Sodium metaperiodate

Naringinase from Aspergillus aculeatus/Aspergillus niger (Novozyme NS 33117) was kindly donated by Novozyme. Naringin (≥95% HPLC), limonin (>90% HPLC), p-nitrophenyl-α-l-rhamnopyranoside (pNPR), and p-nitrophenyl-β-d-glucoside (pNPG) were purchased from Sigma-Aldrich, Santiago, Chile. Acetonitrile, octyl-sepharose 4 Fast Flow, butyl-sepharose 4 Fast Flow, and sodium metaperiodate were purchased from Merck, Santiago, Chile. All other chemicals were of analytical grade. Grapefruits were purchased from the local market.

One volume of cold 0.4 M acetate buffer (pH 4.5, Merck) containing 40 mM sodium metaperiodate (Merck) was added to one volume of SEA fractions. Samples were incubated overnight at 4°C while rotating. Quenching was performed by adding one volume of cold 50 mM sodium borohydride solution (Fluka) to the different samples and incubated for 30 min on ice. Treated SEA fractions were dialyzed against PBS using Slide-A-Lyzer MINI Dialysis Devices (3,5K MWCO, Thermo Fisher Scientific, according to the manufacturer’s instructions). Mock samples were subjected to the same treatment without the presence of sodium metaperiodate in the acetate buffer.

Bacteria were first labeled with biotin hydrazide after periodate oxidation (21 (link)). Around 1 g (wet weight) of bacteria was washed twice with PBS and resuspended in 200 μl of 0.1 M ammonium acetate buffer (pH 5.5) containing 15 mM sodium metaperiodate (Merck). After a 20-min incubation at 4°C in the dark with gentle rotation, the oxidation reaction was quenched by adding 200 μl of 0.1 M ammonium acetate buffer (pH 5.5) containing 30 mM sodium bisulphite (Sigma-Aldrich). After centrifugation, bacteria were resuspended in 400 μl of PBS containing 5 mM biotin hydrazide (Sigma-Aldrich). After a 2-hour incubation at RT with gentle rotation, cells were washed three times with PBS. Biotin-labeled bacteria were resuspended in PBS and added to 2 × 10⁵ HEK cells at the indicated MOI in a total volume of 1 ml of cold DMEM medium. After a 4-hour incubation at 4°C under gentle rotation, the cell suspension was centrifuged at 300g for 10 min. The pellet was suspended in 50 μl of allophycocyanin-conjugated streptavidin (BD Pharmingen). After 20 min at 4°C in the dark, cells were washed twice with PBS, resuspended in 400 μl of PBS, and analyzed by confocal microscopy (fig. S9) and flow cytometry using a FACSCalibur CellQuest Pro (BD Biosciences) software.

Alginic acid sodium salt from brown algae was purchased from Sigma-Aldrich (Bangalore, India), gelatin was purchased from Gelita (Eberbach, Germany), sodium tetraborate (borax) was purchased from Fisher Scientific (United Kingdom), and sodium metaperiodate (EMSURE) was purchased from Merck (Mumbai, India). PRP was isolated from blood samples taken from healthy volunteers after Institutional Ethics Committee l approval (IEC number: SCT/IEC/1366/APRIL-2019), and L929 cell line was obtained from American Type Culture Collection (Manassas, VA, United States). All cell culture related reagents and consumables were obtained from Thermo-Scientific (Bangalore, India).

sodium metaperiodate treatment of colostral IgG was performed as previously described by Alemka et al. (2010 (link)). IgG (24 mg/mL) was incubated with 0.011 mM sodium metaperiodate (Merck) dissolved in PBS. The mixture was then incubated at room temperature for 30 min. Excess sodium metaperiodate was removed by centrifugal filtration using a 3-kDa molecular weight cut-off with 3 × 1 mL PBS, pH 7.4, washes and the retentate containing meta-periodate treated IgG was diluted to its original volume and stored at − 20 °C. The adhesion assay was then carried out with the metaperiodate-treated IgG and B. longum 8809 as described above.

The samples of known concentrations of E. coli contaminated water were prepared using the serial dilution method. The microbial count of these samples is determined using Spread Plate Method. These samples were inoculated to the functionalized GO nanomaterial in various molar ratios, and the voltage values were noted. For ensuring better calibration, the optical densities of these samples were also measured simultaneously. The water samples from various water matrices were collected and tested using the developed sensor for real water analysis. Conc. H₂SO₄, sodium meta periodate, ethanol, tetrahydrofuran, H₂O₂ (30%), Diethylenetriamine (DETA), and phthalic anhydride were purchased from Sigma-Aldrich. Polydimethylsiloxane (PDMS) is a silicone-based organic polymer used for the fabrication of nanosensors^{34 (link)}. The silicone elastomer curing agent and its elastomer base were purchased from Sigma Aldrich. The protected DETA functionalized GO and free amine-functionalized GO (fGO) were prepared by the previously reported procedure³⁵ .

Assessment of the impact of periodate treatment on antigens followed a published method [39 ]. Antigen-coated microtitre plates were incubated with 200 μl per well of 25 mM sodium metaperiodate (Sigma-Aldrich) in 50 mM sodium acetate buffer (pH 4.5), or with buffer only for controls, and kept in the dark at 4°C for the required time. Plates were then washed, blocked and developed as for standard ELISA.