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\begin{document}

\title{Rearranging Fluorescence-Magneto Spatiality for ``Win-Win'' Dual
Functions to Enhance Point-of-Care Diagnosis}



\author[1]{Yu Su}%
\author[1]{Xirui Chen}%
\author[2]{Huan Huang}%
\author[1]{Yuhao Wu}%
\author[1]{Xuan-ang Shen}%
\author[1]{Xiangkai Lin}%
\author[1]{Kun Sun}%
\author[2]{Xiao-Yong Fan}%
\author[1]{Xiaolin Huang}%
\author[1]{Yonghua Xiong}%
\affil[1]{Nanchang University}%
\affil[2]{Fudan University}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Fluorescent-magneto nanoemitters have gained considerable attention for
their applications in mechanical controlling-assisted optical signaling.
However, the incompatibility between magnetic and fluorescent components
often leads to functional limitations in traditional
magneto@fluorescence nanostructure. Herein, we introduce a new
compact-discrete spatial arrangement on a ``fluorescence@magneto''
core--shell nanostructure consisting of a close-packed
aggregation-induced emission luminogen (AIEgen) core and a discrete
magnetic shell. This structural design effectively eliminates the
optical and magnetic interferences between the dual components by
facilitating AIEgens loading in core region and reducing the magnetic
feeding amount through effective exposure of the magnetic units.
Thereby, the resulting magneto-AIEgen nanoparticle (MANP) demonstrates
``win-win'' performances: (i) high fluorescent intensity contributed by
AIEgens stacking-enhanced photoluminescence and reduced photons loss
from the meager magnetic shell; (ii) marked magnetic activity due to
magneto extraposition-minimized magnetic shielding. Accordingly, the
dual functions-retained MANP provides a proof of concept for
construction of an immunochromatographic sensing platform, where it
enables bright fluorescent labeling after magnetically enriching and
separating procalcitonin and lipoarabinomannan in clinical human serum
and urine, respectively, for the clinical diagnosis of bacterial
infections-caused inflammation and tuberculosis. This study not only
inspires the rational design of magnetic-fluorescent nanoemitter, but
also highlights promising potential in magneto-assisted point-of-care
test and biomedicine applications.%
\end{abstract}%



\sloppy


\par\null

\textbf{Rearranging Fluorescence-Magneto Spatialityfor ``Win-Win'' Dual
Functions to Enhance Point-of-Care Diagnosis}

Yu Su\textsuperscript{1, 2}, Xirui Chen\textsuperscript{1, 2}, Huan
Huang\textsuperscript{4, 5}, Yuhao Wu\textsuperscript{1, 2}, Xuan-ang
Shen\textsuperscript{1, 2}, Xiangkai Lin\textsuperscript{1, 2}, Kun
Sun\textsuperscript{1, 2}, Xiao-Yong Fan\textsuperscript{4, *}, Xiaolin
Huang\textsuperscript{1, 2, *} and Yonghua Xiong\textsuperscript{1, 2,
3, *}

{[}1{]} Dr. Y. Su, Dr. X. Chen, Y. Wu, X. Shen, X. Lin, K. Sun, Prof. X.
Huang, Prof. Y. Xiong

State Key Laboratory of Food Science and Resources, Nanchang University,
Nanchang 330047, P. R. China

E-mail: xiaolin.huang@ncu.edu.cn; xiongyonghua@ncu.edu.cn

{[}2{]} Dr. Y. Su, Dr. X. Chen, Y. Wu, X. Shen, X. Lin, K. Sun, Prof. X.
Huang, Prof. Y. Xiong

School of Food Science and Technology, Nanchang University, Nanchang
330047, P. R. China

{[}3{]} Prof. Y. Xiong

Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang
330047, P. R. China

H. Huang, Prof. X.-Y. Fan

Shanghai Institute of Infectious Disease and Biosecurity, Shanghai
Public Health Clinical Center, Fudan University, Shanghai 200032, China

E-mail: xyfan008@fudan.edu.cn

H. Huang

Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention,
Department of Environmental Science \& Engineering, Fudan University,
Shanghai 200438, China

Keywords: fluorescent-magneto nanoemitters, aggregation-induced
emission, self-assembly, point-of-care diagnosis, bacterial infection
diagnosis

Abstract: Fluorescent-magneto nanoemitters have gained considerable
attention for their applications in mechanical controlling-assisted
optical signaling. However, the incompatibility between magnetic and
fluorescent components often leads to functional limitations in
traditional magneto@fluorescence nanostructure. Herein, we introduce a
new compact-discrete spatial arrangement on a ``fluorescence@magneto''
core--shell nanostructure consisting of a close-packed
aggregation-induced emission luminogen (AIEgen) core and a discrete
magnetic shell. This structural design effectively eliminates the
optical and magnetic interferences between the dual components by
facilitating AIEgens loading in core region and reducing the magnetic
feeding amount through effective exposure of the magnetic units.
Thereby, the resulting magneto-AIEgen nanoparticle (MANP) demonstrates
``win-win'' performances: (i) high fluorescent intensity contributed by
AIEgens stacking-enhanced photoluminescence and reduced photons loss
from the meager magnetic shell; (ii) marked magnetic activity due to
magneto extraposition-minimized magnetic shielding. Accordingly, the
dual functions-retained MANP provides a proof of concept for
construction of an immunochromatographic sensing platform, where it
enables bright fluorescent labeling after magnetically enriching and
separating procalcitonin and lipoarabinomannan in clinical human serum
and urine, respectively, for the clinical diagnosis of bacterial
infections-caused inflammation and tuberculosis. This study not only
inspires the rational design of magnetic-fluorescent nanoemitter, but
also highlights promising potential in magneto-assisted point-of-care
test and biomedicine applications.

1. Introduction

The emergence of multifunctional materials (MFMs) that integrate
multiple functions into a single hybrid has revolutionized
nano-operation and expanded the range of
applications.\textsuperscript{{[}1-4{]}}Ideal MFMs should meet two main
criteria: (i) maximize synthesis efficiency and minimize inactive
components by adhering to the principle of ``atom economy'', and (ii)
achieve maximal integrated functionalities, known as ``multifunctional
efficiency''.\textsuperscript{{[}5,6{]}} However, achieving excellent
multiperformance in a single composite remains challenging due to the
mismatch of physicochemical properties and mutual interferences among
functional building blocks.\textsuperscript{{[}7,8{]}}

