Upload
ashley-anthony
View
222
Download
6
Tags:
Embed Size (px)
Citation preview
Biosensors
Peter C. Doerschuk Biomedical Engineering
and Electrical and Computer EngineeringCornell University
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.
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
Bioanalytical Microsystems & Biosensors Laboratory
Department of Biological & Environmental Engineering145 Riley-Robb Hall
Cornell University, Ithaca, NY
Antje J. Baeumner (PI)/Katie A. Edwards
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– ~105-106 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
Comparison to other detection methods
LOD (bkgd+3*stdev) Maximum S:N at maximum
Fluorescein-labeled antibody 13.3 ng/mL 500 ng/mL 3.35
HRP-labeled antibody 2.05 ng/mL 50 ng/mL 1.95
Antibody-tagged liposomes 0.45 ng/mL 500 ng/mL 14.95
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
0.01 0.1 1 10 100 1000 10000
[CTB] (ng/mL)
Signal:Noise
Fluorescein-labeled antibody
Antibody-tagged dye-encapsulating liposomes
HRP-labeled antibody
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.
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-analyteanalysis proof of principle
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
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
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0.001 0.01 0.1 1 10 100 1000 10000 100000
[CTB] (ng/mL)
Fluorescence Signal
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
mRNA detection
0
10000
20000
30000
40000
50000
60000
70000
80000
0.001 0.01 0.1 1 10 100
[Synthetic DNA Target] (nM)
Fluorescence (RFU)
• 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)
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 GSerotype 1Serotype 2Serotype 3Serotype 4Negative 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
Antje J. Baeumner (PI)/Katie A. Edwards
3 Post-doctoral associates3 Ph.D. students
1 Research support Specialist4 Undergraduate students
Present technical capabilities:Dynamic light scattering
Sequential injection analysesMicrofluidic device developmentLateral flow assay development
Microtiter plate assay developmentNucleic Acid Based Sequence Amplification (NASBA), PCR
Liposome preparationBlood handling
Lab information
Ethanol Biosensor: Models and Signal Processing Jae-Joon Han, Martin Plawecki,
Peter Doerschuk, and Sean O’Connor