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).