Nanostructured electrochemical immunosensor for detection of serological alkaline phosphatase

Alkaline phosphatase (ALP) is an enzyme that plays an important role in bone mineralization and skeletal growth. Variations in physiological levels of ALP have been correlated to diseases such as osteomalacia, Paget's disease and arterial calcifications. In this context, the integration of carbon nanotubes (CNT) within osteointegration implants has shown to increase ALP's mineralization activity in virtue of their surface chemistry and their morphological resemblance to collagen nanofibers. In this study we present the development and analytical application of an impedimetric immunosensor based in gold nanoparticle-decorated CNT, which characteristics are desirable in implantable biosensors. The device effectively detects ALP within blood serum, a complex biological fluid where most expressed proteins can be found. Robustness and high sensitivity were attained by immobilizing covalently anti-ALP antibody as a specific probe towards ALP. Cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy were used to characterize the sensor system throughout mounting steps and real sample testing. Impedimetric responses were adjusted to a theoretical electrical circuit and charge transfer resistance showed to be an adequate parameter to evaluate the biorecognition process of the analyte. Additionally, amperometrical current variation and changes in topography found over the surface after positive samples evidenced biorecognition. The final biosensor showed excellent performance with two linear ranges from 0.5 to 50 IU.L-1 and from 100 to 600 IU.L-1; limits of detection were calculated as 0.25 and 84.6IU.L-1 respectively with a relative standard deviation lower than 5%. The device was found to be selective, avoiding protein c, a potential interferer occurring during inflammatory processes. The proposed strategy is promising for osteogenic applications where it can improve osteointegration implants by monitoring ALP activity.