Fluorescent-magneto nanoemitters (FMNs), as a typical type of MFM,
combine luminous and magnetic properties, enabling brilliant optical
signaling and mechanical magnetic manipulation. FMNs find widespread
applications in
sensing,\textsuperscript{{[}9-11{]}}imaging,\textsuperscript{{[}12,13{]}}biomedicine,\textsuperscript{{[}14,15{]}}
information decoding,\textsuperscript{{[}16{]}} environmental
treatment,\textsuperscript{{[}17,18{]}} nanorobot
operation\textsuperscript{{[}19,20{]}} and
catalysis.\textsuperscript{{[}21{]}} Over the past decades, various
synthesis strategies have been proposed for
FMNs.\textsuperscript{{[}22-25{]}} For example, a layer-by-layer
assembly approach has been applied to synthesize FMNs by
electrostatically attracting oppositely charged polyelectrolytes and
CdTe quantum dots (QDs) on the surface of
Fe\textsubscript{3}O\textsubscript{4}nanoparticles.\textsuperscript{{[}26{]}}
However, the weak binding affinity resulting from electrostatic
interactions often leads to detachment of the exterior fluorescent
particles from the FMNs. Another approach, introduced by Huang \emph{et
al.} , involves the chemical coupling of
Fe\textsubscript{3}O\textsubscript{4} core@silica shell@QDs
satellites,\textsuperscript{{[}27{]}} which improves colloid stability
but compromises the loading capacity of fluorescent materials. In a
remarkable advancement, Hu's lab reported an affinity-driven assembly of
Fe\textsubscript{3}O\textsubscript{4} nanoparticles and QDs in radial
dendritic silica colloids, achieving high packing
density.\textsuperscript{{[}28{]}} These developed MFMs exhibit a
typical ``magneto@fluorescence'' core--shell structure, where the
exterior QD layer causes severe magnetic shielding effects on the
interior magnetic core.\textsuperscript{{[}29,30{]}} Therefore,
increasing the loading of magnetic units becomes necessary to maintain
magnetic manipulation capability. However, excessive magnetic material
loading can lead to fluorescent quenching due to the inner-filter effect
(IFE) because the broad absorption of the magnetic component in the
ultraviolet--visible (UV--vis) light region overlaps with the excitation
and emission spectra of the existing fluorescent
emitter.\textsuperscript{{[}31{]}} In applications, such as lateral flow
immunoassay (LFIA) detection, the mutual interference within FMNs poses
a longstanding challenge. When a large amount of magnetic components
stacks on a nitrocellulose (NC) membrane in the strip, it can cause
nonlinear increments between the fluorescence signal and FMN content on
the test and control line zones, thereby reducing the accuracy of the
strip and potentially resulting in false negative results.

In theory, the rational spatial arrangement of FMNs with a novel
``fluorescence@magneto'' core--shell structure can effectively expose
the magnetic units and reduce magnetic shielding, thereby decreasing the
demand for magnetic loading and alleviating fluorescent IFE.
Consequently, a worthy attempt is to decrease the exterior magneto
loading while increasing the interior fluorophore content to achieve
dual-retained activities. Moreover, ensuring a discrete spatial
distribution of the magnetic layer is crucial to provide sufficient
space for efficient photoluminescence process. Therefore, controlled
phase separation in the nanometer regime through co-assembly becomes
highly desirable.\textsuperscript{{[}32,33{]}} For instance, in our
previous work,\textsuperscript{{[}34{]}} oleic acid-modified CdSe/ZnS
QDs and Fe\textsubscript{3}O\textsubscript{4} nanoparticles were
successfully encapsulated into two polymer matrixes with different
hydrophobic properties using a simple phase separation assembly. In this
case, the magnetic and fluorescent components were mainly distributed in
the outer layer, achieving a discrete co-display distribution that
maintained high saturation magnetization due to the outward location of
magnetic subunits. However, this approach sacrificed the large packing
space inside the particle for fluorescent material because the maximized
fluorophore coverage on the exterior interface was still not abundant
enough to achieve a more enhanced fluorescent signal. Moreover, spatial
separation of the two components is advantageous in minimizing
multicomponent interference.\textsuperscript{{[}35{]}} In addition,
organic fluorescent emitters or inorganic fluorescent materials often
suffer from the aggregation-caused quenching effect at high fluorophore
concentrations or in the solid state, which hinders the enhancement of
fluorescent brightness in compactly packed FMN
hybrids.\textsuperscript{{[}36-38{]}} By contrast, luminogens with
aggregation-induced emission
(AIEgens),\textsuperscript{{[}39,40{]}}characterized by propeller-shaped
structures, provide an excellent choice for remarkably increasing
molecule loading and enhancing fluorescent intensity.

In this study, we present a novel compact-discrete
``fluorescence@magneto'' spatial arrangement by incorporating AIEgens
into the core and hydrophobic Fe\textsubscript{3}O\textsubscript{4}
nanoparticles into the polymer shell (\textbf{Scheme 1} ). This rational
spatial design of the magneto-AIE nanoparticle (MANP) largely reduces
magnetic shielding on the exterior magnetic shell, enabling a lower
magnetic feeding amount. Consequently, the fluorescent performance is
effectively maintained because of AIE-enhanced photoluminescence, a
large AIEgen loading capacity, and reduced IFE by the meager magnetic
shell. Capitalizing on the dual-retained magnetic and fluorescent
properties, the MANP was employed as a bifunctional LFIA nanoprobe
(MANP-LFIA) to magnetically enrich, separate, and fluorescently label
procalcitonin (PCT) in serum samples and lipoarabinomannan (LAM) in
urine samples of tuberculosis (TB) patients with remarkably enhanced
sensitivity. We believe that the proposed MANP offers a new approach for
the design of highly performing FMNs and holds great potential for
applications in point-of-care test (POCT), fluorescent-magnetic
resonance dual-mode imaging, and magnetically manipulated biomedicine.

Results and Discussion

\textbf{2.1. Synthesis and Characterization of the Compact-Discrete
MANP.}

The fabrication of MANP involved a one-pot emulsification and
evaporation method, where superparamagnetic
Fe\textsubscript{3}O\textsubscript{4} NPs@OA (\textbf{Figure S1} ) and
red-emitting AIEgens (\textbf{Figure S2} ) were co-assembled as
functional units, along with poly(maleicanhydride-alt-1-octadecene)
(PMAO) polymer as the supporting skeleton. In this process, 6 mg of
hydrophobic Fe\textsubscript{3}O\textsubscript{4} NPs@OA, 6 mg of
AIEgens, and 5 mg of PMAO were dissolved in chloroform and underwent
micellar encapsulation in an aqueous phase using sodium dodecyl sulfate
(SDS) as a surfactant. During the evaporation of chloroform, the
components (Fe\textsubscript{3}O\textsubscript{4} NPs@OA, AIEgens, and
PMAO) self-assembled and co-constructed a spherical nanocomposite called
MANP\textsubscript{6:6}. Scanning electron microscopy (SEM) image
(\textbf{Figure 1} a) and transmission electron microscopy (TEM) image
(\textbf{Figure} \textbf{1} b) demonstrate the homogeneous spherical
morphology of MANP\textsubscript{6:6}. The magnified TEM image
(\textbf{Figure1} c) clearly shows a core--shell nanostructure with a
compact AIE core and a discretely packed
Fe\textsubscript{3}O\textsubscript{4}NPs@OA shell, indicating a phase
separation process within MANP\textsubscript{6:6} due to the different
polarities of the building blocks. By contrast, PMAO polymer
nanoparticles (PNP), AIE nanoparticles (ANP\textsubscript{6}), and
magnetic nanoparticles (MNP\textsubscript{6}) were synthesized as single
functional component analogs. TEM image in\textbf{Figure} \textbf{1} d
reveals the solid spherical structure of PNP. However,
ANP\textsubscript{6} displays a distinct AIE core-PMAO shell structure
due to the polarity difference between AIEgens and the polymer
(\textbf{Figure} \textbf{1} e). \textbf{Figure} \textbf{1} f shows that
MNP\textsubscript{6} exhibits a spherical geometry with
Fe\textsubscript{3}O\textsubscript{4} NPs@OA homogeneously distributed
throughout the nanoparticle, owing to the high compatibility between OA
and PMAO. These results support the reasonable formation mechanism of
MANP: (i) the high mutual affinity between OA, PMAO, and SDS leads to
the uniform assembly of Fe\textsubscript{3}O\textsubscript{4} NPs@OA on
the exterior PMAO shell, close to the SDS aqueous phase, with a discrete
distribution; (ii) the aromatic AIEgens tend to migrate to the particle
center and aggregate together due to their incompatible molecular
affinity with the shell components.

