4; 0.01 M), to remove the extravidin peroxidase solution in excess.3.2. Anti-lactoferrin immobilization on Immobilon membraneThe Immobilon Ny+ Membrane was cut into approximately 1 cm2 surface area disks and 100 ��L of a 1.0 mg/mL anti-lactoferrin was directly deposited on the membrane surface. The membrane was then dried at room temperature for about 24 h and selleck stored at 4�� C.3.3. Immunosensor assemblyThe transducer Inhibitors,Modulators,Libraries consisted of an amperometric electrode for H2O2 determination, with a Pt anode and an Ag/AgCl/Cl? Inhibitors,Modulators,Libraries cathode, provided with a plastic cap filled Inhibitors,Modulators,Libraries with 0.1 M KCl solution and screwed onto the body of the electrode, at the lower end of which a dialysis membrane was positioned. The Immobilon membrane with the immobilized anti-lactoferrin overlapped the dialysis membrane.

Finally, a nylon net overlapped the latter membrane. The two membranes and the net were secured by a rubber O-ring to the plastic cap of the electrode as shown in Figure 2.Figure 2.Amperometric immunosensor for lactoferrin determination using hydrogen peroxide Inhibitors,Modulators,Libraries electrode as transducer.3.4. Determination of lactoferrin by immunosensorCompetition procedure: competition between lactoferrin biotin-avidin-peroxidase conjugated and non conjugated lactoferrin, both free in solution, for anti-lactoferrin immobilized in membrane. To this end, the Immobilon membrane, on which the anti-lactoferrin was immobilized, was fixed to the head of the amperometric electrode for hydrogen peroxide as described in Section 3.3. Before measurement, the immunosensor was dipped into a Tris-HCl buffer solution, (pH 8.0; 0.

1 M), containing 0.05 % Tween?-20 by weight and 2.5% BSA by weight (bovine albumin was used to minimize non specific absorption on the membrane). The lactoferrin sample to be determined was added in 5 mL of Tris-HCl buffer solution (pH 8.0; 0.1 M) contained in the measurement cell, together with a fixed supply of lactoferrin biotin-avidin-peroxidase Cilengitide conjugated, i.e. 20 ��L (2.0 mg/mL) of conjugated lactoferrin. The peroxidase-conjugated lactoferrin was allowed to compete with the non-conjugated lactoferrin, both free in solution, in binding with the anti-lactoferrin immobilized on the Immobilon membrane. After washing with the same buffer solution to remove all the unbound lactoferrin, the specific substrate of the enzyme, i.e.

20 ��L of H2O2 selleckchem Tofacitinib solution 1% v/v, was added to the renewed buffer solution in which the immunosensor was dipped, under stirring. The measured signal (as nA) of the transducer correlated directly with the lactoferrin concentration to be measured. In this case, the higher the concentration of non conjugated lactoferrin free in solution, the stronger the signal produced by the hydrogen peroxide. Indeed, the lower the conjugated lactoferrin bound to the antibody immobilized on Immobilon membrane, the lower the H2O2 consumed in the enzymatic reaction, and therefore the higher the signal of the H2O2 oxidized at the amperometric electrode.