1. Biosensors Peter C. Doerschuk Biomedical Engineering and Electrical and Computer Engineering Cornell University

2.  Extensive activity spread throughout Engineering and Science.  Organization: Within departments, e.g., BME, ECE, CBE, BEE, AEP. Within centers, especially the Nanobiotechnology Center (NBTC), a National Science Foundation, Science and Technology Center.  Goals: Biosensor devices (really biointerface devices since both sensing and actuating are of interest). Inference and control algorithms for use with such devices. Basic science to clinical medicine.

3.  Examples Professor Harold Craighead, Applied and Engineering Physics, Nanobiotechnology Center (NBTC) (http://www.nbtc.cornell.edu/): Professor Antje Baeumner, Biological & Environmental Engineering, Bioanalytical Microsystems & Biosensors Lab (http://hive.bee.cornell.edu/bmb_lab/index.html): devices for the detection of hazardous biological and chemical substances in the environment, in food, and in medical diagnostics. Professor Peter Doerschuk, Biomedical Engineering and Electrical and Computer Engineering: mathematical and statistical models, signal and image processing, high performance computing; sketch work on an implanted biosensor for ethanol. research in biomolecular devices & analysis, cellular microdynamics, cell-surface interactions, and nanoscale cell biology

4. Bioanalytical Microsystems & Biosensors Laboratory Department of Biological & Environmental Engineering 145 Riley-Robb Hall Cornell University, Ithaca, NY Antje J. Baeumner (PI)/Katie A. Edwards

5. Advantages of Liposomes Liposomes can serve as a substitute for fluorophore, colloidal gold, or enzymatic signal enhancement Interior cavity can encapsulate many hydrophilic signaling molecules –~10 5 -10 6 dye molecules Hydrophobic molecules can be bi-layer incorporated Lipid bilayers can be conjugated to biorecognition elements –Functional groups available for post-formation conjugation –Direct incorporation Facile control over analytical aspects: –Liposome size, degree of conjugation, concentration of encapsulants Long-term stability Instantaneous signal amplification

6. Comparison to other detection methods LOD (bkgd+3*stdev)MaximumS:N at maximum Fluorescein-labeled antibody13.3 ng/mL500 ng/mL3.35 HRP-labeled antibody2.05 ng/mL50 ng/mL1.95 Antibody-tagged liposomes0.45 ng/mL500 ng/mL14.95 Sandwich immunoassay for cholera toxin, subunit B using fluorescein, HRP, or dye- encapsulating liposome labeled antibody. Results are plotted in terms of signal to noise.

7. Recent Work Development of rapid lateral flow assays for: –CD4 cells from human blood –Cryptosporidium parvum –Pathogenic bacteria (i.e.-Bacillus anthracis, Escherichia coli) –Dengue virus (serotype specific) –Herbicides (Alachlor, imazethapyr) Development of microtiter plate assays for cell culture supernatants: –Cholera toxin –Insulin Visualization and quantification of cholera toxin binding to epithelial cells Encapsulation of DNA oligonucleotides for detection of protective antigen from B. anthracis – allowed for multi-analyte analysis proof of principle

8. Assay Overview Biorecognition elements can be conjugated to liposomal bilayer: –Antibodies –Streptavidin or Protein A/G, Enzymes, Other Proteins –Small-molecule analytes –Fluorophores Hydrophilic molecules can be encapsulated within interior cavity –Enzymes –Fluorophores –Electrochemical markers –Oligonucleotides Assay types –Sandwich immunoassays –Sandwich hybridizations –Competitive assays Assay formats –Lateral-flow assays –Microfluidic devices –Sequential-injection analysis –Microtiter plates

9. Cholera toxin detection Methods to detect on-cell binding of Cholera toxin and its production in culture supernatants were developed Used to visualize and quantify the binding of CT to Caco-2 epithelial cells co- cultured with V. cholerae Detected by sandwich immunoassay for detection by a fluorescence microtiter plate reader and microscopy Analytical Biochemistry, vol. 368 (1), p. 39 – 48 (2007) Caco-2 epithelial cells grown in microtiter plates and incubated with cholera toxin (CT) standards or V. cholerae. GM1 tagged fluorophore labeled liposomes were used to visualize bound CT. Sandwich immunoassay using GM1-tagged liposomes. Limit of detection (bkgd+3xStDev) = 0.34 ng/mL, Assay range: ~1-500 ng/mL, CV ≤ 3.7%, Assay time: 3.5 hours

10. mRNA detection mRNA extracted from culture and amplified using NASBA Sandwich-hybridization of amplified RNA target between reporter probe- tagged liposomes and immobilized capture probes Synthetic DNA analogue used for development work Assay proven successful for the detection of mRNA from E. coli, B. anthracis, Dengue virus and C. parvum DNA-tagged liposomes in a sandwich hybridization assay for B. anthracis atxA mRNA. Limit of detection (bkgd+3xStDev) = 0.11 nM, Assay range: ~0.5-50 nM, CV ≤ 4.4%, Assay time: 1.75 hours Analytical Bioanalytical Chemistry, vol. 386 (6), p. 1613 – 1623 (2006)

11. Dengue virus detection Sandwich hybridization detection of amplified mRNA using LFA with capture probes immobilized in different zones Allows for distinction between 4 serotypes Sensitivity: 10 pfu/mL 2 4 (4) 3 1 G Serotype 1 Serotype 2 Serotype 3 Serotype 4 Negative control Analytical Bioanalytical Chemistry, vol. 380 (1), p. 46 – 53 (2004) DNA-tagged liposomes in a sandwich hybridization assay for Dengue virus mRNA. Serotype-specific capture probes were immobilized in spatially different zones

12. Antje J. Baeumner (PI)/Katie A. Edwards 3 Post-doctoral associates 3 Ph.D. students 1 Research support Specialist 4 Undergraduate students Present technical capabilities: Dynamic light scattering Sequential injection analyses Microfluidic device development Lateral flow assay development Microtiter plate assay development Nucleic Acid Based Sequence Amplification (NASBA), PCR Liposome preparation Blood handling Lab information

13. Ethanol Biosensor: Models and Signal Processing Jae-Joon Han, Martin Plawecki, Peter Doerschuk, and Sean O’Connor