Fig 1.
LOV device configured for micro-bead injection spectroscopy immunoaffinity extraction.
A 1.0 mL syringe pump was used to aspirate and dispense reagents and carrier solution through the LOV ports (not shown for the sake of simplicity). Close-up on the LOV multiposition valve: TentaGel HL suspension in storage (port 1), and packed as immunoaffinity microcolumn inside the flow cell (port 4). A PEEK plug (0.13 mm ID, 3 mm long) was placed immediately below the light beam to retain the microspheres between the fiber optics cables while the liquid flows out. CBZ-HRP, carbamazepine-horseradish peroxidase tracer; TMB, 3,3’,5,5’-tetramethylbenzidine.
Table 1.
Automated micro-bead injection spectroscopy LOV steps for the immunoaffinity extraction of CBZ.
Fig 2.
Experimental workflow for the production of anti-CBZ IgG decorated TentaGel HL-COOH beads.
The applied strategy started off by the study of general reaction parameters (step I), such as the buffer system and concentration of activation agents, using the model protein OVA labeled with fluorescein (as FITC): OVA-FITC. Then the influence of more specific parameters, which depend on the final coupling target, was evaluated, namely the loading capacity of to the beads and reaction times (steps II and III). Finally, the viability of the final material for immunoassay development was tested (step IV).
Fig 3.
Influence of the concentration of activation reagents and buffer solution in fluorescence intensity (a.u.) obtained by coupling 100 μg OVA-FITC per 1000 μg of beads.
Asterisks indicate statistically significant differences between 2 eq. and 10 eq. within each buffer system (p < 0.05). For all buffer systems, fluorescence intensity values obtained after incubating OVA-FITC to beads activated (dashed columns) with DCC/NHS and to non-activated beads (dotted columns) were statistically significantly different (p < 0.05).
Fig 4.
Comparison of fluorescence intensity (exc. 635 nm; em. 665–685 nm) of TentaGel (TG) beads incubated with Alexa647 IgG a) with and b) without previous NHS-activation.
The mass ratio of antibody to beads was 10 μg to 500 μg. Blank beads were also scanned as negative control (c). Average fluorescence intensity values were calculated for two independent populations (n = 50) from the same batch of beads. The respective fluorescence scanning images are displayed below the chart (Resolution: 5μm pixel-1).
Fig 5.
Relative fluorescence intensity (%) versus mass of anti-CBZ IgG-Cy5 (μg) added to 500 μg of TentaGel beads.
Data were obtained from two independent runs (run one red square, run two blue dots, 20 h activation, 240 min coupling). Linear Least-Squares fit yielded a slope of 8.9% RFI μg-1 and an intercept of 13% RFI (R2 = 0.983) for Run 1, and a slope of 8.5% RFI μg-1 and an intercept of 15% RFI (R2 = 0.990).
Fig 6.
Effect of coupling time on relative fluorescence intensity (%) of the TentaGel beads.
Red dots correspond to 20 h of activation with DCC/NHS and incubation with 2.5 μg anti-CBZ IgG-Cy5 conjugate per 500 μg of beads. The calculated (calc.) theoretical amount of hydrolyzed PEG-NHS ester is also presented in blue squares and dashed-line.
Fig 7.
Effect of activation time on relative fluorescence intensity (%) provided by the TentaGel beads, after incubation with 5 μg anti-CBZ IgG-Cy5 conjugate per 500 μg of beads for 240 min.
Table 2.
Results of screening ELISA.
Fig 8.
Schematic representation of the main steps of immunoextraction of carbamazepine (CBZ).
Steps comprising automatic sorbent column assembly, retention of CBZ-HRP conjugate, and confirmation of CBZ immobilization by oxidation of TMB substrate (λ = 370 nm). HRP, horseradish peroxidase.