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In this study, two methods were examined to optimize the immobilization of antibodies on paper when conducting a paper-based enzyme-linked immunosorbent assay (P-ELISA). Human IgG, as a test-capture protein, was immobilized on paper via the formation of Schiff bases. Aldehyde groups were introduced onto the surface of the paper via two methods: NaIO 4 and 3-aminopropyltriethoxysilane (APTS) with glutaraldehyde (APTS-glutaraldehyde). In the assay, horseradish peroxidase-conjugated anti-human IgG (HRP-anti-IgG) binds to the immobilized human IgG, and the colorimetric reaction of 3,3',5,5'-tetramethylbenzyzine (TMB) produces a blue color in the presence of H 2 O 2 and HRP-anti-IgG as a model analyte. The immobilization of human IgG, the enzymatic reaction conditions, and the reduction of the chemical bond between the paper surface and immobilized human IgG all were optimized in order to improve both the analytical performance and the stability. In addition, the thickness of the paper was examined to stabilize the analytical signal. Consequently, the APTS-glutaraldehyde method was superior to the NaIO 4 method in terms of sensitivity and reproducibility. Conversely, the reduction of imine to amine with NaBH 4 proved to exert only minimal influence on sensitivity and stability, although it tended to degrade reproducibility. We also found that thick paper was preferential when using P-ELISA because a rigid paper substrate prevents distortion of the paper surface that is often caused by repeated washing processes., (© 2024. The Author(s).)

Glass slides have been widely used for DNA immobilization in DNA microarray and numerous bioassays for decades, whereas they are faced with limitations of low probe density, time-consuming modification steps, and expensive instruments. In this work, a simple one-step surface modification method using 3-aminopropyl trimethoxysilane (APTMS) has been developed and applied to graft DNA codes on paper. Higher DNA immobilization efficiency was obtained in comparison with that in a conventional method using glass slides. Fluorescence detection, X-ray photoelectron spectroscopy (XPS), infrared spectra (FT-IR), and pH influence studies were employed to characterize the surface modification and subsequent DNA immobilization, which further reveals a mechanism in which this method lies in ionic interactions between the positively charged APTMS-modified paper surface and negatively charged DNA probes. Furthermore, an APTMS-modified paper-based device has been developed to demonstrate application in low-cost detection of a foodborne pathogen, Giardia lamblia , with high sensitivity (the detection limit of 22 nM) and high specificity. Compared with conventional methods using redundant cross-linking reactions, our method is simpler, faster, versatile, and lower-cost, enabling broad applications of paper-based bioassays especially for point-of-care detection in resource-poor settings.

Many lanthanide ions-based probes have been widely used for detecting anthrax spores biomarker-dipicolinic acid (DPA). However, little work has realized detection of bacillus anthrax spores in real environmental samples. In this work, a novel ratiometric fluorescent nanoprobe based on europium (Eu)-doped silicon nanoparticles (Eu@SiNPs) was fabricated for the first time by one-pot method without post-modification for determination of the DPA in bacillus subtilis spores (simulant bacillus anthrax spores). Based on Eu(III) in the Eu@SiNPs could be sensitized by DPA to emit intrinsic fluorescence and the fluorescence intensity of SiNPs in the Eu@SiNPs almost remained stable, a new ratiometric fluorescent method for determination of micro DPA in bacillus subtilis spores and bacillus subtilis spores in real environmental samples, such as Yellow river water, tap water and soil was established. Under the optimum conditions, the limit of detection (LOD) of the method toward bacillus subtilis spores was as low as 2.38×10 4 spore/mL. Simple, fast and visual DPA and bacillus subtilis spores determination was also achieved by the Eu@SiNPs-based test paper. Therefore, the newly established method was expected to be a powerful tool for efficiently determination of bacillus anthrax spores to avoid anthrax threats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)