Luminol electrochemiluminescence-based biosensor for total cholesterol determination in natural samples

A new cholesterol flow injection analysis biosensor is described. It is based on a luminol/hydrogen peroxide electrochemiluminescence (ECL) reaction induced by a glassy carbon electrode polarised at + 425 mV vs a Pt pseudo reference. The cholesterol biosensor sensing layer is based on cholesterol oxidase (COD) immobilised on either UltraBind(TM) membrane or Immunodyne membrane. The physicochemical properties of the UltraBind(TM) type membranes enable a high-performance biosensor to be obtained. A high salt concentration. 3 M NaCl, is shown to induce a 400% increase of the immobilised COD activity, and intermediate salt concentrations, from 1 to 1.5 M NaCl, induce a 230% increase of the H2O2 sensor performances. Both phenomena enable the achievement of a sensitive ECL cholesterol biosensor which exhibited a 800% increase of its performance in the presence of 2 M NaCl. in those optimised conditions, the determination of free cholesterol could be performed with a detection limit of 0.6 nmol and a detection ranging over at least two decades. When used in conjunction with a cholesterol esterase in solution, the biosensor enables the detection of free and total cholesterol in human sera, in 15 minutes with a good accuracy. The biosensor operational stability was satisfactory either with buffered standard solutions or with complex matrices since a total of 55 and 43 assays, respectively, could be performed with good reproducibility (CV = 4.8% and 8.3%) and without detectable loss of sensitivity.