Diabetes is a serious lifestyle-related disease that can result in a myriad of complications. 1 Exact, rapid and quantitative determinations of glucose are thus essential in the diagnosis and management of diabetes. At present, several assays have been reported for the determination of glucose, including chemiluminescence (CL), 2-7 spectrophotometry, [8][9][10] electrochemical methods, 11,12 fluorescence methods 13,14 and others. [15][16][17] A sensitive CL method was reported for the determination of 2 × 10 -8 -1 × 10 -4 mol/L glucose, and some metal ions catalyzed the luminol CL reaction. 2 A flow injection spectrophotometric method was used to detect 1 × 10 -5 -1 × 10 -3 mol/L glucose with immobilized enzyme reactors. The method was simple and convenient but exhibited only a low sensitivity. 6 A voltammetric method has a good selectivity with a linear range of 8 × 10 -7 -2 × 10 -5 mol/L glucose and a detection limit of 3.5 × 10 -7 mol/L, but the assay course was complex. 12 A precise intrinsic fluorescence method was proposed to detect 5 × 10 -4 -2 × 10 -2 mol/L glucose, with only low sensitivity. 14 RS spectral analysis is simple and sensitive, and was applied in the analysis of trace inorganic substances and organic substances 18-24 with good results. In order to improve the selectivity and the sensitivity of the RS spectral method, applications of catalytic reaction, especially enzymatic reaction are effective. Recently, a combination of the catalytic effect of metal ions and the RS technique was utilized for analysis of some inorganic substances. 25 Based on the catalytic reaction of enzymes, an enzyme catalytic-RS assay for lysozyme activity was proposed. 26 Coupling the enzyme catalytic reaction of HRP with the association and aggregation reactions, an enzyme catalytic-RS assay for HRP activity was reported. 27 Up to date, however there is no report about RS assay for glucose, or about double enzyme catalytic-RS assay. In this work, the glucose-GOD-HRP-CS-KI catalytic system was studied by RS technique. A new double enzymatic catalytic-RS bioassay is proposed for determination of trace glucose in human serum, with high sensitivity, good selectivity and simplicity. Furthermore, the substrate was easy to obtain and was nontoxic.
experimental
Reagents and apparatusA model of a Cary Eclipse fluorescence spectrophotometer (Varian Company, Palo Alto, CA) was used to record the RS spectra by means of synchronous scanning excited wavelength λex and emission wavelength λem (λex -λem = Δλ = 0) and the RS intensity. A TU-1901 double beams UV-visible spectrophotometer (Beijing Purkinje General Instrument Ltd. Co., China), and a Model of a NaNo-ZS90 nano-sized and zeta potential analyzer (Malvem, GB) were used.A 0.50 mg/mL sample of horseradish peroxidase (HRP) was prepared by dissolving 5.0 mg of HRP (300 U/mg, Huamei Biological Engineering Co., China) in 10 mL of double-distilled water. The mixture was stored at 4 C in a refrigerator; a 2.0 × 10 -7 g/mL HRP solution was prepared daily by diluting the HRP store sol...