Furthermore, \textbf{Figure} \textbf{1} g shows that
MANP\textsubscript{6:6} exhibits the characteristic UV--vis spectrum of
ANP\textsubscript{6} and demonstrates an absorption lift in the 200--600
nm range due to the incorporation of the magnetic component. By
contrast, PNP exhibits negligible light absorption, indicating that the
polymer matrix does not contribute to optical interference in
MANP\textsubscript{6:6}. The energy dispersive X-ray (EDX) elemental
mapping images display the distribution of C, Fe, O, and S in
MANP\textsubscript{6:6}. \textbf{Figure} \textbf{1} h shows that the C
elements are primarily concentrated in the core position, Fe and O
elements are located on the exterior shell, and S elements are uniformly
distributed throughout MANP\textsubscript{6:6}. Further EDX line scan
(\textbf{Figure} \textbf{1} i) across the center of
MANP\textsubscript{6:6} confirms the presence of a nuclear C core and an
out-layer Fe, verifying the distinct assembling spatiality of
Fe\textsubscript{3}O\textsubscript{4} NPs@OA and AIEgens in the
core--shell magnetic-fluorescent MANP\textsubscript{6:6}. X-ray
photoelectron spectroscopy (XPS) analysis (\textbf{Figures 1} j,
k,\textbf{Figure} \textbf{S3} ) implies the co-existence of C, O, and Fe
in MANP\textsubscript{6:6}. In addition, the powder X-ray diffraction
(XRD) analysis (\textbf{Figure} \textbf{1} l) reveals characteristic
diffraction peaks corresponding to the (220), (311), (400), (511), and
(440) planes of superparamagnetic
Fe\textsubscript{3}O\textsubscript{4}crystal, suggesting the successful
loading of Fe\textsubscript{3}O\textsubscript{4} NPs@OA in
MANP\textsubscript{6:6}. Dynamic light scattering measurements show that
MANP\textsubscript{6:6}is well-monodispersed with a uniform hydrodynamic
size of \textasciitilde{}320 nm (\textbf{Figure} \textbf{1} m).
Moreover, the surface of MANP\textsubscript{6:6} becomes predominantly
negatively charged after the carboxylation process, which is beneficial
for subsequent biomodification (\textbf{Figure 1} n). The fluorescent
spectra demonstrate that MANP\textsubscript{6:6} exhibits maximum
excitation wavelengths at 365 and 465 nm, with a maximum emission peak
at 600 nm (\textbf{Figure S4} ). This red-light emitter property enables
MANP\textsubscript{6:6} to effectively resist biological interference.
Collectively, these results provide strong evidence for the successful
construction of MANP.

\textbf{2.2. ``Win-Win'' Magneto-Fluorescent Performances of MANP.}

The magneto-fluorescent performance of MANP is influenced by its
well-defined spatial structure, including the compact AIE core, discrete
magneto shell, and the composition ratio of its functional components.
Therefore, a rational regulation of the optical and magnetic integration
in MANP was conducted to achieve dual-retained performances. SEM and TEM
images shown in \textbf{Figures 2} a--c and \textbf{Figure}
\textbf{S5}demonstrate that by increasing the feeding amount of
Fe\textsubscript{3}O\textsubscript{4} NPs@OA from 2 mg to 16 mg while
keeping the AIEgens mass at 6 mg, the MANP\textsubscript{2:6},
MANP\textsubscript{4:6}, MANP\textsubscript{6:6},
MANP\textsubscript{8:6}, and MANP\textsubscript{16:6} maintain the
core--shell nanostructure. However, an increase in
Fe\textsubscript{3}O\textsubscript{4} NPs@OA content results in gradual
surface roughness and denser magnetic inlay. The increased magnetic
feeding also leads to higher UV--vis absorbance of the synthesized MANP
due to the intrinsic optical absorption of
Fe\textsubscript{3}O\textsubscript{4} NPs@OA (\textbf{FigureS6} ).
Fluorescent lifetime measurements show negligible lifetime decay when
incorporating 6 mg or 16 mg Fe\textsubscript{3}O\textsubscript{4} NPs@OA
into MANP in comparison to ANP\textsubscript{6} (\textbf{Figure}
\textbf{2} d). These results imply the occurrence of a fluorescent IFE
in MANP, which inevitably quenches the final fluorescent emission. As
validated in \textbf{Figure2} e, a higher proportion of magnetic
component corresponds to a gradually decreased fluorescent signal.
Additionally, \textbf{Figures 2} f and \textbf{S7} show that the
magnetic recovery of MANP under an external magnetic field increases
with the amount of Fe\textsubscript{3}O\textsubscript{4} NPs@OA and
reaches a plateau at a Fe\textsubscript{3}O\textsubscript{4} NPs@OA
content of 6 mg. On the basis of these observations, the fluorescent
intensity of MANP\textsubscript{6:6} exhibits a much higher retention
rate of 82\%, which is much higher than the traditionally reported
``magneto@fluorescence'' core-shell nanostructure possessing a
fluorescent retention rate below 50\%.\textsuperscript{{[}41{]}} This
can be attributed to the outstanding luminescence property of AIEgens in
an aggregated state and the discrete distribution of
Fe\textsubscript{3}O\textsubscript{4} NPs@OA that enables effective
passage of photons through the polymer shell layer. Therefore,
simultaneous high magneto-fluorescent activities can be achieved by
constructing MANP\textsubscript{6:6} with magnetic and fluorescent of 6
mg each.

The magnetic performance of the well-emitted MANP\textsubscript{6:6} was
evaluated. As shown in \textbf{Figure} \textbf{3} a,
MNP\textsubscript{6}and ANP\textsubscript{6} exhibit either magnetic or
fluorescent responsiveness, respectively, due to their single functional
component. By contrast, MANP\textsubscript{6:6} can be readily collected
and exhibits bright fluorescence under an external magnetic field and UV
light source. The magnetization curves in \textbf{Figure} \textbf{3} b
quantify that MANP\textsubscript{6:6} exhibits superparamagnetism with a
saturation magnetization of 24.8 emu g\textsuperscript{-1}, which is
remarkably higher than that of MNP\textsubscript{6} (20.9 emu
g\textsuperscript{-1}). This indicates that the solid structure of
MNP\textsubscript{6}, with Fe\textsubscript{3}O\textsubscript{4} NPs@OA
filling the interior of the nanoparticle, can suffer from a magnetic
shielding effect caused by the nonmagnetic dielectric polymer
matrix.\textsuperscript{{[}42{]}} In the contrastive case of
MANP\textsubscript{6:6}, the well-defined core--shell nanostructure
allows the magnetic constituent to spread over the exterior shell while
the AIEgens occupy the inner core space. This leads to a highly retained
saturation magnetization. In other words, the rational spatial
distribution of Fe\textsubscript{3}O\textsubscript{4} NPs@OA on the
exterior shell layer effectively guarantees the magnetic performance of
MANP\textsubscript{6:6}, curtails magnetic constituent to suppress
fluorescent quenching, and improves optical sensitivity. Thus, the
constructed MANP\textsubscript{6:6} possesses excellent dual
functionality with simultaneous high magnetic manipulation and optical
output activity.

