An Experimental Research Of A Dry-reagent Microassay For Simultaneous Determination Of Multiple Biochemical Items Based On A Miniature Biochemistry Analyzer

Objective:With the rapid and constant development of the society, the demand for the diversity of medical laboratory examination is also escalating. Laboratory examinations, which used to be performed exclusively in the central or stat laboratory of large hospitals, extend to non-laboratory settings such as district clinics, patients'bedsides and homes now. In order to determinate routine biochemical items more easily outside of the confines of the clinical laboratories of health care facilities, we studied seven biochemical items including Glu, TG, TC, TP, Alb, TBil and ALT, and established a precise dry-reagent microassay with unified key assay parameters. This make it possible to perform multiple tests simultaneously with a self-designed dry reagent card based on a miniature biochemistry analyzer. Such assays will contribute to the emergence of an improved model of miniature biochemistry analyzer which will be more compact, easily portable, and user-friendly.Since the whole research program is a complicated system, this thesis just focuses on the establishment of dry-reagent microassays of Glu, TG, TC, TP, Alb, TBil and ALT.Methods:1. Select for each item an appropriate method and reagent kit. The reagent should be mixable as a single working solution allowing for successful lyophilization, and the wavelength ranges from 380nm to 780nm.2. Add 200μl single reagent solution to each well of a microplate, and then dry the reagent in a thermostatic drier or lyophilizer. Screen proper lyopretector when necessary.3. Explore the ability to redissolve by adding water to wells with dry reagent and the reactivity of the dry reagent by adding diluted standards, and then evaluate the linear range and sensitivity.4. Increase the sample/reagent ratio or replace the chromogen or even the whole reagent kit to improve the linear range and sensitivity when necessary.5. Analyze clinical samples, and compare the results with that of automatic biochemical analyzer to evaluate the correlation of two methods.Results:1. The phenol was replaced by TBHBA as chromogen for the determination of glucose. The excipient was 10g/L albumin and the sample/reagent ratio was adjusted to 1:200.This optimized method have the advantages of higher reaction velocity, higher sensitivity, wider linear range, and lower detection limit. There was a good correlation between the results and that of biochemical analyzer.2. The reagent of triglyceride was lyophilized by the addition of 10g/L glycine. The sample/reagent ratio was increased to 1:50. The detection mode balanced linear range and sensitivity, and met the requirement of clinical application.3. The reagent of total cholesterol was lyophilized by the addition of 10g/L albumin. The sample/reagent ratio was increased to 1:50. The detection mode balanced linear range and sensitivity, and met the requirement of clinical application.4. No suitable bulking agent was found to freeze-dry the reagent of total protein and albumin. The upper limit of linear range of TP and Alb were 80g/L , 40g/L respectively when the reagents were lyophilized without the addition of excipients. The sensitivity was to be improved.5. Total bilirubin was measured by the diazo method. The chromogen powder was dissolved with water instead of the solvent in the kit, and the solution was lyophilized on microplates. The sample was diluted with the solvent in the kit. The established method met the requirement of clinical application.6. Alanine aminotransferase was measured by the kinetic method of pyruvate oxidase. The reagent was was lyophilized by the addition of 10g/L albumin. The maximal limit of linear range for measuring ALT by dry reagent analysis could reach 336IU/L, and there was a good correlation between the results and that of biochemical analyzer.7. The results of simultaneous determination were in good agreements with that of biochemical analyzer(r>0.95), and the proportional and constant systematic errors were in acceptable range. Conclusion:1. The improved methods can meet the requirements for one-step measurement of the items with dry reagent cards.2. The reagents of the established dry-reagent microassays are easily portable and can be preserved for a longer period, which can simplify measurement procedure and meet the demand of point of care testing.3. The unification of key assay parameters successfully enables simultaneous multiple tests, which results in decreased turnaround time and rapid availability of data. It also paves the way for manufacture and application of a miniature biochemistry analyzer with dry-reagent cards for multiple items.