A Prototype Antibody-based Biosensor for Measurement of Salivary MMP-8 in Periodontitis using Surface Acoustic Wave Technology

Abstract

Periodontitis is an economically important disease which is highly prevalent worldwide. Current diagnostic approaches are time-consuming and require interpretation of multiple aspects of clinical and radiographic assessment. Chair-side monitoring of inflammatory mediators of periodontitis could provide immediate information about disease activity, which can inform patient management. We aimed to develop a novel prototype biosensor to measure salivary matrix metalloproteinase-8 (MMP-8) using specific antibodies and surface acoustic wave (SAW) technology. The analytical performance of the prototype biosensor was compared to standard enzyme-linked immunosorbent assay (ELISA) using unstimulated saliva samples obtained from patients with periodontitis before and after non-surgical treatment (N = 58), patients with gingivitis (N = 54) and periodontally healthy volunteers (N = 65). Receiver operator characteristic (ROC) analysis for distinguishing periodontitis from health revealed an almost identical performance between the sensor and ELISA assays (area under curve values (AUC): ELISA 0.93; SAW 0.89). Furthermore, both analytical approaches yielded readouts which distinguished between heath, gingivitis and periodontitis, correlated identically with clinical measures of periodontal disease and recorded similar post-treatment decreases in salivary MMP-8 in periodontitis. The assay time for our prototype device is 20 minutes. The prototype SAW biosensor is a novel and rapid method of monitoring periodontitis which delivers similar analytical performance to conventional laboratory assays.

Figure 1

Response of the biosensor to hrMMP-8. Biosensor phase change response to increasing concentrations of hrMMP-8 (0–1000 ng/ml). Overlaid plots of raw data from a single experiment showing phase shifts recorded as a function of time after the addition of the detection antibody at t = 0 (a). Graph illustrating the relationship of relative phase change between samples containing MMP-8 and controls (Δφ) (b). Phase change measurements were made after final buffer wash (see Methods). Data are derived from 3 independent experiments each comprising parallel duplicate measurements on separate microchips and are presented as means ± SD (n = 6).

Figure 2

Analytical performance of the biosensor for the assay of salivary MMP-8 in periodontal disease. Analysis of salivary MMP-8 in healthy volunteers (N = 56), patients with gingivitis (N = 47) and patients with periodontitis (n = 65) using ELISA (a) and the biosensor assay (b). Data are means of duplicate measurements and are presented as box and whisker plots: boxes represent median (line) and interquartile ranges and whiskers the minimum and maximum range. Data was analysed using Kruskal-Wallis one-way ANOVA with Mann-Whitney U post hoc test, *P < 0.001. Correlation of salivary MMP-8 analysis of clinical samples (n = 168) using ELISA and biosensor assays (c). Data were analysed using Spearman’s Rank correlation, Rho = 0.795, P < 0.001.

Figure 3

Diagnostic performance of salivary MMP-8 analysis using the biosensor and ELISA in periodontal disease. Comparative receiver operator characteristic (ROC) plots for salivary MMP-8 measurements using ELISA and the biosensor assay (BS) assays. Sensitivity and specificity of gingivitis diagnosis (vs health) (a) and periodontitis diagnosis (vs health) (b) are presented and AUC data and P values included.

Figure 4

Comparative biosensor analysis of salivary MMP-8 before and after treatment for periodontitis. Analysis of salivary MMP-8 in a sub-group of patients (n = 20) before and after 6 months after non-surgical treatment for periodontitis. (a) Data presented as ‘box and whisker plots’: boxes represent median (line) and interquartile ranges and whiskers the minimum and maximum range. Data were analysed using a paired samples t-test *P = 0.026. (b) Figure showing the changes in levels on MMP-8 before and after treatment in saliva samples from individual patients.

Figure 5

Molecular interactions at the surface of the biosensor biochip. Schematic illustrating the functionalisation of the gold-coated biochip with firstly anti-IgG antibodies (red), and secondly with anti-MMP-8 capture antibodies (blue), followed by detection by successive addition of sample (containing MMP-8) and anti-MMP-8 detection antibody (green). The binding of the anti-MMP-8 detection antibody to MMP-8 bound to the immobilised complex results in a perturbation of mass at the biochip surface sufficient to generate a phase change (Δφ) in the surface acoustic wave detected by the biosensor control box. In later experiments, the anti-MMP-8 capture antibody was bound directly to the biochip, omitting the anti-IgG antibody step. A full description of the prototype biosensor structure, microelectronics and detection system has been recently published.