Immobilization enzyme fluorescence capillary analysis for determination of lactic acid

Abstract

A new method for the determination of lactic acid based on the immobilization enzyme fluorescence capillary analysis (IE-FCA) was proposed. Lactic dehydrogenase (LDH) was immobilized on inner surface of a capillary with glutaraldehyde, and an immobilized enzyme lactate capillary bioreactor (IE-LCBR) was formed for the determination of lactic acid. After nicotinamide adenine dinucleotide (NAD(+)) is mixed with lactic acid solution, it was sucked into the IE-LCBR and was detected at lambda(ex) 353 nm/lambda(em) 466 nm. Optimized conditions are as follows: the temperature is 38 degrees C; the reaction time is 15 min; the concentrations of Tris buffer (pH 8.8) and NAD(+) are 0.1 mol L(-1) and 4 mmol L(-1), respectively; the concentration of LDH used for immobilization is 15 kU L(-1). The concentration of lactic acid is directly proportional to the fluorescence intensity measured from 0.50 to 2.0 mmol L(-1); and the analytical recovery of added lactic acid was 99-105%. The minimum detection limit of the method is 0.40 mmol L(-1) and sensitivity of the IE-CBR is 4.6 F mmol(-1) L(-1) lactate. Its relative standard deviation (R.S.D.) is < or =2.0%. This IE-FCA method was employed for determination of lactate in milk drink.