/7 clinical samples. These findings underscore the potential of our approach to enhance the efficiency and performance of a tuberculosis (TB) clinical diagnosis.