To further evaluate the fluorescent feasibility of the nanoparticles on
paper test strips, we also investigated the IFE of
MANP\textsubscript{6:6} on the strip by spraying a series of particle
concentration of MANP\textsubscript{6:6} on the NC membrane as the T
line. As presented in \textbf{Figures 3} c, d, the fluorescent
brightness of MANP\textsubscript{6:6} shows a synchronized tendency with
that of ANP\textsubscript{6}, with a limit of detection (LOD) of 42 ng
mL\textsuperscript{-1} particle concentration. Moreover, negligible
fluorescent decay of MANP\textsubscript{6:6} and ANP\textsubscript{6}
occurs at a high particle concentration of 2.8 mg
mL\textsuperscript{-1}(\textasciitilde{}0.8 \selectlanguage{greek}μ\selectlanguage{english}g/strip). This indicates
that the particle stacking process on the NC membrane does not generate
an obvious fluorescent IFE owing to the reduced magnetic loading amount
in MANP\textsubscript{6:6}. The effective avoidance of IFE is thus
crucial in LFIA detection (\textasciitilde{}0.6 \selectlanguage{greek}μ\selectlanguage{english}g/strip in further POCT
sensors). Furthermore, the versatility of the synthesis strategy was
confirmed by individually incorporating green or yellow AIEgens
(\textbf{Figures S8} a, b) into the nanoparticles. As shown
in\textbf{Figures S8} c--f, the obtained green, yellow, and red
MANP\textsubscript{6:6} emit characteristic fluorescence signals at 500,
545, and 600 nm. Confocal laser scanning microscope imaging results
(\textbf{Figure} \textbf{S8} g) demonstrate that multicolor spheres with
single-particle brightness can be visualized simultaneously under a
single excitation. This indicates that the proposed MANP holds great
potential for multicolor fluorescent imaging and high-throughput
multiplex detection.

Given the excellent combination of magnetic and fluorescent performance,
MANP\textsubscript{6:6} shows promise as a dual-functional nanoprobe for
bioanalytical applications. The following investigations were conducted
to ensure its stability in further bioapplications. As shown
in\textbf{Figure} \textbf{3} e, MANP\textsubscript{6:6} exhibits slight
fluorescent decrease and hydrodynamic size increase in acidic or basic
conditions, with negligible change in magnetic recovery across pH values
ranging from 2 to 11. These results indicate that
MANP\textsubscript{6:6} maintains good optical, magnetic, and colloid
stability over a wide pH range, making it suitable for various
biochemical applications. Furthermore, the thermal stability of
MANP\textsubscript{6:6} was evaluated by storing the particles at 60 \selectlanguage{ngerman}°C
for one week. As depicted in \textbf{Figure} \textbf{3} f,
MANP\textsubscript{6:6} demonstrates excellent optical, magnetic, and
colloid stability under elevated temperatures, indicating superior
long-term stability. In addition, the stability of
MANP\textsubscript{6:6} in biological matrices was examined by
incubating the particles in PB, human serum, and human urine for 7 days.
As shown in \textbf{Figures 3} g--i, MANP\textsubscript{6:6} exhibits
stable fluorescent signals, consistent magnetic recoveries, and high
colloid stability in PB, serum, and urine media over the course of 7
days. Taken altogether, these results demonstrate the excellent colloid,
optical, magnetic, and thermal stability of MANP\textsubscript{6:6},
which is vital for its multifunctional bioanalytical application,
enabling simultaneous magnetic separation, enrichment, and fluorescent
signal output.

\textbf{2.3. POCT Applications of MANP in Ultrasensitive
Immunochromatographic Detection.}

The remarkable magneto-fluorescence retention and prominent stability of
MANP\textsubscript{6:6} make it practical for biomedical applications.
In this study, we further investigated the feasibility of
MANP\textsubscript{6:6} as a dual-functional magnetic separation and
fluorescent nanoprobe in the field of POCT, specifically using LFIA as
the detection platform. \textbf{Scheme 1} depicts the construction
principle of the MANP-FLIA platform for ultrasensitive detection of
targets in human serum/urine. The assay is based on a double-antibody
sandwich immunoassay format. When the targets are present in serum or
urine, the antigens are initially bound to monoclonal antibodies
modified MANP\textsubscript{6:6} (MANP\textsubscript{6:6}@mAbs), forming
a MANP\textsubscript{6:6}@mAbs immunocomplex. The immunocomplex is then
collected by an external magnetic field and resuspended in buffer
solution. The complex solution was then applied to the LFIA test strip,
which has antibodies immobilized on the NC membrane. This results in the
formation of a nanoprobe/antigen/antibody sandwich architecture on the T
line with high fluorescent signals (FI\textsubscript{T}). Additionally,
excess MANP\textsubscript{6:6}@mAbs are captured by the goat anti-mouse
IgG on the C line, producing a corresponding fluorescent signal
(FI\textsubscript{C}). In the absence of the targets, the
MANP\textsubscript{6:6}@mAbs run to the C line and react with the IgG.
The presence or absence of fluorescent lines provide a direct visual
diagnosis result: one line (C) indicates a negative result for the
targets, whereas two lines (T + C) indicate a positive result. In
addition, the ratio of FI\textsubscript{T}/FI\textsubscript{C} is used
to accurately quantify the target concentration, which effectively
circumvents the intensity fluctuations by employing a ratiometric
strategy.

\emph{2.3.1. PCT Detection in Human Serum Samples.}

PCT was selected as the serum target, which is a biomarker of sepsis and
pyemia caused by bacterial infection. Accurate monitoring of PCT at low
concentration ranges is of great importance for indicating the degree of
inflammation and guiding antibiotic
therapy.\textsuperscript{{[}43,44{]}} To begin with, the coupling
conditions of the MANP\textsubscript{6:6}@PCT-mAbs were adjusted to
optimize the final performance. This optimization included adjusting the
coupling pH of PCT-mAbs, the amount of EDC used, and the saturated
labeling amount of anti-PCT mAbs on MANP\textsubscript{6:6}
(\textbf{Figures S} 9a--c). As shown in \textbf{Figure} \textbf{S9} d,
the as-obtained MANP\textsubscript{6:6}@PCT-mAbs nanoprobes exhibited
good monodispersity with a slight size increase of 20 nm compared with
that of MANP\textsubscript{6:6}. Additionally, the zeta potential of
MANP\textsubscript{6:6}@PCT-mAbs was decreased by 23.7 mV, due to the
presence of the PCT-mAbs with negative charge (\textbf{FigureS9} e).
These observations demonstrate the successful modification of antibodies
on the MANP\textsubscript{6:6}. Moreover, the as-fabricated
MANP\textsubscript{6:6}@PCT-mAbs probes maintained the fluorescent
profile and brightness (\textbf{Figure} \textbf{S9} f), which remarkably
ensured the detection sensitivity of the LFIA strip. Besides, maximum
responses were further obtained by optimizing the strip and running
parameters, such as the sprayed concentration of PCT-mAbs on the T line,
the added amount of MANP\textsubscript{6:6}@PCT-mAbs probes for each
strip, the immunoreaction time, and the magnetic separation volume
(\textbf{Figures S10} a--d). Furthermore, the serum dilution volume was
investigated to achieve the best immunoreaction and magnetic capture
efficiency in practical samples. As revealed in \textbf{Figure}
\textbf{S10} e, the fluorescent intensity increases with the dilution
degree, and a signal plateau is reached with a twofold serum dilution,
effectively eliminating serum matrix interference on the immunoreaction
and LFIA fluorescent reading.

