Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film.

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.

Binding specificity of alpha-bilirubin-imprinted poly(methacrylic acid-co-ethylene glycol dimethylacrylate) toward alpha-bilirubin.

alpha-Bilirubin is an important index to determine the liver's functions. Poly(methacrylic acid-co-ethylene glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was proposed and shown to be able to bind alpha-bilirubin specifically. The extraction condition was also discussed. Polymers prepared by imprinting bilirubin in poly(MAA-co-EGDMA) and in poly(beta-cyclodextrin-co-EGDMA) were compared. In this work, binding specificity of molecularly imprinted polymer (MIP) toward the target template, alpha-bilirubin, is discussed. Adsorption profile of alpha-bilirubin by bilirubin-imprinted poly(MAA-co-EGDMA) was measured as a function of time, from where the equilibrium could be determined. Two hours was determined to be the proper time for adsorption. Biliverdin as a rather similar analog compound of bilirubin was chosen for comparison of binding specificity in this study. Progesterone as well as testosterone was also chosen for study because they both co-exist in serum with bilirubin and might interfere with binding capacity of imprinted poly(MAA-co-EGDMA) toward alpha-bilirubin. Specificity of this polymer for bilirubin was thus confirmed by tasks carried out in mixture solutions comprised of compounds above. It is true that biliverdin contained in the binary mixture did affect the binding capacity of bilirubin. Nevertheless, polymer's binding specificity for bilirubin was essentially sufficient for recognition of alpha-bilirubin in the presence of other compounds. How MIP identified the target template molecule, alpha-bilirubin, is also elucidated.