Timothy M. Blicharz

Multiplexed Sandwich Immunoassays using Electrochemiluminescence Imaging Resolved at the Single Bead Level

A new class of bead-based microarray that uses electrogenerated chemiluminescence (ECL) as a readout mechanism to detect multiple antigens simultaneously is presented. This platform demonstrates the possibility of performing highly multiplexed assays using ECL because all the individual sensing beads in the array are simultaneously imaged and individually resolved by ECL. Duplex and triplex assay results are demonstrated as well as a cross reactivity study.

Fiber-Optic Microsphere-Based Antibody Array for the Analysis of Inflammatory Cytokines in Saliva

Antibody microarrays have emerged as useful tools for high-throughput protein analysis and candidate biomarker screening. We describe here the development of a multiplexed microsphere-based antibody array capable of simultaneously measuring 10 inflammatory protein mediators. Cytokine-capture microspheres were fabricated by covalently coupling monoclonal antibodies specific for cytokines of interest to fluorescently encoded 3.1 microm polymer microspheres. An optical fiber bundle containing approximately 50,000 individual 3.1 microm diameter fibers was chemically etched to create microwells in which cytokine-capture microspheres could be deposited. Microspheres were randomly distributed in the wells to produce an antibody array for performing a multiplexed sandwich immunoassay. The array responded specifically to recombinant cytokine solutions in a concentration-dependent fashion. The array was also used to examine endogenous mediator patterns in saliva supernatants from patients with pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). This array technology may prove useful as a laboratory-based platform for inflammatory disease research and diagnostics, and its small footprint could also enable integration into a microfluidic cassette for use in point-of-care testing.

Use of Colorimetric Test Strips for Monitoring the Effect of Hemodialysis on Salivary Nitrite and Uric Acid in Patients with End-Stage Renal Disease: A Proof of Principle

BACKGROUND Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte concentrations as dialysis progressed. We observed that salivary nitrite (NO(2)(-)) and uric acid (UA) concentrations consistently decreased as dialysis proceeded. METHODS Solution-based colorimetric-detection chemistries for NO(2)(-) and UA were converted to a test strip format to produce a simple method for semiquantitatively measuring NO(2)(-) and UA concentrations in the clinic or at the patients home. We assessed the test strips with saliva samples collected from both ESRD patients undergoing dialysis and healthy control volunteers to qualitatively monitor the effect of dialysis on salivary NO(2)(-) and UA. We used computer software to analyze digital images of the resulting test strip color intensities. RESULTS Test strip measurements showed that mean salivary concentrations of NO(2)(-) and UA were decreased in ESRD patients by 86% and 39%, respectively, compared with 15% and 9% for time-matched controls. Comparison of test strip results with calibrated solution-based assays suggests that the test strips can semiquantitatively measure salivary concentrations of NO(2)(-) and UA. CONCLUSIONS The colorimetric test strips monitored changes in salivary NO(2)(-) and UA concentrations that occurred in ESRD patients during dialysis. The test strips may prove useful for noninvasively evaluating dialysis progress and may also be useful for monitoring renal disease status.

Microsensor arrays for saliva diagnostics.

Abstract:  Optical fiber microarrays have been used to screen saliva from patients with end‐stage renal disease (ESRD) to ascertain the efficacy of dialysis. We have successfully identified markers in saliva that correlate with kidney disease. Standard assay chemistries for these markers have been converted to disposable test strips such that patients may one day be able to monitor their clinical status at home. Details of these developments are described. In addition, saliva from asthma and chronic obstructive pulmonary disease (COPD) patients is being screened for useful diagnostic markers. Our goal is to develop a multiplexed assay for these protein and nucleic acid biomarkers for diagnosing the cause and severity of pulmonary exacerbations, enabling more effective treatment to be administered. These results are reported in the second part of this article.

Optical-fiber bundles.

Optical‐fiber bundles have been employed as a versatile substrate for the fabrication of high‐density microwell arrays. In this minireview, we discuss the application of optical‐fiber‐bundle arrays for a variety of biological problems. For genomics studies and microbial pathogen detection, individual beads have been functionalized with DNA probes and then loaded into the microwells. In addition, beads differentially responsive to vapors have been employed in an artificial olfaction system. Microwell arrays have also been loaded with living cells to monitor their individual response to biologically active compounds over long periods. Finally, the microwells have been sealed to enclose single enzyme molecules that can be used to measure individual molecule catalytic activity.

Detection of inflammatory cytokines using a fiber optic microsphere immunoassay array

A multiplexed fiber optic microsphere-based immunoassay array capable of simultaneously measuring five inflammatory cytokines has been developed. Five groups of amine-functionalized 3.1 micron microspheres were internally encoded with five distinct concentrations of a europium dye and converted to cytokine probes by covalently coupling monoclonal capture antibodies specific for human VEGF, IFN-gamma, RANTES, IP-10, and Eotaxin-3 to the microspheres via glutaraldehyde chemistry. The microspheres were pooled and loaded into a 1 mm diameter fiber optic bundle containing ~50,000 individual etched microwells, producing the multiplexed cytokine immunoassay array. Multiple arrays can be created from a single microsphere pool for high throughput sample analysis. Sandwich fluoroimmunoassays were performed by incubating the probe array in a sample, followed by incubation in a mixture of biotin-labeled detection antibodies that are complementary to the five cytokines. Finally, universal detection of each protein was performed using a fluorescence imaging system after briefly immersing the array in a solution of fluorophore-labeled streptavidin. The multiplexed cytokine array has been shown to respond selectively to VEGF, IFNgamma, RANTES, IP-10, and Eotaxin-3, permitting multiplexed quantitative analysis. Ultimately, the multiplexed cytokine array will be utilized to evaluate the potential of using saliva as a noninvasive diagnostic fluid for pulmonary inflammatory diseases such as asthma.

Microsphere based saliva diagnostics

Saliva presents a minimally invasive alternative medium to blood for performing diagnostics1. Microsphere sensors for ions, small organic molecules, and proteins are currently being developed and optical microarrays containing thousands of these sensors will be used for simultaneous multi-analyte analysis. The fiber bundle platform in use is 1mm in diameter and contains approximately 50,000 individually addressable 3.1μm fibers, each with an etched well capable of housing a single 3.1μm microsphere sensor. Micron-sized bead-based chemistries are produced in house, followed by deposition onto a fiber-optic bundle platform, allowing for multiplexed analysis. The ultimate goal is to develop a universal diagnostic system using saliva as the diagnostic medium. This platform will permit multiplexed analysis of a sample by integrating microfluidics with the optical arrays loaded with sensors capable of detecting relevant biomarkers associated with a wide range of disease states. Disease states that are currently under investigation include end stage renal disease (ESRD) and Sjoegrens Syndrome (SS).