A design of reaction-controlled chemiluminescence imaging and its application

The paper presented a design of reaction-controlled chemiluminescence (CL) imaging analysis method. The design was based on controlling pH to initiate CL reaction and obtain high sensitivity. The pH value of CL reaction was controlled by ammonia, which was produced by injecting NaOH solution to NH4Cl solution, and the amount of ammonia could be adjusted by varying concentration, pumping time and flow rate of NaOH solution or varying concentration of NH4Cl solution. The pH of CL reagents in 96-well microtiter plates increased continuously from the same initial value due to the absorbance of ammonia and the relative CL intensity increased with the increasing pH. Based on above reaction-controlled design, the CL reaction in 96 wells could be initiated at the same time and the total CL signal of each well could be monitored. As results of above operation, a high sensitivity and better reproducibility could be obtained. A luminol, H2O2, CL system for determination of hemoglobin (Hb) was selected to validate the presented design. The CL intensity was proportional with the concentration of Hb in the range of 1.0 × 10−9 to 1.0 × 10−7 mol l−1 and the detection limit was 3.0 × 10−10 mol l−1 (3σ), the relative standard deviation (R.S.D.) for 11 parallel measurements of 1.0 × 10−8 mol l−1 Hb was 2.7%.