The novelty consists in using as transducer not an ordinary electrochemical sensor but an electrochemical biosensor, which gives to the immunosensor explained herein a very high selectivity. Acknowledgments This work has been funded by MIUR, Project co-financed Kaempferol-3-O-glucorhamnoside by PRIN – 2005.. 2y = -2.0( 0.4) log x- 8.5 ( 1.6)r2 = 0.9786run 3y = -1.1( 0.3) log x- 7.8 ( 1.4)r2 = 0.9377 Open in a separate window ( 0.13) log x- 4.7 ( 1.8)r2 = 0.9872run 2y = -0.7( 0.10) log x- 3.5 ( 1.2)r2 = 0.9451run 3y = -0.4( 0.18) log x- 2.5 ( 1.6)r2 = 0.9288 Open in a separate Kaempferol-3-O-glucorhamnoside window The best results have so far been obtained by gently washing with Glycine-HCl buffer, 0.1 M, pH 2.0, containing 2.5 M MgCl2. The number of occasions the membranes can be reused (three at most) is usually indicated in Table 4. Both membranes can be re-used for subsequent steps (as shown in Physique 7). However, although it was possible in all three case to obtain a good calibration curve, a decrease in calibration sensitivity is displayed both using the Pall-Biodyne membrane and the Immobilon one. Lastly, the new biosensor was used to determine immunoglobulin G in human serum. Also the previously developed potentiometric immunosensor [7] had been used for this purpose. In the latter case, however, it was observed that the presence of urea in the serum could lead to non negligible interference [7]. On that occasion, it was attempted to overcome the problem by optimizing the number of washings carried out around the sensor membrane made up of the immunocomplex before performing the enzymatic measurement. This optimization was moderately successful; nevertheless this procedure continued to be rather critical as one more or one less washing could lead respectively to an under – or an over-estimation of immunoglobulin G content. Using the new immunosensor, on the other hand, as was expected, in view of the extreme selectivity of both the immunological reaction and the biosensor used as transducer, urea interference is completely absent and the serum measurement can be carried out without any problem, as shown by the results of the steps displayed in Table 5. Table 5. Results of steps performed in human serum to verify possible urea interference. (a) = Transmission (ppm O2), as a function of Log [HIgG], referring to serum Kaempferol-3-O-glucorhamnoside answer at different dilutions; (b) = Transmission (ppm O2), as a function of Log [HIgG], referring to serum answer at different dilutions (after addition to serum of 0.5 mL of urea solution 0.05 M); (c) = Transmission (ppm O2), as a function of Log [HIgG], referring to washing answer from serum DIAPH2 at different dilutions; (d) = Transmission (ppm O2), as a function of Log [HIgG], referring to washing answer from serum at different dilutions (after addition to serum of 0.5 mL of urea solution 0.05 M). 10-82.3 10-4Human Serum1:1,0001.9 10-71.9 10-4Human Serum1:1002.4 10-62.4 10-4 10-72.0 10-4Human Milk1:1001.8 10-61.8 10-4 Open in a separate window Table 7. Recovery tests by new immunosensor of added immunoglobulin G in human serum and human milk (at different dilutions). 10-73.5 10-75.5 10-7102.4Human Serum1:10024. 10-73.5 10-729.2 10-7103.2 10-712. 10-714. 10-799.5Human Milk1:10013. 10-712. 10-725. 10-797.3 Open in a separate window As can be observed in Furniture 6 and ?and7,7, analyses have been carried out both on sera and on milk samples with somewhat high concentrations of immunoglobulin G; in our case, for example, the test samples displayed a Kaempferol-3-O-glucorhamnoside concentration of about 10-4 mM; usually concentrations of the order of about 10-1 Kaempferol-3-O-glucorhamnoside – 10-3 mM are found in these human biological matrices, as reported in literature [11-13]; the determination of these samples thus necessarily required them to be diluted considerably so that final concentration would lie within the linear range of.