4. Experimental Section
Materials : The red, green, and yellow AIEgens were generously
provided by Jiangxi Weibang Biotechnology Co., Ltd. Ferric chloride
(FeCl3·6H2O), ferrous chloride
(FeCl2·4H2O), 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.
Apparatus and
Characterizations : Transmission
electron microscopy (TEM) images and energy dispersive X-ray (EDX)
images were obtained using an FEI Talos F200× 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 CuKα radiation (λ = 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).
Synthesis of Fe3O4 NPs@OA :
Fe3O4 NPs@OA with a diameter of
~10 nm were synthesized in accordance with a previously
reported method.[42] In detail, a 150 mL aqueous
solution containing 1.59 g of FeCl2·4H2O
and 2.59 g of FeCl3·6H2O was heated to
50 °C with N2 bubbling for 15 min. Subsequently, 12.5 mL
of NH3·H2O (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
N2 protection. Finally, the oily
Fe3O4 NPs@OA product was washed with
ethanol and resuspended in chloroform for further use.
Synthesis of MANP6:6, PNP, ANP6,
and MNP6 : MANP6:6 was prepared by
co-assembling Fe3O4 NPs@OA and AIEgens
into a polymer matrix using our reported
emulsification method with some
modifications.[42] Briefly, 5 mg of PMAO, 6 mg of
Fe3O4 NPs@OA, and 6 mg of AIEgens were
completely dissolved in 150 µL of chloroform to form an oil phase. Then,
400 µL of 0.2 mg mL−1 SDS solution as the aqueous
phase was added to the oil phase. The mixture was emulsified viasonicating 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
Fe3O4 NPs@OA and AIEgens into
MANP6:6. The obtained MANP6:6 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
MANP6:6 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, ANP6, and MNP6followed a similar procedure to that of MANP6:6, with
the only difference being the feeding groups of PMAO, PMAO and AIEgens,
and PMAO and Fe3O4 NPs@OA as the
building blocks, respectively.
Synthesis of MANP6:6@PCT-mAbs and
MANP6:6@LAM-mAbs : The MANP6:6@PCT-mAbs
nanoprobes were synthesized using the EDC method to form an amide bond
between the carboxyl group of MANP6:6 and the amino
group of anti-PCT mAbs. The same procedure was followed for the
synthesis of MANP6:6@LAM-mAbs, with A1 antibody used as
the LAM detective antibody. Briefly, 6
μg of anti-PCT mAbs was added to 400
μL of 0.01 M PB solution (pH 7.4) containing 20 µg of
MANP6:6, and the mixture was incubated for 30 min. Then,
2 μg 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 MANP6:6for 1 h. Finally, the mixture was centrifuged
at 9,000 rpm for 5 min, and the
precipitates were resuspended in 100 µL of 0.01 M PB (pH 7.4) containing
25% w/v saccharose, 1% w/v BSA, and 0.1% w/v NaN3 and
stored at 4 °C for further use.
Fabrication of MANP-LFIA Strips : The
MANP6:6-LFIA strip
fabrication process, including both PCT and LAM detection, is described
as follows: For PCT detection,
anti-PCT pAbs (2 mg mL−1) and goat anti-mouse IgG (1
mg mL−1) were sprayed on the NC membrane at a density
of 0.74 μL cm−1 as the test (T) and control (C) lines,
respectively, using the ZX1000 dispensing platform. The modified NC
membrane was dried at 37 °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−1) and goat
anti-mouse IgG (2 mg mL−1) were sprayed on the NC
membrane.
Detection of PCT in Serum and LAM in Urine Using MANP-LFIA Strip :
For PCT detection, 3 μL of MANP6:6@PCT-mAbs nanoprobe
was added to 280 μL of sample solution containing 140 μL of serum sample
and 140 μL of artificial serum solution.
After incubation for 5 min, the
formed MANP6:6@PCT-mAbs-PCT complex was collected using
an external magnetic field for 10 min and resuspended in 70 μL 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
FIT and FIC) were recorded.
The standard curve was generated
by plotting the correlation between the
FIT/FIC and the target concentration of
PCT.
For LAM, the obtained urine samples were first heated to 100 °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 μL of
MANP6:6@A1 nanoprobe
was added to 350 μL of sample solution containing 175 μL of urine sample
and 175 μL of artificial urine solution. After incubation for 5 min, the
formed MANP6:6@A1-LAM complex was collected using an
external magnetic field for 10 min and resuspended in 70 μL 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.
Statistical Analysis : Data were presented as mean ± standard
deviation (SD). The statistical differences between data were evaluated
using ANOVA. “*,” “**,” and “***” represent p <
0.05, p < 0.01, and p < 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).