Fig. 1. Scheme of the workflow with a microarray biosensor based on imaging SPR system. CMD sensor chip is spotted with different ligands using contact spotter. Then the sensor chip is mounted on a glass prism and assembled with the flow cell. The surface of the sensor chip is illuminated at different light angles and images of the surface are taken by a CCD camera. For each spot the SPR angle is determined from angle versus intensity plots. The change in SPR angle (sensorgram) is monitored in real time and simultaneously on all the spots during buffer, sample and regeneration solution injections.

Fig. 2. SPR images of sensor chips with immobilized anti-bIgG antibody. CMD sensor chip spotted in Microgrid II with 1 mg mL⁻¹ anti-bIgG (a). CM5 sensor chip with different immobilization levels (FC1: 0 RU, FC2: 8892 RU, FC3: 2078 RU and FC4: 543 RU) of anti-bIgG, coated in Biacore 3000 at 0.025 mg mL⁻¹ concentration (b).

Fig. 3. Direct immunoassay for bIgG detection performed in a microarray format using IBIS iSPR and in conventional four FCs format using Biacore 3000. CMD sensor chip was spotted with 1 mg mL⁻¹ anti-bIgG and bIgG calibration curve was measured in duplicate on each spot using IBIS iSPR (black). CM5 sensor chip was coated with different levels of anti-bIgG, and bIgG calibration curve was measured in duplicate using Biacore 3000 (white, FC2: round, FC3: triangle, FC4: square series).

Fig. 4. Sensorgrams recorded during competition immunoassay for gentamicin and neomycin, in a microarray format, using IBIS iSPR. CMD sensor chip was spotted with 3 mg mL⁻¹ gentamicin and neomycin on different spots. Gentamicin and neomycin at several concentrations were mixed in solution with anti-gentamicin and anti-neomycin antibodies and introduced to the sensor chip in triplicate. Sensorgrams measured simultaneously on gentamicin (dark grey), neomycin (black) spots and a reference spot (light grey) during triplicate injections of 1, 5 and 10 ng mL⁻¹ of antibiotics.

Fig. 5. Competition immunoassay for gentamicin and neomycin detection performed in a microarray format using IBIS iSPR. Gentamicin and neomycin at several concentrations were mixed in solution with anti-gentamicin and anti-neomycin antibodies and introduced to the CMD sensor chip spotted with gentamicin and neomycin. Inhibition curves for gentamicin (white) and neomycin (black) were constructed from binding percentage for each antibiotics concentration and IC₅₀ values were calculated.

Summary of the IBIS iSPR optics performance

Δ: Change; n: refractive index of solution; variation = (standard deviation/average response) × 100; LOD: baseline noise plus three standard deviations.