Under the developed conditions mentioned above, PCT standards in serum
solutions with various concentrations were magnetically enriched and
detected using MANP\textsubscript{6:6}@PCT-mAbs probes-immunolabeled
LFIA test strips. As shown in \textbf{Figure} \textbf{4} a, the
fluorescent signal on the T line gradually increases as the
concentration of PCT increases, followed by a decrease in the
fluorescent signal on the C line. The logarithm of the ratiometric
FI\textsubscript{T}/FI\textsubscript{C} linearly increases with the
logarithm of the antigen concentration in the range from 0.0018 ng
mL\selectlanguage{english}\textsuperscript{-1} to 62.5 ng mL\selectlanguage{english}\textsuperscript{-1}, as shown
in\textbf{Figure} 4b. The linear regression equation for PCT in serum
is\emph{Y} = 0.9992 \emph{X} \textsuperscript{0.7798}, where \emph{Y}
represents the logarithm of FI\textsubscript{T}/FI\textsubscript{C}, and
\emph{X} is the logarithm of PCT concentration, with a linear regression
coefficient of 0.9977. The LOD of this method for PCT was determined to
be 0.0012 ng mL\selectlanguage{english}\textsuperscript{-1} (defined as the concentration
corresponding to 20 negative means plus triple standard deviation). In
comparison, direct detection of PCT in serum without magnetic separation
resulted in a liner responding range (0.0075--62.5 ng
mL\selectlanguage{english}\textsuperscript{-1}, \emph{R} \textsuperscript{2} = 0.9947) with a
higher LOD of 0.0068 ng mL\selectlanguage{english}\textsuperscript{-1}(\textbf{Figures 4} b,
\textbf{S11} ). Moreover, a traditional AuNPs-based colorimetric LFIA
(AuNPs-LFIA) was constructed, which showed a much narrower liner
responding range (0.06--15.6 ng mL\selectlanguage{english}\textsuperscript{-1},\emph{R}
\textsuperscript{2} = 0.9826) and a higher LOD of 0.06 ng
mL\selectlanguage{english}\textsuperscript{-1} (\textbf{Figures 4} b, \textbf{S12} ). This
indicates a 5.6-fold and 50-fold higher sensitivity of the
magnetic-assisted MANP-LFIA compared with MANP-LFIA without magnetic
operation and AuNPs-LFIA, demonstrating the remarkable superiority of
the developed sensor in detecting trace targets. Furthermore, the upper
limit of PCT detection by magnetic-assisted MANP-LFIA is lifted by four
times compared with that of AuNPs-LFIA, which is highly important to
severe inflammation diagnosis. Such excellent analytical performance is
attributable to the magnetic operation, which effectively enriches the
PCT protein from a larger sample volume to obtain a higher target amount
and extracts the PCT from the serum to mitigate matrix interference, and
high fluorescent brightness of MANP\textsubscript{6:6} with weak
IFE.\textbf{Figure} \textbf{4} c shows the typical strip prototypes
after the detection of various concentration (0--250 ng
mL\selectlanguage{english}\textsuperscript{-1}) of PCT-spiked serum samples. Clearly, the
brightness of the T lines remarkably increases with an increasing
concentration of the target. The particle location was also confirmed by
SEM. As shown in \textbf{Figure4} d, masses of
MANP\textsubscript{6:6}@PCT-mAbs are immobilized tightly on the NC fiber
in the T line region in the case of a PCT concentration of 100 ng
mL\selectlanguage{english}\textsuperscript{-1}, whereas negligible nonspecific adsorption is
observed on the NC membrane in the PCT-negative sample, which matches
well with the visual result. These results demonstrate that the proposed
MANP-FLIA provided good sensitivity for POCT detection, and the paper
strip platform provides visual analysis of biomarker monitoring. The
detection specificity is also a key issue in immunoassays. Here,
hepatitis B surface antigen (HBsAg), prostate-specific antigen (PSA),
human chorionic gonadotropin (HCG), HIV p24 antigen, carcinoembryonic
antigen (CEA), \selectlanguage{greek}α-\selectlanguage{english}fetoprotein (AFP), N-terminal prohormone of brain
natriuretic peptide (NT-ProBNP), and C-reactive protein (CRP) were
selected as the interfering proteins to evaluate the selectivity of the
immunoassay platform. The FI\textsubscript{T}/FI\textsubscript{C} of PCT
at 1 ng mL\textsuperscript{-1} was compared with those of interference
species at the 1000 ng mL\textsuperscript{-1} concentration level. As
shown in the spider diagram in \textbf{Figure} \textbf{4} e and
photograph in\textbf{Figure} \textbf{S13} , a negligible signal was
detectable in the interference species, whereas a strong fluorescent
signal was observed in the PCT sample. This result demonstrates that the
proposed MANP-FLIA platform exhibits good specificity to the target
protein and has the ability to differentiate it from complex samples. In
addition, the accuracy and precision of the MANP-LFIA method were
investigated by determining the intra- and inter-assay recoveries and
percent coefficients of variation (\%CVs) of seven PCT-spiked serum
samples with concentrations of 0.05, 0.1, 0.5, 1, 5, 10, and 50 ng
mL\textsuperscript{-1}. As revealed in \textbf{Table S1} , the average
recoveries of the intra-assay ranged from 84.7\% to 105.6\% with CVs
ranging from 3.49\% to 9.66\%, and the average recoveries of the
inter-assay ranged from 85.2\% to 96.3\% with CVs ranging from 7.3\% to
14.4\%. These results indicate that the proposed MANP-LIFA is suitable
for accurate PCT quantification with satisfactory precision.

Furthermore, we demonstrated the reliability and practicability of the
proposed method by measuring 34 human blood plasma samples from
patients. As shown in \textbf{Figure} \textbf{4} f and \textbf{Table S2}
, the detection results showed a high level of agreement compared with
those measured by commercial chemiluminescence immunoassay (CLIA) kits,
without any false-positive or false-negative signals. On this basis, a
regression analysis was conducted between the proposed method and the
standard CLIA by the hospital. \textbf{Figure} \textbf{4} g demonstrates
a strong positive correlation between the MANP-FLIA method and the
standard CLIA, with correlation coefficients of 0.966. These results
confirm that the proposed method is accurate and valid for clinical
diagnosis.

\emph{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.\textsuperscript{{[}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
MANP\textsubscript{6:6}, and the immunoreaction conditions, such as
sprayed concentration of A12 mAbs on the T line, added amount of
MANP\textsubscript{6:6}@A1 mAbs probes for each strip, immunoreaction
time, running buffer, magnetic separation volume, and urine dilution
volume (\textbf{Figure} \textbf{S14} ).

