Development of an in vitro microassay for glucose quantification in submicrolitre volumes of biological fluid

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Abstract
Glucose quantification in serum or plasma is traditionally based on a colourimetric enzymatic assay using commercially available assay kits. Sample volumes of blood or serum are usually in the range of a few microlitres to a few millilitres. However, for biological fluids such as gingival crevicular fluid (GCF), which can only be sampled in submicrolitre volumes, such assays have proven unsuitable. The aim of this study was to develop a reliable and reproducible assay for quantifying glucose in submicrolitre samples of GCF. The assay involved modification of a commercially available kit for glucose quantification. Test solutions of (i) serum and (ii) serum with added glucose at known concentrations (range 50–400 mg/dl) were prepared to simulate GCF and GCF enriched with glucose, respectively. Submicrolitre volumes (range 0.2 µl to 0.8 µl) of the test solutions were added to the reagent solution (200 µl) using a Hamilton syringe. The reaction was performed under standard conditions of time and temperature. The colour change was assayed spectrophotometrically at 492 nm. The results showed that this microassay is sufficiently sensitive to detect 50 mg/dl glucose in 0.2 µl of sample and indicate that the accuracy and sensitivity of this assay make it suitable for glucose quantification in submicrolitre volumes of GCF, particularly relevant to investigations of the relationship between diabetes mellitus and chronic inflammatory periodontal disease. In vivo evaluation of this novel microassay was performed using GCF samples taken from periodontally healthy and chronic periodontitis patients. Using non‐parametric analysis, the results showed that the assay detected statistically significant differences in glucose concentrations between the two patient groups (p < 0.05). Higher glucose levels were detected at the periodontally diseased sites. For each patient, the GCF‐glucose : blood‐glucose ratio was calculated. The results show that this ratio was higher in the periodontitis group (1 : 2) when compared to the healthy group (1 : 9). In conclusion, the results of this investigation have shown that this microassay can quantify glucose in GCF and that GCF‐glucose levels are higher at periodontitis sites.