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... by sandwich immunoassay for detection by a fluorescence microtiter plate reader and microscopy ... liposomes in a sandwich hybridization assay for Dengue ... – PowerPoint PPT presentation

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Title: Biosensors

Peter C. Doerschuk Biomedical Engineering
and Electrical and Computer Engineering Cornell
  • Extensive activity spread throughout Engineering
    and Science.
  • Organization
  • Within departments, e.g., BME, ECE, CBE, BEE,
  • 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
  • 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//
  • Professor Antje Baeumner, Biological
    Environmental Engineering, Bioanalytical
    Microsystems Biosensors Lab
  • (http//
  • 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
  • 145 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
  • Interior cavity can encapsulate many hydrophilic
    signaling molecules
  • 105-106 dye molecules
  • Hydrophobic molecules can be bi-layer
  • Lipid bilayers can be conjugated to
    biorecognition elements
  • Functional groups available for post-formation
  • 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
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.
LOD (bkgd3stdev) Maximum SN 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
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

Assay Overview
  • Biorecognition elements can be conjugated to
    liposomal bilayer
  • Antibodies
  • Streptavidin or Protein A/G, Enzymes, Other
  • 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.
  • Detected by sandwich immunoassay for detection by
    a fluorescence microtiter plate reader and

Sandwich immunoassay using GM1-tagged liposomes.
Limit of detection (bkgd3xStDev) 0.34 ng/mL,
Assay range 1-500 ng/mL, CV 3.7, Assay time
3.5 hours
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.
Analytical Biochemistry, vol. 368 (1), p. 39 48
mRNA detection
  • mRNA extracted from culture and amplified using
  • 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.
  • DNA-tagged liposomes in a sandwich hybridization
    assay for B. anthracis atxA mRNA. Limit of
    detection (bkgd3xStDev) 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

Serotype 1 Serotype 2 Serotype 3 Serotype
4 Negative control
DNA-tagged liposomes in a sandwich hybridization
assay for Dengue virus mRNA. Serotype-specific
capture probes were immobilized in spatially
different zones
Analytical Bioanalytical Chemistry, vol. 380 (1),
p. 46 53 (2004)
Lab information
  • 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

Ethanol Biosensor Models and Signal Processing
Jae-Joon Han, Martin Plawecki, Peter Doerschuk,
and Sean OConnor
(No Transcript)
(No Transcript)