On the basis of these optimal conditions, a series of urine solutions
spiked with LAM in the concentration range of 0 ng
mL\textsuperscript{-1} to 20 ng mL\textsuperscript{-1} were determined
using our developed MANP-LFIA platform. As displayed in typical strip
prototypes in \textbf{Figure} \textbf{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
FI\textsubscript{T}/FI\textsubscript{C} signals were observed in
MANP-LFIA with magnetic operation (\textbf{Figure} \textbf{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\textsuperscript{-1} to 20 ng
mL\textsuperscript{-1} with a correlation coefficient of 0.997. The
linear regression equation for LAM in urine is \emph{Y} = - 0.30791 +
0.68521 \emph{X} , where \emph{Y} represents the logarithm of
FI\textsubscript{T}/FI\textsubscript{C}, and \emph{X} is the logarithm
of LAM concentration, with an LOD of 0.016 ng
mL\textsuperscript{-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\textsuperscript{-1}
(\textbf{Figure5} c). In addition, we constructed a traditional
AuNPs-LFIA and only obtained a LOD of 0.7 ng mL\textsuperscript{-1}
(\textbf{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 \emph{Mycobacterium tuberculosis} H37Rv and other
non-tuberculosis mycobacterial strains (\emph{M. simiae} , \emph{M.
scrofulaceum} , \emph{M. parascrofulaceum} , \emph{M. moelleri} ,
\emph{M. intracellulare} , \emph{M. paraJortuitum} , \emph{M. triviale}
, \emph{M. peregrinum} , \emph{M. abscessus} , \emph{M. septium} ,
\emph{M. mucogenicum} , \emph{M. avium} , \emph{M. gilvum} , \emph{M.
chelonei} , and\emph{M. smegmatis} ) with a bacterial concentration of
2.5\selectlanguage{ngerman}×10\textsuperscript{4} CFU mL\selectlanguage{english}\textsuperscript{-1}. As shown
in\textbf{Figure} \textbf{5} d, all rapid-growing-mycobacteria (RGM) and
the majority of slow-growing-mycobacteria (SGM) species showed
negligible signal, whereas a few SGM include \emph{M. simiae} , \emph{M.
scrofulaceum} , \emph{M. parascrofulaceum} , \emph{M. moelleri} ,
and\emph{M. intracellulare} exhibited varying degrees of
cross-reactivity with \emph{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\selectlanguage{english}\textsuperscript{-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 \textbf{Figure5} e,
the batches of ratiometric signals corresponding to 100, 500, and 1000
pg mL\selectlanguage{english}\textsuperscript{-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
\textbf{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
Fe\textsubscript{3}O\textsubscript{4}@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.

\textbf{4. Experimental Section}

\emph{Materials} : The red, green, and yellow AIEgens were generously
provided by Jiangxi Weibang Biotechnology Co., Ltd. Ferric chloride
(FeCl\textsubscript{3}*6H\textsubscript{2}O), ferrous chloride
(FeCl\textsubscript{2}*4H\textsubscript{2}O), oleic acid (OA), SDS,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), (PMAO, MW =
30,000-50,000 Da), casein, and bovine serum albumin (BSA) were purchased
from Sigma-Aldrich. The sample pad, NC membrane, and absorbent pad were
obtained from Schleicher and Schuell GmbH (Dassel, Germany). Anti-PCT
monoclonal antibodies (mAbs), anti-PCT polyclonal antibodies (pAbs), PCT
standards, and goat anti-mouse IgG antibodies were obtained from Hua Kui
Jin Pei Biotechnology Inc. (Shanghai, China). Human serum containing PCT
was collected from Jiangxi Provincial People's Hospital (Nanchang,
China). Anti-LAM mAbs (A12 and A1) and LAM standards were provided by
the Shanghai Public Health Clinical Center (Shanghai, China). All
chemicals were of analytical grade and used as received without further
purification.

\emph{Apparatus and Characterizations} : Transmission electron
microscopy (TEM) images and energy dispersive X-ray (EDX) images were
obtained using an FEI Talos F200x at an accelerating voltage of 200 kV.
Scanning electron microscopy (SEM) images were acquired using a Hitachi
SU8100 scanning electron microscope (Tokyo, Japan). The hydrodynamic
size distributions and zeta potentials were measured using a scientific
NP analyzer (Malvern Nano ZSE, London, UK). Small angle powder X-ray
diffraction (XRD) measurements were conducted on a Bruker D8 ADVANCE
diffractometer using CuK\selectlanguage{greek}α \selectlanguage{english}radiation (\selectlanguage{greek}λ \selectlanguage{english}= 0.15405 nm). X-ray
photoelectron spectroscopy (XPS) analysis was conducted using an
ESCALAB25OXi spectrometer (Thermo Fisher Scientific Co., USA). UV--vis
spectra were recorded using a Hitachi U-3900 spectrophotometer (Tokyo,
Japan). Fluorescence spectra were collected using an F-380 fluorescence
spectrophotometer (Tianjin, China). The magnetic properties were
characterized using a superconducting quantum interference device at 300
K. The BioDot XYZ platform, equipped with a motion controller, a BioJet
Quanti3000k dispenser, and an AirJet Quanti3000k dispenser for solution
dispensing, was supplied by BioDot (Irvine, CA). The portable
fluorescent strip reader was obtained from Fenghang Laboratory
Instrument Co. Ltd (Hangzhou, China).

\emph{Synthesis of Fe\textsubscript{3}O\textsubscript{4} NPs@OA} :
Fe\textsubscript{3}O\textsubscript{4} NPs@OA with a diameter of
\textasciitilde{}10 nm were synthesized in accordance with a previously
reported method.\textsuperscript{{[}42{]}} In detail, a 150 mL aqueous
solution containing 1.59 g of FeCl\textsubscript{2}\selectlanguage{ngerman}·4H\textsubscript{2}O
and 2.59 g of FeCl\textsubscript{3}·6H\textsubscript{2}O was heated to
50 °C with N\textsubscript{2} bubbling for 15 min. Subsequently, 12.5 mL
of NH\textsubscript{3}·H\textsubscript{2}O (25\% v/v) was quickly added
to the mixture under vigorous stirring at 500 rpm for 30 min, resulting
in a color change from yellow to black. The formed precipitate was
collected under an additional magnetic field and washed with water five
times to achieve a neutral pH. The precipitate was then ultrasonically
dispersed in 100 mL of water. Following that, 1.2 mL of OA was added to
the dark suspension, which was stirred for 3 h at 70 °C under
N\textsubscript{2} protection. Finally, the oily
Fe\textsubscript{3}O\textsubscript{4} NPs@OA product was washed with
ethanol and resuspended in chloroform for further use.

\emph{Synthesis of MANP\textsubscript{6:6}, PNP, ANP\textsubscript{6},
and MNP\textsubscript{6}} : MANP\textsubscript{6:6} was prepared by
co-assembling Fe\textsubscript{3}O\textsubscript{4} NPs@OA and AIEgens
into a polymer matrix using our reported emulsification method with some
modifications.\textsuperscript{{[}42{]}} Briefly, 5 mg of PMAO, 6 mg of
Fe\textsubscript{3}O\textsubscript{4} NPs@OA, and 6 mg of AIEgens were
completely dissolved in 150 \selectlanguage{greek}µ\selectlanguage{english}L of chloroform to form an oil phase. Then,
400 \selectlanguage{greek}µ\selectlanguage{english}L of 0.2 mg mL\textsuperscript{-1} SDS solution as the aqueous
phase was added to the oil phase. The mixture was emulsified
\emph{via}sonicating for 5 min (9.9 s working and 5.5 s pausing) at an
ultrasound power of 114 W. The resulting solution was then vortexed and
evaporated for 20 min to remove the chloroform, resulting in the
assembly of Fe\textsubscript{3}O\textsubscript{4} NPs@OA and AIEgens
into MANP\textsubscript{6:6}. The obtained MANP\textsubscript{6:6} was
centrifuged at 8,000 rpm for 15 min, and the pellet was resuspended in
alkaline water (pH 9) overnight to hydrolyze the anhydride of PMAO and
generate carboxyl groups for further antibody coupling. The carboxylated
MANP\textsubscript{6:6} was washed three times with distilled water,
adjusting the pH to neutral each time, by centrifugation at 8,000 rpm
for 15 min. The final product was dispersed in ultrapure water for
further use.

