1��PBS was 0.01 mol/L phosphate buffer, 0.8% saline solution and unless otherwise indicated the pH was 7.4. 5��PBS and 10��PBS is 5 times and 10 times concentrated 1��PBS. 1 mg/L MC-LR stock solutions were prepared in 0.01 mol/L PBS and stored at 4 ��C.2.2. EWAI instrumentationThe slightly modified EWAI immunosensor used in this study was previously described in . The pulse laser beam from a 635-nm pulse diode laser was directly launched into the single-mode fiber of the single-multi mode fiber coupler. The laser light then entered the multi-mode fiber with the diameter of 600 ��m and numerical aperture of 0.22 from the single-mode fiber. Afterwards, the excitation light from the laser, through the fiber connector, was coupled to a fiber probe.
The incident light propagates along the length of the probe via total internal reflection. The evanescent wave generated at the surface of the probe then interacted with the surface-bound fluorescently labelled analyte complexes, and causes excitation of the fluorophores. The collected fluorescence was subsequently filtered by means of a bandpass filter and detected by photodiodes through lock-in detection. The probe was embedded in a flow glass cell with a flow channel having a nominal dimension of 70 mm in length and 2 mm in diameter. All reagents were delivered by a flow analysis system operated with a peristaltic pump.2.3. Probe preparationCombination tapered fiber optic probes were prepared as previously described . The hapten-carrier conjugate MC-LR-OVA, used as recognition element, were covalently attached to the sensing surface of the probes with a heterobifunctional reagent.
Employing a modified procedure originally described by Bhatia et al. , the hapten-carrier conjugate was immobilized onto the probe surface. Briefly, the probes were initially cleaned with piranha reagents (concentrated H2SO4/H2O2 2:1), rinsed with distilled deionized water, and dried in N2. Next, the probe was placed in 2% MTS in toluene for 2 hours, under an inert atmosphere. Excess MTS was eliminated with dry toluene to assure the order and uniformity of the SAM. The thiol group of the silane was allowed to react for 1 hour with a heterobifunctional crosslinker, 2 mM GMBS in ethanol. After rinsing with ethanol and PBS, the succinimide group on the GMBS was then used to covalently bind the epsilon amino groups on proteins.
Immersion of the probe for 20 min in 2 mg/mL BSA was then carried out to block its non-specific binding sites.2.4. Immunoassay procedureThe indirect competitive inhibition method was developed for MC-LR determination. Free analyte (MC-LR, 240 ��L) of different concentrations was mixed with a fixed (0.6 ��g/mL) concentration of antibody in PBS (240 ��L) supplemented with BSA (2.0 mg/mL), Entinostat which reduce non-specific binding of antibody, and allowed for incubation at room temperature for 6 min.