A new colorimetric lactate biosensor based on CUPRAC reagent using binary enzyme (lactate-pyruvate...

A novel optical lactate biosensor is presented that utilizes a colorimetric interaction between H₂O₂ liberated by a binary enzymatic reaction and bis(neocuproine)copper(II) complex ([Cu(Nc)₂]²⁺) known as CUPRAC (cupric reducing antioxidant capacity) reagent. In the first step, lactate oxidase (LOx) and pyruvate oxidase (POx) were separately immobilized on silanized magnetite nanoparticles (SiO₂@Fe₃O₄ NPs), and thus, 2 mol of H₂O₂ was released per 1 mol of the substrate due to a sequential enzymatic reaction of the mixture of LOx-SiO₂@Fe₃O₄ and POx-SiO₂@Fe₃O₄ NPs with lactate and pyruvate, respectively. In the second step, the absorbance at 450 nm of the yellow-orange [Cu(Nc)₂]⁺ complex formed through the color reaction of enzymatically produced H₂O₂ with [Cu(Nc)₂]²⁺ was recorded. The results indicate that the developed colorimetric binary enzymatic biosensor exhibits a broad linear range of response between 0.5 and 50.0 µM for lactate under optimal conditions with a detection limit of 0.17 µM. The fabricated biosensor did not respond to other saccharides, while the positive interferences of certain reducing compounds such as dopamine, ascorbic acid, and uric acid were minimized through their oxidative removal with a pre-oxidant (NaBiO₃) before enzymatic and colorimetric reactions. The fabricated optical biosensor was applied to various samples such as artificial blood, artificial/real sweat, and cow milk. The high recovery values (close to 100%) achieved for lactate-spiked samples indicate an acceptable accuracy of this colorimetric biosensor in the determination of lactate in real samples. Due to the increase in H₂O₂ production with the bienzymatic lactate sensor, the proposed method displays double-fold sensitivity relative to monoenzymatic biosensors and involves a neat color reaction with cupric-neocuproine having a clear stoichiometry as opposed to the rather indefinite stoichiometry of analogous redox dye methods.