Saliva Diagnostics Accomplishments and Future Research

The microchip efforts described here remain unique worldwide in terms of functional lab-on-a-chip (LOC) methods having a demonstrated capacity to meet or exceed the analytical characteristics (sensitivity, selectivity, precision, limit of detection) of mature macroscopic instrumentation for a large variety of analyte systems. Listed below are some of the major accomplishments that have been made in the area of salivary diagnostics.

Bead Array Multiplexed Assays

Perhaps the most powerful characteristic of the bead-array chip is its capacity to detect

multiple analytes simultaneously. The array multiplexing capacity is demonstrated here in a colorimetric mode for the two cardiac risk factors, CRP and IL-6. Detection of 3 analytes (CRP, IL-6 and TNF-a) in the fluorescence mode has also been achieved. These results demonstrate the capacity of the bead array system to detect simultaneously multiple analytes without a compromise in sensitivity or specificity.

Ultra-sensitive Saliva Assay Methods Developed

A particularly notable achievement was the detection of CRP in saliva samples at ultra-low concentrations. The bead array approach yields a much lower (by at least 5 orders of magnitude) limit of detection than that exhibited by the standard ELISA procedure (see Figure below).

Indeed, the limit of detection and assay range exhibited by the bead array LOC approach are lower and wider than all other CRP assays reported in the literature to date. It is possible, with as low as 10 µL of sample, to detect accurately CRP concentrations between 10 pg/mL to 100,000 ng/mL using this 12 minute assay procedure.

Correlation with ELISA

As the majority of saliva CRP levels are undetectable with ELISA, validation studies of the LOC approach were first performed using serum samples (see Figure below). Data shown here represent CRP values from 9 serum donors evaluated in parallel by ELISA and the bead array method. With such serum measurements there is excellent agreement between the two methods.

Recovery Studies

Further, the bead array was also validated and compared to ELISA in recovery studies. Saliva samples were spiked with known amounts of CRP (10 and 100 pg/mL) and measured results were compared with the expected value based on the amount added to the sample. Recovery of salivary CRP by bead array was determined to be between 94-100%, while that of ELISA, presumably because of its lack in sensitivity in that CRP concentration range was much lower at ~70%.

First to Establish Patho-Physiological Range for Important Inflammation marker, CRP in Saliva

Saliva samples of healthy individuals, edentulous subjects and periodontitis patients were measured for CRP. Healthy and edentulous groups demonstrated significantly lower CRP levels (92 and 65 pg/mL, respectively) compared to the periodontitis counterpart (mean CRP levels at 2,001pg/mL). Furthermore, the microchip system efficiently detected differences in CRP levels between and within each of the groups tested. The CRP values below 225 pg/mL define the majority (93.3 %) of healthy individuals, whereas CRP levels above this concentration discriminate 86% of periodontitis subjects. Further, patients with high CRP levels (over 225 pg/mL) were associated with an odds ratio (OR) of 91 for periodontitis (95% confidence interval, 7.35 to 1100, P < 0.0001). In contrast, when the same samples were analyzed with the hs-CRP ELISA, with a detection limit at 2 ng/ml, CRP was detected in only 2 of the 30 samples tested, both of which were from the periodontitis group.

This work, funded by NIH, represents the first study in which CRP was efficiently measured in saliva of both healthy and periodontitis patients, and was recently published in and featured on the cover of the March 2005 issue of the Lab on a Chip journal and selected as a Hot Article in the journal's web page upon its publication.

This work demonstrates clearly the advantages offered by bead-array approach over established methods and illustrates the strong utility of ultra-sensitive assays for use in saliva-based diagnostic testing. Our ultra-sensitive saliva assay methods allow us to investigate the link between local and systemic disease through the detection of CRP and other biomarkers in saliva with an expanded biomarker panel in conjunction withy the development of customized instrumentation.

Biomarker Discovery

Marrying our experience in LOC assay systems with biomarker discovery achieved by our collaborators at the University of Kentucky, we develop novel assays targeting potentially discriminatory salivary biomarkers of periodontitis, identified in saliva, serum, GCF, and gingival tissues through ELISA, Luminex, and microarray technologies. This screening process led to identification of a smaller panel of biomarkers, associated with local oral disease as well as with systemic disease, such as heart disease. In this context, even though historically periodontitis has been considered a disease with ramifications localized to the oral cavity, recent data support that this chronic infection with continued stimulation of the inflammatory responses of the host communicates with the systemic circulation and may contribute to cardiovascular disease. The table below provides an updated list of such biomarkers that could potentially be associated with both periodontal inflammation and systemic disease.

Handheld Laboratory Prototypes and Microfluidic Cartridges

A commercial flow cytometer now being used in the McDevitt laboratory at UT for correlation studies is shown in the background of Figure A with an image of a prototype handheld analysis card in the forefront. In part B of the image below, we show a handheld optical imaging system which includes embedded light source, customized lenses, mechanical structure and integrated video chip. This fully functional miniaturized imaging system has been compared to and validated against the commercial microscope system shown in the background. While additional work is required to optimize these handheld units, the initial studies here appear to be quite encouraging.