Use of saliva-based nano-biochip tests for acute myocardial infarction at the point of care: a feasibility study.

BACKGROUND: For adults with chest pain, the electrocardiogram (ECG) and measures of serum biomarkers are used to screen and diagnose myocardial necrosis. These measurements require time that can delay therapy and affect prognosis. Our objective was to investigate the feasibility and utility of saliva as an alternative diagnostic fluid for identifying biomarkers of acute myocardial infarction (AMI). METHODS: We used Luminex and lab-on-a-chip methods to assay 21 proteins in serum and unstimulated whole saliva procured from 41 AMI patients within 48 h of chest pain onset and from 43 apparently healthy controls. Data were analyzed by use of logistic regression and area under curve (AUC) for ROC analysis to evaluate the diagnostic utility of each biomarker, or combinations of biomarkers, in screening for AMI. RESULTS: Both established and novel cardiac biomarkers demonstrated significant differences in concentrations between patients with AMI and controls without AMI. The saliva-based biomarker panel of C-reactive protein, myoglobin, and myeloperoxidase exhibited significant diagnostic capability (AUC = 0.85, P < 0.0001) and in conjunction with ECG yielded strong screening capacity for AMI (AUC = 0.96) comparable to that of the panel (brain natriuretic peptide, troponin-I, creatine kinase-MB, myoglobin; AUC = 0.98) and far exceeded the screening capacity of ECG alone (AUC approximately 0.6). En route to translating these findings to clinical practice, we adapted these unstimulated whole saliva tests to a novel lab-on-a-chip platform for proof-of-principle screens for AMI. CONCLUSIONS: Complementary to ECG, saliva-based tests within lab-on-a-chip systems may provide a convenient and rapid screening method for cardiac events in prehospital stages for AMI patients.

Lab-on-a-chip methods for point-of-care measurements of salivary biomarkers of periodontitis.

Salivary secretions contain a variety of molecules that reflect important pathophysiological activities. Quantitative changes of specific salivary biomarkers could have significance in the diagnosis and management of both oral and systemic diseases. Modern point-of-care technologies with enhanced detection capabilities are needed to implement a significant advancement in salivary diagnostics. One such promising technology is the recently described lab-on-a-chip (LOC) assay system, in which assays are performed on chemically sensitized beads populated into etched silicon wafers with embedded fluid handling and optical detection capabilities. Using this LOC system, complex assays can be performed with small sample volumes, short analysis times, and markedly reduced reagent costs. This report describes the use of LOC methodologies to assess the levels of interleukin-1beta (IL-1beta), C-reactive protein (CRP), and matrix metalloproteinase-8 (MMP-8) in whole saliva, and the potential use of these biomarkers for diagnosing and categorizing the severity and extent of periodontitis. This study demonstrates that the results achieved by the LOC approach are in agreement with those acquired with standard enzyme-linked immunosorbent assay (ELISA), with significant IL-1beta and MMP-8 elevations in whole saliva of periodontitis patients. Furthermore, because of the superior detection capacities associated with the LOC approach, unlike those with ELISA, significant differences in CRP levels between periodontitis patients and normal subjects are observed. Finally, principal component analysis (PCA) is performed to yield an efficient method to discriminate between periodontally healthy and unhealthy patients, thus increasing the diagnostic value of these biomarkers for periodontitis when examined with the integrated LOC sensor system.

A microchip-based assay for interleukin-6.

The electronic taste chip (ETC) assay system is a lab-on-a-chip technology that offers a microchip platform on which bead-based immunoassays are performed. Each bead within the array serves as its own independent self-contained "microreactor" system, with its selectivity determined by the specificity of the antibody that it hosts. The bead-loaded chip is sandwiched between two optically transparent polymethylmethacrylate inserts, packaged within a metal casing described here as the "flow cell." This flow cell allows for delivery of sample and detecting reagents to the microchip and the associated beads. Images of fluorescent beads are captured with a digital video chip and analyzed to facilitate detection and, ultimately, quantitation of analytes in complex fluids. This chapter describes the application of the ETC system for the detection and measurement of interleukin (IL)-6.

Disposable polydimethylsiloxane/silicon hybrid chips for protein detection.

This paper presents disposable protein analysis chips with single- or four-chamber-constructed from poly(dimethylsiloxane) (PDMS) and silicon. The chips are composed of a multilayer stack of PDMS layers that sandwich a silicon microchip. This inner silicon chip features an etched array of micro-cavities hosting polymeric beads. The sample is introduced into the fluid network through the top PDMS layer, where it is directed to the bead chamber. After reaction of the analyte with the probe beads, the signal generated on the beads is captured with a CCD camera, digitally processed, and analyzed. An established bead-based fluorescent assay for C-reactive protein (CRP) was used here to characterize these hybrid chips. The detection limit of the single-chamber protein chip was found to be 1 ng/ml. Additionally, using a back pressure compensation method, the signals from each chamber of the four-chamber chip were found to fall within 10% of each other.

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

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.