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.