The synthesis of PNP, ANP\textsubscript{6}, and
MNP\textsubscript{6}followed a similar procedure to that of
MANP\textsubscript{6:6}, with the only difference being the feeding
groups of PMAO, PMAO and AIEgens, and PMAO and
Fe\textsubscript{3}O\textsubscript{4} NPs@OA as the building blocks,
respectively.

\emph{Synthesis of MANP\textsubscript{6:6}@PCT-mAbs and
MANP\textsubscript{6:6}@LAM-mAbs} : The MANP\textsubscript{6:6}@PCT-mAbs
nanoprobes were synthesized using the EDC method to form an amide bond
between the carboxyl group of MANP\textsubscript{6:6} and the amino
group of anti-PCT mAbs. The same procedure was followed for the
synthesis of MANP\textsubscript{6:6}@LAM-mAbs, with A1 antibody used as
the LAM detective antibody. Briefly, 6 \selectlanguage{greek}μ\selectlanguage{english}g of anti-PCT mAbs was added to
400 \selectlanguage{greek}μ\selectlanguage{english}L of 0.01 M PB solution (pH 7.4) containing 20 \selectlanguage{greek}µ\selectlanguage{english}g of
MANP\textsubscript{6:6}, and the mixture was incubated for 30 min. Then,
2 \selectlanguage{greek}μ\selectlanguage{english}g of EDC was added to the solution and stirred for 30 min at room
temperature. This step was repeated for three times. Subsequently, 7 mg
of casein was used to block the unbound part of
MANP\textsubscript{6:6}for 1 h. Finally, the mixture was centrifuged at
9,000 rpm for 5 min, and the precipitates were resuspended in 100 \selectlanguage{greek}µ\selectlanguage{english}L of
0.01 M PB (pH 7.4) containing 25\% w/v saccharose, 1\% w/v BSA, and
0.1\% w/v NaN\textsubscript{3} and stored at 4 \selectlanguage{ngerman}°C for further use.

\emph{Fabrication of MANP-LFIA Strips} : The
MANP\textsubscript{6:6}-LFIA strip fabrication process, including both
PCT and LAM detection, is described as follows: For PCT detection,
anti-PCT pAbs (2 mg mL\selectlanguage{english}\textsuperscript{-1}) and goat anti-mouse IgG (1
mg mL\selectlanguage{english}\textsuperscript{-1}) were sprayed on the NC membrane at a density
of 0.74 \selectlanguage{greek}μ\selectlanguage{english}L cm\textsuperscript{-1} as the test (T) and control (C) lines,
respectively, using the ZX1000 dispensing platform. The modified NC
membrane was dried at 37 \selectlanguage{ngerman}°C overnight. Then, the absorbent pads, treated
NC membrane, and sample pads were assembled in sequence onto the PVC
backing card with an overlap, ensuring secure attachment. The assembled
strip was divided into pieces with a width of 3.9 mm. The as-prepared
strips were packaged in a sealed bag and stored in dry and cool
conditions for further use.

For LAM detection, A12 mAbs (1 mg mL\selectlanguage{english}\textsuperscript{-1}) and goat
anti-mouse IgG (2 mg mL\selectlanguage{english}\textsuperscript{-1}) were sprayed on the NC
membrane.

\emph{Detection of PCT in Serum and LAM in Urine Using MANP-LFIA Strip}
: For PCT detection, 3 \selectlanguage{greek}μ\selectlanguage{english}L of MANP\textsubscript{6:6}@PCT-mAbs nanoprobe
was added to 280 \selectlanguage{greek}μ\selectlanguage{english}L of sample solution containing 140 \selectlanguage{greek}μ\selectlanguage{english}L of serum sample
and 140 \selectlanguage{greek}μ\selectlanguage{english}L of artificial serum solution. After incubation for 5 min, the
formed MANP\textsubscript{6:6}@PCT-mAbs-PCT complex was collected using
an external magnetic field for 10 min and resuspended in 70 \selectlanguage{greek}μ\selectlanguage{english}L of
artificial serum buffer solution. Subsequently, the solution was
pipetted into the sample well of the strip. After running for 20 min,
the strip was scanned using a commercial fluorescence strip reader, and
the fluorescence intensities (FI) at the T and C lines (denoted as
FI\textsubscript{T} and FI\textsubscript{C}) were recorded. The standard
curve was generated by plotting the correlation between the
FI\textsubscript{T}/FI\textsubscript{C} and the target concentration of
PCT.

For LAM, the obtained urine samples were first heated to 100 \selectlanguage{ngerman}°C for 10
min and then centrifuged at 12,000 rpm for 2 min to remove the excess
non-LAM proteins. In the detection process, 3 \selectlanguage{greek}μ\selectlanguage{english}L of
MANP\textsubscript{6:6}@A1 nanoprobe was added to 350 \selectlanguage{greek}μ\selectlanguage{english}L of sample
solution containing 175 \selectlanguage{greek}μ\selectlanguage{english}L of urine sample and 175 \selectlanguage{greek}μ\selectlanguage{english}L of artificial
urine solution. After incubation for 5 min, the formed
MANP\textsubscript{6:6}@A1-LAM complex was collected using an external
magnetic field for 10 min and resuspended in 70 \selectlanguage{greek}μ\selectlanguage{english}L of artificial serum
buffer solution. Subsequently, the solution was pipetted into the sample
well of the strip and run for 20 min for subsequent signal reading.

\emph{Statistical Analysis} : Data were presented as mean \selectlanguage{ngerman}± standard
deviation (SD). The statistical differences between data were evaluated
using ANOVA. ``*,'' ``**,'' and ``***'' represent \emph{p} \textless{}
0.05, \emph{p} \textless{} 0.01, and \emph{p} \textless{} 0.001,
respectively, indicating significance or very high significance.

Acknowledgements

This work was supported by the National Natural Science Foundation of
China (32172296, 32160598, 82171815), Key Research and Development
Program of Jiangxi Province (20232BBG70030, 20232BCD44004),
Double-Thousand Plan of Jiangxi Province (jxsq2023201113), Jiangxi
Provincial Natural Science Foundation (20224BAB215040, 20224ACB205012,
20192BAB204021, and 20202BAB216021), Technological Planning Project of
Jiangxi Provincial Health Commission (202310455 and 20201034),
Preferential Funding for Scientific Research Projects of Postdoctoral
Researchers in Jiangxi Province, Shanghai Hygiene and Health Outstanding
Leader Project (2022XD060), Shanghai Science and Technology Commission
(20Y11903400) and State Key Laboratory of Analytical Chemistry for Life
Science (SKLACLS2304).

