Cardiac Diagnostics - Research Accomplishments

In the past several years, our research team has made significant advances in the field of cardiac diagnostics.  In 2002, we reported the first fully developed and tested microchip array for cardiac risk assessment and pioneered methodologies for multiplexing cardiac biomarkers using fingerprinting techniques.  In 2004, we established the lowest limit of detection reported to date for measurement of C-reactive protein (CRP) in saliva, far exceeding the sensitivity of all current FDA approved CRP detection kits.  This increased sensitivity enabled definition of the physiological range of CRP in saliva of normal healthy, edentulous, and periodontitis patients as published and featured on the cover of Lab-on-a-Chip journal in 2005. 

In addition, we have designed and implemented the first dual platform system for simultaneous protein detection and cellular analysis of CRP and total white blood cell (WBC) counts, respectively.  According to some reports, the combined measurement of CRP and WBCs represents one of the most accurate methods to assess an individual's risk of heart disease.  Our initial data in this area was recently published in Clinical Chemistry and validation of the miniaturized analysis system is currently underway using clinical samples from healthy and heart disease patients.

Below, we provide summaries of publications relevant to the cardiac theme:

A Microchip-Based Multianalyte Assay System for the Assessment of Cardiac Risk
Anal. Chem. 2002, 74, 3030-3036

Nick Christodoulides, Maiyen Tran, Pierre N. Floriano, Marc Rodriguez, Adrian Goodey, Mehnaaz Ali, Dean Neikirk, and John T. McDevitt

The development of a novel chip-based multianalyte detection system with a cardiac theme is reported. This work follows the initial reports of "electronic taste chips" whereby multiple solution-phase analytes such as acids, bases, metal cations, and biological cofactors were detected and quantified.

The newly fashioned "cardiac chip" exploits a geometry that allows for isolation and entrapment of single polymeric spheres in micromachined pits while providing to each bead the rapid introduction of a series of reagents/washes through microfluidic structures. The combination of these miniaturized components fosters the completion of complex assays with short analysis times using small sample volumes. Optical signals derived from single beads are used to complete immunological tests that yield outstanding assay characteristics.The power and utility of this new methodology is demonstrated here for the simultaneous detection of the cardiac risk factors, C-reactive protein and interleukin-6, in human serum samples. This demonstration represents the first important step toward the development of a useful cardiac chip that targets numerous risk factors concurrently and one that can be customized readily for specific clinical settings.

Application of microchip assay system for the measurement of C-reactive protein in human saliva.
Lab Chip 2005, 261-269

Nicolaos Christodoulides, Sanghamitra Mohanty, Craig S. Miller, M. Chris Langub, Pierre N. Floriano, Priya Dharshan, Mehnaaz F. Ali, Bruce Bernard, Dwight Romanovicz, Eric Anslyn, Philip C. Fox and John T. McDevitt.

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 21 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.

Toward the Development of a Lab-on-a-Chip Dual-Function Leukocyte and C-Reactive Protein Analysis Method for the Assessment of Inflammation and Cardiac Risk
Clin Chem 2005 51: 2391-2395

Nicolaos Christodoulides, Pierre N. Floriano, Shelley A. Acosta,Karri L. Michael Ballard, Shannon E. Weigum, 1 Sanghamitra Mohanty, 1 Priya Dharshan, Dwight Romanovicz,and John T. McDevitt

Recent reports have concluded that the combined measurement of both CRP and leukocytes provides one of the most accurate methods available to date to assess an individual's risk for heart disease. This information is useful both in the context of future risk profile for apparently healthy individuals as well as for prognosis of individuals with recent occurrences of AMI. Likewise, the availability of convenient methods to measure these two disparate quantities in the same setting would be of significant importance in the management of cardiac disease, the number one health issue in developed countries.

In this report, we demonstrate the capacity of miniaturized LOC methods to perform both protein and cellular analysis of bodily fluids. While further work is required to produce practical and ruggedized instrumentation to support these tests, the basic methodologies here described are suitable for full automation and allow for multiplexing of theme-specific groups of analytes, thus promoting a more comprehensive approach to disease risk assessment and diagnosis. The use of bead- and membrane-based assay platforms and the implementation of dual function LOC structures promise the development of a flexible miniaturized total analysis system to be used at point-of-care settings. Larger scale studies are currently in progress using the newly fashioned miniaturized analysis system to test inflammatory markers CRP and leukocyte counts in healthy and heart disease patients.