Introduction:: Electrospinning is a cost-effective method to create fibers with diameters ranging from microscale to nanoscale that have been used in various fields such as tissue engineering, biosensors, and drug delivery. Electrospun fibers have a large surface area and porous structure, making them an ideal substrate for biosensors due to their rapid diffusion of target molecules. However, controlling fiber density and matrix size remains a challenge. The use of surface-enhanced Raman scattering (SERS) technology in biosensors has been highly selective and sensitive in detecting molecules of interest, but few studies have focused on detecting large molecules such as proteins using fibrous substrates. This study introduces a nanofiber-based microparticle that combines electrospinning and photolithography techniques to create an array of micropatterned nanofibers that are coated with silver nanoparticles to serve as capture substrates for the SERS-based immunoassay. The shape-coded multiplex immunoassay's sensing performance and capability were investigated using SERS tags made with Au-Ag alloy nanocubes. This study provides a new platform for biosensors that can detect large molecules and demonstrates the potential of combining electrospinning and photolithography techniques in creating complex and highly sensitive biosensor substrates.
Materials and Methods:: Materials PVA-SbQ (Mw=45,000, with 4.1 mol % SbQ) was purchased from Polysciences Inc. (Warrington, PA, UK). Also, 2-hydroxy-2-methylpropiophenone (HOMPP), silver nitrate (AgNO3), HS-PEG 7.5k-COOH, ammonium hydroxide (NH4OH), potassium hydroxide (KOH), D-(+)-glucose, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4∙3H2O), bovine serum albumin (BSA), ascorbic acid (AA), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), human alkaline phosphatase (ALP), osteocalcin (OC), hemoglobin, human serum albumin (HSA), fibronectin, thrombin, anti-osteocalcin, and anti-human alkaline phosphatase were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Photomasks for photopatterning were made on transparent film using a LaserJet printer (LaserWriter 16/600 PS; Apple, Inc., Cupertino, CA, USA). Methods Researchers have used photolithography to create PVA-SbQ nanofiber microparticles (NF-MPs) that were used to create a Surface-enhanced Raman spectroscopy (SERS) substrate for use in immunoassays. The team then immobilized antibodies onto the AgNP-coated NF-MPs, which were immersed in a 1 μg/mL antibody solution. The team also prepared SERS tags by allowing Au-Ag alloy nanocubes to react with HS-PEG7.5k-COOH and DTNB for 6 h, before immobilizing antibodies on them via the EDC-NHS reaction. The amount of immobilized antibodies was determined by measuring the initial and final concentration of anti-ALPs.
Results, Conclusions, and Discussions:: The article describes a new method of creating shape-coded nanofiber microparticles (NF-MPs) for use as a suspension array in bioanalysis. The method involves creating a nanofibrous matrix using a material containing styrylpyridinium side chains, which can be crosslinked by UV irradiation. Micropatterned nanofibers are then created using photolithography, which detach from the substrate when immersed in water to form the NF-MPs. The shape, lateral dimensions, and thickness of the NF-MPs can be controlled through photomask design and electrospinning time. The NF-MPs are coated with AgNPs to create SERS capture substrates for ultrasensitive detection of targets via the SERS signals of the Raman reporters. The article concludes that shape-coded NF-MPs can provide a low-cost, productive, and versatile platform for multiplex bioanalysis
Acknowledgements (Optional): : This work was supported by National Research Foundation (NRF) grants funded by the Ministry of Science, ICT, and Future Planning (MSIP) (grant numbers 2017M3D1A1039289, 2022M3H4A4085936).
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