\textbf{Conflict of Interests}

The authors declare no conflict of interests.

\textbf{Ethics Statement}

All serum and urine samples were obtained from healthy volunteers and
patients who provided written informed consent before participation.
This study was approved by the Medical Ethics Committee of The First
Affiliated Hospital of Nanchang University and the Ethics Committee
Review Boards of Shanghai Public Health Clinical Center and Shanghai
Pulmonary Hospital.

\textbf{Supporting Information}

Supporting Information is available from the Wiley Online Library or
from the author.

Received: ((will be filled in by the editorial staff)) Revised: ((will
be filled in by the editorial staff)) Published online: ((will be filled
in by the editorial staff))

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Scheme 1.} Schematic of (a) the construction process of the
MANP, (b) the magnetic pretreatment of blood and urine samples using the
MANP nanoprobe, and (c) the mechanism of the MANP-based LFIA for
ultrasensitive POCT detection.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 1.} (a) SEM image, (b) TEM image, and (c) magnified TEM
image of MANP\textsubscript{6:6}. Magnified TEM images of (d) PNP, (e)
ANP\textsubscript{6}, and (f) MNP\textsubscript{6}. The insets show
structural illustrations of the nanoparticles. (g) UV--vis spectra of
PNP, ANP\textsubscript{6}, MNP\textsubscript{6}, and
MANP\textsubscript{6:6}. (h) EDX elemental mapping images of C, Fe, O,
and S on MANP\textsubscript{6:6}, along with the merge image. (i) EDX
line scan across the center of MANP\textsubscript{6:6}, showing the
atomic relative counts of C and Fe. (j) XPS spectrum of
MANP\textsubscript{6:6} and (k) high-resolution XPS spectrum of Fe 2p.
(l) XRD pattern of MANP\textsubscript{6:6}. (m) Hydrodynamic size
distribution of MANP\textsubscript{6:6}. (n) Zeta potential evolution of
MANP\textsubscript{6:6} before and after carboxylation.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 2.} (a) SEM images, (b) TEM images, and (c) Magnified TEM
images of (i) MANP\textsubscript{2:6}, (ii) MANP\textsubscript{4:6},
(iii) MANP\textsubscript{6:6}, (iv) MANP\textsubscript{8:6}, and (v)
MANP\textsubscript{16:6}. (d) Fluorescent lifetimes of
ANP\textsubscript{6}, MANP\textsubscript{6:6}, and
MANP\textsubscript{16:6}. (e) Fluorescent spectra and (f) Fluorescence
retention rate, magnetic recovery, and AIE mass ratio of MANP at
different feeding amounts of
Fe\textsubscript{3}O\textsubscript{4}NPs@OA.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 3.} (a) Structural and performance illustration of
MNP\textsubscript{6}, ANP\textsubscript{6}, MANP\textsubscript{6:6}, and
actual photographs of MNP\textsubscript{6}, ANP\textsubscript{6},
MANP\textsubscript{6:6}with/without applying an external magnetic field
under day light and UV light. (b) Magnetic hysteresis loops of
MNP\textsubscript{6} and MANP\textsubscript{6:6} obtained on a SQUID
system at 300 K. (c) Fluorescence photos of ANP\textsubscript{6} and
MANP\textsubscript{6:6}and the corresponding color maps in different
particle concentrations. (d) Fluorescent intensities of different
concentrations of ANP\textsubscript{6} and MANP\textsubscript{6:6}
sprayed on paper strips. (e) Fluorescent intensity, magnetic recovery,
and hydrodynamic size of MANP\textsubscript{6:6} dispersed in aqueous
solutions with pH values ranging from 2 to 11. (f) Fluorescent
intensity, magnetic recovery, and hydrodynamic size of
MANP\textsubscript{6:6} stored in water at 60 \selectlanguage{ngerman}°C for 7 days. Fluorescent
intensity, magnetic recovery, and hydrodynamic size of
MANP\textsubscript{6:6} incubated in (g) 10 mM PB (pH 7.4), (h) human
serum, and (i) human urine for 7 days.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 4.} (a) Integrated scanning curves of MANP-FLIA with
different PCT concentrations in serum. (b) Calibration curves of
MANP-FLIA with magnetic operation (red), MANP-FLIA without magnetic
operation (black), and AuNPs-LFIA (blue) with different PCT
concentrations in serum. (c) Fluorescent photographs of MANP-FLIA test
strips in response to different concentrations of PCT in serum after
magnetic enrichment and separation (0--250 ng mL\textsuperscript{-1}).
(d) SEM images of the T line region on the NC membrane after running
with MANP\textsubscript{6:6}@mAbs at PCT concentrations of (i) 0 ng
mL\textsuperscript{-1} and (ii) 100 ng mL\textsuperscript{-1}. (e)
Specificity evaluation of the proposed MANP-FLIA platform toward various
inspected species. The concentration of PCT is 1 ng
mL\textsuperscript{-1}, and the concentration of all the other
interfering species is 1000 ng mL\textsuperscript{-1}. (f) Comparison of
the proposed MANP-FLIA method with the standard clinical CLIA method in
actual serum samples. (g) Correlation analysis between the proposed
method and the standard CLIA method.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 5.} (a) Fluorescent photographs, (b) signal responses,
and (c) calibration curves of MANP-FLIA test strips in response to
different concentrations of LAM (0--20 ng mL\textsuperscript{-1}) in
urine with/without magnetic enrichment and separation. (d) Specificity
evaluation of the proposed MANP-FLIA platform toward various
mycobacteria species with a bacterial concentration of
2.5\selectlanguage{ngerman}×10\textsuperscript{4} CFU mL\selectlanguage{english}\textsuperscript{-1}. (e) Fluorescent
signal of MANP-FLIA when adding different concentration levels of LAM
into urine samples provided by 10 individual healthy people. (f)
Diagnosis result of clinical TB-positive patients and healthy
individuals obtained using MANP-FLIA.

\textbf{The table of contents entry:}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

A new compact-discrete spatial arrangement is introduced on a
``fluorescence@magneto'' core-shell nanostructure with a close-packed
AIEgen core and a meager magnetic shell. The rational design endows the
magneto-AIE nanoparticle (MANP) with ``win-win'' performances: highly
retained fluorescent intensity and remarkedly enhanced magnetic
activity. The MANP demonstrates great potential in ultrasensitive
point-of-care bacterial infection diagnosis on the immunochromatographic
assay platform.

\textbf{Keywords:} fluorescent-magneto, aggregation-induced emission,
self-assembly, point-of-care test, bacterial infection diagnosis

\textbf{Authors:} Yu Su\textsuperscript{1, 2}, Xirui
Chen\textsuperscript{1, 2}, Huan Huang\textsuperscript{4, 5}, Yuhao
Wu\textsuperscript{1, 2}, Xuanang Shen\textsuperscript{1, 2}, Xiangkai
Lin\textsuperscript{1, 2}, Kun Sun\textsuperscript{1, 2}, Xiao-Yong
Fan\textsuperscript{4, *}, Xiaolin Huang\textsuperscript{1, 2, *} and
Yonghua Xiong\textsuperscript{1, 2, 3, *}

\textbf{Title:} Rearranging Fluorescence-Magneto Spatiality for
``Win-Win'' Dual Functions to Enhance Point-of-Care Diagnosis

\selectlanguage{english}
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\end{document}

