2.3.2. LAM Detection in Human Urine Samples.
Encouraged by the excellent analytical performance of MANP-LFIA in serum matrix, we further evaluated its universality in another noninvasive body fluid, urine samples. Lipoarabinomannan (LAM), a mycobacterial-specific glycolipid shed into urine during active tuberculosis (TB), has been confirmed as a promising TB biomarker that can facilitate TB diagnosis and guide TB treatment.[45−47] We selected LAM as the target for the urinary diagnosis of TB to demonstrate the superiority of our developed MANP-LFIA. The non-sputum POCT method was based on the specific recognition of LAM in urine using a pair of monoclonal antibodies (A12 and A1) on the sensing strip. To optimize the magnetic separation-assisted MANP-LFIA for achieving the best sensitivity for LAM detection on the strip nanobiosensor, we systematically investigated and optimized the probe fabrication parameters, including the coupling pH of A1 mAbs, EDC amount, saturated labeling amount of A1 mAbs on MANP6:6, and the immunoreaction conditions, such as sprayed concentration of A12 mAbs on the T line, added amount of MANP6:6@A1 mAbs probes for each strip, immunoreaction time, running buffer, magnetic separation volume, and urine dilution volume (Figure S14 ).
On the basis of these optimal conditions, a series of urine solutions spiked with LAM in the concentration range of 0 ng mL−1 to 20 ng mL−1 were determined using our developed MANP-LFIA platform. As displayed in typical strip prototypes in Figure 5 a, the fluorescent brightness of the T lines gradually increased as the concentrations of the target LAM increased, showing target concentration-dependent fluorescent signal changes. The MANP-LFIA with magnetic operation presented a more readily visual signal on the T line at low target concentration level compared with that without magnetic assistance. Higher ratiometric FIT/FIC signals were observed in MANP-LFIA with magnetic operation (Figure 5 b), indicating the unique superiority of the dual-functional MANP in enriching LAM and avoiding urine interference. Our magnetic operation-assisted strip nanobiosensor exhibited a broad linear detection range for LAM from 0.01 ng mL−1 to 20 ng mL−1 with a correlation coefficient of 0.997. The linear regression equation for LAM in urine is Y = − 0.30791 + 0.68521 X , where Y represents the logarithm of FIT/FIC, and X is the logarithm of LAM concentration, with an LOD of 0.016 ng mL−1(defined as the concentration corresponding to 20 negative means plus triple standard deviation). By contrast, the direct running of spiked urine without magnetic operation resulted in lower sensitivity with a higher LOD of 0.088 ng mL−1 (Figure5 c). In addition, we constructed a traditional AuNPs-LFIA and only obtained a LOD of 0.7 ng mL−1 (FigureS15 ). This indicates a 5.5-fold and 44-fold higher sensitivity of the magnetic-assisted MANP-LFIA compared with MANP-LFIA without magnetic operation and AuNPs-LFIA, revealing the remarkable advantage of the developed MANP-LFIA in LAM determination.
The specificity of our developed strip biosensor toward LAM was studied by recording the responses against several common mycobacterial strains, including the target Mycobacterium tuberculosis H37Rv and other non-tuberculosis mycobacterial strains (M. simiae , M. scrofulaceum , M. parascrofulaceum , M. moelleri , M. intracellulare , M. paraJortuitum , M. triviale , M. peregrinum , M. abscessus , M. septium , M. mucogenicum , M. avium , M. gilvum , M. chelonei , andM. smegmatis ) with a bacterial concentration of 2.5×104 CFU mL−1. As shown inFigure 5 d, all rapid-growing-mycobacteria (RGM) and the majority of slow-growing-mycobacteria (SGM) species showed negligible signal, whereas a few SGM include M. simiae , M. scrofulaceum , M. parascrofulaceum , M. moelleri , andM. intracellulare exhibited varying degrees of cross-reactivity with M. tuberculosis H37Rv. Furthermore, a series of urine sample solutions collected from 10 individual healthy individuals with LAM spiking concentrations of 0, 100, 500, and 1000 pg mL−1 were analyzed to explore the feasibility of our fabricated analytical platform for determining the presence of targets at different concentration levels. As presented in Figure5 e, the batches of ratiometric signals corresponding to 100, 500, and 1000 pg mL−1 LAM concentrations showed significant statistical differences compared with the blank group, indicating the application potential of the developed MANP-LFIA for clinical TB diagnosis. This objective was straightforwardly achieved by analyzing clinical urine samples from TB patients who have been diagnosed positive through the standard sputum test. The MANP-LFIA diagnosis results in Figure 5 f indicate that our method possesses the ability to differentiate cohorts of TB-positive patients and healthy individuals. These findings demonstrate a highly sensitive response of our dual functional MANP-FLIA test strip to LAM in urine, providing promising potential for the early diagnosis of TB in resource-limited settings.
3. Conclusion
In this study, we successfully addressed the challenge of performance retention in FMN by introducing a novel compact-discrete “fluorescence@magneto” spatial arrangement on the MANP. The extraposition of a magnetic shell effectively circumvents magnetic shielding, allowing for a reduced requirement of Fe3O4@OA amount and consequently decreasing the fluorescent IFE. Additionally, the bright fluorescence can be attributed to the high loading content of AIEgens in the core and the AIE-enhanced photoluminescence. This rational spatial arrangement enables efficient mechanical control-assisted optical signaling in a LIFA platform for PCT and LAM detection, achieving a “win-win” situation where both magnetic and fluorescent activities are performed and validated. The analytical results demonstrate that the MANP probe minimizes interference from human serum and urine matrix while enriching the targets, resulting in remarkably increased sensitivity for the diagnosis of bacterial infection diseases, such as inflammation and TB. We suggest that the as-proposed MANP provides an alternative strategy to guide the design of FMN and holds great promise for multifunctional utilization in areas, such as POCT, fluorescence-magnetic resonance dual-mode bioimaging, and magnetically manipulated biomedicine.