title: P450-based porous silicon biosensor for arachidonic acid detection.
abstract: A porous silicon biosensor based on P450 enzyme for arachidonic acid detection was developed. A new transduction method is presented with a simultaneous measurement of refractive index and fluorescence intensity changes when the analyte is binding to an enzyme on the porous silicon surface. A fluorophore bound to a cysteine residue in an allosteric position of the haem domain (BMP) of cytochrome P450 BM3 enhances its fluorescence intensity upon interaction with its substrate arachidonic acid, involved in diseases such as Alzheimer's, liver cancer and cellular inflammation processes. BMP has been anchored on porous silicon surface and the new transduction method has been successfully exploited to develop a biosensor for arachidonic acid, reaching a detection limit of 10 μM arachidonic acid in a dynamic range of 10-200 μM. Moreover, the change of the refractive index has been also monitored at the same time, displaying a higher detection limit of 30 μM. Preliminary test were also conducted in plasma proving the high specificity and selectivity of the sensor even in presence of interferents in the range of 50-100 μM. Here we suggest these two detection systems could be used simultaneously to increase the accuracy and the dynamic range of the sensor avoiding a false positive response.
authors.affiliation: Thermodynamic Division, Istituto Nazionale di Ricerca Metrologica, Strada delle Cacce 91, 10135 Torino, Italy. a.giovannozzi@inrim.it
authors.forename: A M
authors.initials: AM
authors.lastname: Giovannozzi
authors.forename: V E V
authors.initials: VE
authors.lastname: Ferrero
authors.forename: F
authors.initials: F
authors.lastname: Pennecchi
authors.forename: S J
authors.initials: SJ
authors.lastname: Sadeghi
authors.forename: G
authors.initials: G
authors.lastname: Gilardi
authors.forename: A M
authors.initials: AM
authors.lastname: Rossi
mesh_terms: D016718:Arachidonic Acid / Q000032:analysis*; D001426:Bacterial Proteins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D016062:Porosity; D012031:Refractometry; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001426:Bacterial Proteins; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D012825:Silicon