Application of microchip assay system for the measurement of C-reactive protein in human saliva

Abstract

In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in this study to provide direct experimental evidence that chronic periodontal disease may be associated with higher levels of salivary CRP.

Mixing with bubbles: a practical technology for use with portable microfluidic devices

“…40 The mixer is compatible with viscosities corresponding to those of biological fluids that are of potential interest: the viscosity of human blood serum (y2 mPa s), 41 whole blood, which has a viscosity of approximately 5-6 mPa s at low rates of shear, 41 urine (y1 mPa s), 42 tears (2 to 9 mPa s), 43 and saliva (2 to 9 mPa s). 44 …”

Salivary Biomarkers, Oral Inflammation, and Functional Status in Patients With Heart Failure

“…Although prior studies have reported that saliva can be used to measure biomarkers in patients with cardiovascular disease (CVD) and acute myocardial infarction (Christodoulides et al, 2005;Jones et al, 1994;Miller et al, 2010;Mirzaii-Dizgah & Jafari-Sabet, 2011), only one study of HF patients has examined salivary biomarkers. In a study comparing 40 outpatients with HF to 45 healthy controls, Offenbacher and Beck (2014) found that salivary N-terminal pro-BNP (NT-proBNP) had 82.2% sensitivity and 100% specificity for the presence of HF.…”

Novel possibilities in the study of the salivary proteomic profile using SELDI-TOF/MS technology

“…In addition, saliva contains analytes at concentrations 1,000 times lower than in plasma (33). Thus, sensitive detection systems are required when using saliva as diagnostic material (9).…”