2. Colorimetric Analysis
The principle of colorimetric analysis is based on the interaction between an enzyme and substrate to form and estimate colorimetrically a colored, light-absorbing complex by adding other reagent after stopping the enzyme reaction. Meanwhile, calibration curves must be made using measurand. By comparison and calculation, the generation amount of product or consumption amount of substrate can be reached. It can also be called end-point method, two-point method, sampling method, or fixed-time method. In most references, colorimetric analysis was known as fixed-time method.
Babson et al. [21] presented a colorimetric assay of serum GOT activity using a stabilized diazonium salt that reacts specifically with oxalacetic acid to yield a red colored compound. The authors suggested that the color reaction was more specific and sensitive for the reaction product, and the reagent blank was minimized. Matsuzawa and Katunuma [22] developed a rapid and accurate method for the determination of GPT and lactic dehydrogenase in serum and tissue using the diazonium salt and the coupling enzymes. They studied and discussed several factors affecting the coupling reactions in detail, and suggested that it was possible to measure the ranges of 0-150 mU in GPT assay and 0-1500 mU in lactic dehydrogenase assay by the present methods. It took about 5 min at 37 °C in a final volume of 2 ml to detect the mixtures. Lippi and Guidi [23] reported a colorimetric ultramicromethod for serum GOT and GPT determination. This method was based on the use of glutamate dehydrogenase for the enzymatic estimation of the glutamate formed. The dehydrogenation of the glutamate gave rise to the reduction of a diazonium salt, and it was possible to perform a photometric reading of the colored compound at 520 nm. Their experimental results showed that 20 μl serum and only 45 min incubation time at the temperature of 37 °C were necessary. The normal values never exceeded 54.5 U for the serum GOT and 52 U for GPT. Under conditions of viral hepatitis values of 390 U for GPT and 310 U for serum GOT were obtained. Bailey et al. [24] determined whether the ingestion of isoniazid interfered with the colorimetric reaction used in most autoanalyzers to measure serum GOT. Colormetric (autoanalytic) and noncolormetric (enzymatic) measurements of GOT were made on a venous blood sample from each of 100 hospital employees who were taking isoniazid to prevent tuberculosis. It was concluded that autoanalytic (colormetric) measurements of GOT in isoniazid recipients are reliable. A general colorimetric procedure for measuring GOT and GPT which were linked to the NADH/NAD+ system was described by Whitaker [25]. Two color reactions for determining the reduced coenzyme NADH were discussed. A colored formazan was produced by the reduction of the tetrazolium salt INT with NADH, electron transfer being facilitated by PMS; the formazan was stable and the extinction was read at 500 nm. The colored ferrous dipyridyl complex was produced following the reduction of a ferric salt with NADH in the presence of PMS; the colored complex was stable for 30 min, the extinction was read at 520 nm. An important extension on colorimetric method was reported by Oki et al.[26], who suggested a biochip which can analyze hepatic function quickly from the bedside or at home. The chip essentially consisted of two chips, one was used for mixing the substrate buffer solution with serums using a centrifugal method and the other was used for measuring the amounts of γ-glutamyltranspeptidase (γ-GTP), GOT and GPT in the serums employing a colorimetric method. The mixing and measurement channels were fabricated by molding their reverse patterns onto a poly (ethylene terephthalate) plate. The inner wall of the measurement channel was performed by a hydrophobic treatment to efficiently propagate the light efficiently. Calibration curves were obtained based on an end-point method for γ-GTP and a rate assay for GOT and GPT. Figure 1 shows a cross-sectional view of the measurement chip equipped with peripheral components and the actual mixing process. The mixing chip pattern was suggested by taking account of the concept that multiple-chambers with several branch channels were formed to ensure efficient contact of the solution with the inner walls of the mixing chambers, thus enhancing the mixing on the wall surfaces. The mixing of the stored solution in the chambers was carried out by means of a stirring process in one chamber group based on the chip rotation and the solution was transferred from one chamber group to another in which the same process was carried out. This series process was then repeated.