Rapid PCR-free detection of paucibacillary mycobacteria using nanoparticle-enabled phenomics-to-genomic biosensing assay

Tuberculosis (TB) remains a leading global health challenge, accounting for approximately 1.4 million deaths annually and disproportionately affecting low-and middle-income countries. Despite advances in diagnostics, rapid and affordable TB detection remains limited in resource-constrained settings. Culture-based methods, while highly sensitive, require prolonged incubation, and sputum smear microscopy, though widely used, is low in sensitivity, particularly at low bacterial loads. Molecular assays such as GeneXpert offer improved sensitivity but are costly and infrastructure-dependent, restricting their accessibility in high-burden regions. This study developed a rapid, stepwise, low-cost diagnostic workflow that integrates glycan-functionalized magnetic nanoparticles (gMNPs) for mycobacterial isolation and enrichment with a gold nanoparticle (GNP)-based plasmonic biosensor for molecular specificity. Using Mycobacterium smegmatis (Msm) as a validated biosafety level 1 surrogate for Mycobacterium tuberculosis (Mtb), gMNPs were optimized to capture and concentrate mycobacteria from liquid suspension and Ziehl-Neelsen visualization. Following magnetic enrichment and DNA extraction, plasmonic biosensing was performed using an IS6110-targeting TB probe and an Msm-specific probe. Analytical sensitivity and selectivity were assessed using absorbance ratio measurements (A 520 /A 620 ), serial DNA dilutions, and comparisons against non-mycobacterial respiratory species, including Pseudomonas aeruginosa , Streptococcus pneumoniae , Klebsiella pneumoniae , Streptococcus spp. , Escherichia coli , and Staphylococcus aureus . Magnetic enrichment significantly increased bacterial recovery, extending the analytical detection range and enabling visualization of acid-fast bacilli (AFB) as low as 10² CFU/mL, which are otherwise undetectable by conventional smear microscopy. The plasmonic biosensor successfully differentiated mycobacterial samples from non-mycobacterial controls without PCR amplification. Statistically supported limits of detection were determined as 2.5 ng/µL for the Msm-specific probe and 5 ng/µL for the IS6110-targeting TB probe. Sequence alignment demonstrated 87% identity with an IS6110-related region, which likely contributed to measurable hybridization under low-stringency plasmonic conditions. This proof-of-concept study demonstrates a potential rapid, scalable, and low-cost diagnostic platform that combines magnetic enrichment with plasmonic biosensing for mycobacterial detection. By improving bacterial recovery, enhancing microscopic visualization, and enabling molecular discrimination without PCR amplification or complex instrumentation, the integrated gMNP-GNP workflow shows strong potential for translational research toward accessible TB diagnostics in resource-limited settings. However, clinical validation using Mtb and patient specimens remains necessary.