Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Schematic illustration of the stepwise fabrication and operating principle of the electrochemical dual immunosensor for the simultaneous determination of iFABP and FC. The procedure includes (i) electrochemical grafting of p -aminobenzoic acid diazonium salt onto SPdCEs to introduce carboxylic groups, followed by EDC/sulfo-NHS activation; (ii) covalent immobilization of capture antibodies (anti-iFABP and anti-FC); (iii) surface blocking with BSA to minimize nonspecific adsorption; (iv) formation of the sandwich immunocomplex through antigen binding and subsequent incubation with biotinylated detection antibodies; and (v) signal amplification using the V 2 O 5 /MWCNTs-HRP–streptavidin nanocomposite via biotin–streptavidin interaction. The analytical signal is generated by the catalytic reduction in H 2 O 2 , combining the peroxidase-like activity of V 2 O 5 and the enzymatic activity of HRP, and recorded amperometrically for each biomarker.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques: Introduce, Activation Assay, Blocking Assay, Adsorption, Binding Assay, Incubation, Amplification, Generated, Activity Assay, Biomarker Discovery

Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Optimization of the different experimental variables involved in the preparation of the electrochemical immunosensor for iFABP. Dependence of the amperometric responses measured in the absence (light purple, N) or in the presence (dark purple, S) of 1 ng mL −1 iFABP standards and the resulting signal-to-blank ratio (red lines, S/N) with the following: anti-iFABP concentration and incubation time ( A , B ); BSA concentration and incubation time ( C , D ); incubation time for iFABP standard ( E ); concentration of Biotin-anti-iFABP and incubation time ( F , G ); dilution of nanocomposite V 2 O 5 /MWCNTs/HRP-Strep ( H ). Unless otherwise specified, the concentration of iFABP used in the time-dependent panels ( B , D , E , G ) was fixed at 1 ng mL −1 . The background signal (N) arises from the intrinsic electrochemical activity of the nanocomposite and nonspecific adsorption processes, as confirmed by control experiments in the absence of antigen. After each incubation step, electrodes were rinsed with PBS to remove unbound species and minimize nonspecific contributions. Incubation time = 20 min. Error bars estimated as triple of the standard deviation of three replicates.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques: Concentration Assay, Incubation, Activity Assay, Adsorption, Control, Standard Deviation

Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Cyclic voltammograms ( A , B ) and Nyquist plots ( C , D ) recorded for 5 mM Fe(CN) 6 3−/4− in 0.1 mol L −1 PBS of pH 7.4 (scan rate 50 mV·s −1 ) at the following: ( A , C ) SPCE (1); HOOC-Phe-SPCE (2); HOOC-Phe-SPCE after EDC/sulfo-NHS activation (3); anti-iFABP-SPCE (4). ( B , D ) blocked anti-iFABP-SPCE (5); iFABP-anti-iFABP-SPCE (6); Biotin-anti-iFABP-iFABP-anti-iFABP-SPCE (7); V 2 O 5 /MWCNT-HRP-Strept-Biotin-anti-iFABP-iFABP-anti-iFABP-SPCE (8). The progressive modification of the electrode surface leads to changes in the electrochemical response, characterized by a decrease in peak currents in CV and an increase in charge transfer resistance (Rct) in EIS, confirming the stepwise assembly of the immunosensor. The equivalent circuits used to adjust the experimental results are shown within the figure, including Rs (solution resistance), Rct (charge transfer resistance), CPE (constant phase element), and Zw (Warburg impedance), allowing accurate fitting of the impedance data.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques: Activation Assay, Modification

Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Calibration plot constructed with the developed dual immunosensor for the amperometric determination of iFABP and FC standards in the concentration range studied under optimized experimental conditions. The plots represent the steady-state current responses (i, nA) obtained after addition of H 2 O 2 , showing the simultaneous and independent detection of both biomarkers. Error bars are estimated as triple of the standard deviation of three replicates. Insets show representative chronoamperometric responses for increasing concentrations of FC (top) and iFABP (bottom), illustrating the stepwise increase in signal with analyte concentration.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques: Construct, Concentration Assay, Standard Deviation

Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Amperometric responses provided by the developed immunosensor for the following: ( A ) 0 (light pink) and 10 ng mL −1 (dark pink) iFABP or ( B ) 0 (light blue) and 10 ng mL −1 (dark blue) FC in the presence of the following non-target compounds: 10 ng mL −1 FC ( A ) or iFABP ( B ), 5 mg mL −1 hemoglobin (HB), 50 mg mL −1 human serum albumin (HSA), 100 pg mL −1 interferon gamma (INF-γ), 1 mg mL −1 human immunoglobulin G(IgG), 200 pg mL −1 tumoral necrosis factor alpha (TNF-α) and 100 μg mL −1 uric acid (UA). The dashed lines represent the mean signal ±2 standard deviations obtained in the absence of interferents, providing a reference for evaluating potential interference effects. The results demonstrate that the presence of non-target species does not significantly affect the analytical signal, confirming the high selectivity of the immunosensor.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques:

Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Schematic illustration of the stepwise fabrication and operating principle of the electrochemical dual immunosensor for the simultaneous determination of iFABP and FC. The procedure includes (i) electrochemical grafting of p -aminobenzoic acid diazonium salt onto SPdCEs to introduce carboxylic groups, followed by EDC/sulfo-NHS activation; (ii) covalent immobilization of capture antibodies (anti-iFABP and anti-FC); (iii) surface blocking with BSA to minimize nonspecific adsorption; (iv) formation of the sandwich immunocomplex through antigen binding and subsequent incubation with biotinylated detection antibodies; and (v) signal amplification using the V 2 O 5 /MWCNTs-HRP–streptavidin nanocomposite via biotin–streptavidin interaction. The analytical signal is generated by the catalytic reduction in H 2 O 2 , combining the peroxidase-like activity of V 2 O 5 and the enzymatic activity of HRP, and recorded amperometrically for each biomarker.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques: Introduce, Activation Assay, Blocking Assay, Adsorption, Binding Assay, Incubation, Amplification, Generated, Activity Assay, Biomarker Discovery

Journal: Biosensors

Article Title: Nanocomposite-Based Dual Electrochemical Immunosensor for Simultaneous Detection of Intestinal Barrier Biomarkers: Intestinal Fatty Acid Binding Protein and Fecal Calprotectin

doi: 10.3390/bios16040199

Figure Lengend Snippet: Optimization of the different experimental variables involved in the preparation of the electrochemical immunosensor for iFABP. Dependence of the amperometric responses measured in the absence (light purple, N) or in the presence (dark purple, S) of 1 ng mL −1 iFABP standards and the resulting signal-to-blank ratio (red lines, S/N) with the following: anti-iFABP concentration and incubation time ( A , B ); BSA concentration and incubation time ( C , D ); incubation time for iFABP standard ( E ); concentration of Biotin-anti-iFABP and incubation time ( F , G ); dilution of nanocomposite V 2 O 5 /MWCNTs/HRP-Strep ( H ). Unless otherwise specified, the concentration of iFABP used in the time-dependent panels ( B , D , E , G ) was fixed at 1 ng mL −1 . The background signal (N) arises from the intrinsic electrochemical activity of the nanocomposite and nonspecific adsorption processes, as confirmed by control experiments in the absence of antigen. After each incubation step, electrodes were rinsed with PBS to remove unbound species and minimize nonspecific contributions. Incubation time = 20 min. Error bars estimated as triple of the standard deviation of three replicates.

Article Snippet: Anti-iFABP capture antibody, human iFABP standard, and biotinylated anti-iFABP detection antibody were obtained from the FABP2/iFABP DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA, Cat. No. DY3078).

Techniques: Concentration Assay, Incubation, Activity Assay, Adsorption, Control, Standard Deviation

Journal: Journal of Physiology and Biochemistry

Article Title: Modulation of SIRT1/PPARγ pathways and tight junction proteins by nicotinamide riboside under chronic variable stress

doi: 10.1007/s13105-026-01153-7

Figure Lengend Snippet: The effects of different nicotinamide riboside doses on the levels of PepT1 ( A ), LAT2 ( B ), EAAT3 ( C ), FABP2 ( D ), and FATP4 ( E ) proteins in the jejunum tissue of rats under normal and chronic variable stress (CVS) conditions. Western blot analysis was performed using β-actin as a loading control to ensure equal protein loading. All bands are shown in (G). Different letters (a–e) indicate statistically significant differences between groups ( p < 0.05) according to Tukey’s post hoc test following two-way ANOVA. Two-way ANOVA revealed significant effects for PepT1 (Condition p < 0.0001; NR p = 0.0001; Condition x NR p < 0.0001), LAT2 (Condition p < 0.0001; NR p = 0.0002; Condition x NR p = 0.0007), EAAT3 (Condition p < 0.0001; NR p < 0.0001; Condition x NR p < 0.0001), FABP2 (Condition p < 0.0001; NR p < 0.0001; Condition x NR p = 0.0236), and FATP4 (Condition p < 0.0001; NR p < 0.0001; Condition x NR p = 0.3521). Comprehensive statistical data are presented in Supplementary Table and full immunoblots images are shown in supplementary Fig.

Article Snippet: Membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies as follows: PPARγ (sc-271392), GLUT2 (sc-518022), IRS-1 (sc-8038), SIRT1 (sc-74465), FASN (sc-48357), claudin-1 (sc-166338), claudin-4 (sc-376643), occludin (sc-133256), ZO-1 (sc-33725), MUC2 (sc-53381), PepT1 (sc-373742), LAT2 (sc-293242), FABP2 (sc-374482), FATP4 (sc-393309) and β-Actin (sc-517582) (all from Santa Cruz Biotechnology); SGLT1 (DF7202-100; Affinity Biosciences); EAAT3 (orb1161904; Biorbyt Ltd., Cambridge, UK).

Techniques: Western Blot, Control