Material and methods

Twenty patients were enrolled at the Department of Orthodontics of the dental school affiliated with Capital Medical University in Beijing, China. They were orthodontic patients receiving treatment for 8 to 12 months, including 12 female and 8 male subjects (age range, 13-24 years). Inclusion criteria were good oral hygiene, no periodontal disease, no bleeding on probing, no caries, and no systemic diseases. The type of orthodontic displacement of the incisors was not considered in the inclusion criteria because it was not supposed to affect the comparison of the 2 detection methods.

This project was approved by the institutional review board of Beijing Stomatological Hospital, and informed consents were obtained from the patients.

For each patient, 2 teeth were chosen for GCF collection: the left and right maxillary central incisors. The teeth were gently washed with water, dried, and isolated with cotton rolls to prevent saliva contamination. GCF was collected from the mesial and distal sides of each tooth with a filter paper strip (Tianjin Zhongjin Biological Technology, Tianjin, China), which was inserted 1 to 2 mm into the gingival sulcus for 30 seconds. The same procedure was repeated at a 1-minute interval. As a result, 160 strips of samples were obtained, 2 for each position. After removal, each strip was immediately sealed in a microcentrifuge tube with phosphate-buffered saline solution containing 0.1 m mol/L of phenylmethylsulphonyl fluoride. To retrieve the sample from the paper strip, the GCF was eluted by centrifugal filtration at 15,000 g for 5 minutes with 100 μL aliquots of buffer. Two GCF samples from the same position were then pooled to give a total volume of 200 μL and stored at −70°C. Therefore, 80 GCF samples were prepared, and each was assessed with both electrochemical and spectrophotometric ELISA.

The sandwich ELISA procedure was used for the detection of DSPP in the standard solution of all GCF samples. In general, 50 μL of the DSPP sample was mixed with biotin labeled DSPP antibody and added into the microplate, which was precoated with DSPP monoclonal antibody. Then 50 μL of streptavidin labeled horseradish peroxidase solution was added into each well. After incubating at 37°C for 60 minutes, the wells of the microplate were washed 5 times with 0.02 mol/L of phosphate-buffered saline solution containing 0.05% Tween-20. After washing, 200 μL of substrate solution was added into each well and incubated at 37°C for 30 minutes. Then 50 μL of Britton-Robinson buffer (0.2 mol/L, pH 5.0) was added to each well.

For the electrochemical analysis of the samples, a miniature 3-electrode system was directly inserted into the well, and the second-order derivative linear sweep voltammetric reduction peak was measured with a JP-303 voltammetric analyzer (Chengdu Apparatus, Chengdu, China). The experimental procedure is shown in Figure 1 .

Procedures for the sandwich ELISA for DSPP.

Using horseradish peroxidase as the labeled enzyme, o-tilidine was used as the enzymatic reaction substrate in this research; it can be oxidized by hydrogen peroxide to give an electroactive azo product. Under the optimal conditions, the proposed o-tilidine–hydrogen peroxide–horseradish peroxidase system was combined with double-antibody sandwich ELISA and further used to detect DSPP in GCF. In the electrochemical ELISA procedure described above, the more DSPP in the solution of GCF, the more the labeled horseradish peroxidase was coupled on the microplate and more of the enzymatic product could be formed; this gave a higher reduction peak current.

The prepared GCF solution of each sample was also analyzed with the traditional spectrophotometric ELISA procedure.

In the spectrophotometric ELISA procedure, the more DSPP in the solution of GCF, the more the labeled horseradish peroxidase was coupled on the microplate and more of the enzymatic product could be formed; this gave a darker color.