Microfluidic immunoassays as rapid saliva-based clinical diagnostics
- Amy E. Herr†,‡,
- Anson V. Hatch†,
- Daniel J. Throckmorton†,
- Huu M. Tran†,
- James S. Brennan†,
- William V. Giannobile§, and
- Anup K. Singh†
- †Biosystems Research Department, Sandia National Laboratories, Livermore, CA 94550; and
- §Michigan Center for Oral Research, School of Dentistry, University of Michigan, Ann Arbor, MI 48106
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Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved January 11, 2007 (received for review August 21, 2006)
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Fig. 1.μCEI device layout. Fluid wells are labeled according to contents as follows: S, sample; B, buffer; SW, sample waste; BW, buffer waste; mAb*, fluorescently labeled monoclonal antibody to MMP-8. Polyacrylamide gel composition is indicated by grayscale shading (%T and %C are percentage of total acrylamide and bis-acrylamide cross-linker, respectively). Inset shows a 40× bright-field image of the size-exclusion membrane. (Scale bar, 100 μm.)
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Fig. 2.On-chip sample enrichment. (A) Schematic depicting operation of the μCEI device. After a buffer priming step (not depicted), the detection mixture is loaded against the size-exclusion membrane. Saliva sample is then loaded, resulting in coenrichment of saliva and αMMP-8* at the size-exclusion membrane. An electric potential is applied across the membrane, causing the enriched species to elute into the separation channel, thus initiating the electrophoretic immunoassay. Subsequently, the electric potential is switched to omit the membrane from the current path. Current flow is indicated by i. (B) Electropherograms and gel-like plots show that, with all other conditions held fixed, substantial complex is observed after a 5-min enrichment. Protein internal standard is marked with an asterisk on the electropherograms.
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Fig. 3.Calibration of the MMP-8 μCEI using pooled saliva from healthy controls. (A) Electropherograms and corresponding gel-like plots for MMP-8 standard sample analyses demonstrate detection of recombinant human MMP-8 spiked into saliva at known concentrations. (B) MMP-8 dose–response curve generated by μCEI. The MMP-8 complex peak areas of the standard samples (black filled symbols) are plotted as a function of known MMP-8 concentration. A four-parameter logistic model (4PL, solid line) of the form y = β2 + (β1 − β2)/(1 + (x/β3)β4) was fit by the method of nonlinear least-squares to the standard samples. The 4PL fit was used to calculate the concentration of endogenous MMP-8 in the patient saliva samples (gray filled symbols).
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Fig. 4.μCEI measurement of endogenous MMP-8 in saliva. (A) Linear regression analysis for the MMP-8 concentrations measured by μCEI and commercial ELISA. A linear relationship (y = 0.91x + 181.7, r 2 = 0.979) was observed over the operating range investigated. (B) μCEI MMP-8 measurements from a healthy patient and patients clinically diagnosed as having moderate to severe periodontitis reveal differences in MMP-8 levels.
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Fig. 5.Multistep photopolymerization process enables fabrication of μCEI device. (A) μCEI chip layout with size-exclusion membrane. (B) Separation gel precursor solution loaded into channels. UV photomasking is used to define an 8% total acrylamide plug near the end of the separation channel. (C) Loading gel precursor solution is used to define the loading gel (3.5%) and the smaller pore-size separation gel. (D) Flood UV exposure polymerizes the separation and loading gels.
Footnotes
- ‡To whom correspondence should be addressed at: Sandia National Laboratories, P.O. Box 969, MS 9292, Biosystems Research Department, Livermore, CA 94550. E-mail: aeherr{at}sandia.gov
- © 2007 by The National Academy of Sciences of the USA
