Mission Administration Events News Publications Seminar Series Training & Employment
Biomolecular Devices Cell-Surface Interactions Nanoscale Cell Biology
Academia Industry Government Sciencenter
Nanobiotechnology Course K-12 Undergraduates Graduates
Cornell University Princeton University Wadsworth Center Oregon Health Sciences U. Clark Atlanta University Howard University
NBTC
nbtc logo  Program  †  Research Areas     Partners     Education     Members     Facilities 
Project Title
Biosensor Based on Electrochemical/Gravimetric Detection of Intrinsic Antibody Catalysis
 

Research Program
Biomolecular Devices and Analysis

 
Project #
BDA14
 
Participating Faculty: B. Baird, H. Abruña, C. Ober, J. Appleton, T. Clark
NBTC Students/Postdocs: N. Smith, S. Nad, A. Rastogi, L. Blum, Y. Bisharyan
Other Students/Postdocs:  N. DaMota, M. Tanaka
 

Objectives
Novel devices are being developed for electrochemical/gravimetric detection of antibodies, in particular, antibodies specific for the avian influenza virus (H5N1) hemagglutinin protein (H5). This approach takes advantage of the intrinsic antibody-catalyzed water oxidation pathway (ACWOP) that was previously characterized. This activity will be detected on a specifically modified electrode or within a quartz crystal microbalance (QCM). These detection methods, within microscale devices, are expected to be more specific and more sensitive than current enzyme-linked-immuno-sorbant-assays (ELISAs).

Methods
Intrinsic ACWOP activity of antibodies is confirmed by measuring direct production of hydrogen peroxide (H2O2) using a colorimetric (Amplex Red) assay that is also used for optimization. Measurements of antibodies bound to polymer brushes can be compared to electrochemical/gravimetric measurements of H2O2 produced by the same substrates in the development of the proposed device.

Assembly of general platform, electrochemical/gravimetric devices includes determination of the surface coverage of the various components in the assembly: polymer brushes, hapten and antibody, using a combination of electrochemical (cyclic voltammetry), gravimetric (QCM), fluorescence and other techniques. Detection of ACWOP is based on H2O2 reduction to water, using amperometry or gravimetry as the analytical signal.

Polymer Brushes consisting of poly(acrylic acid) are modified with haptenic groups and used to bind specific antibodies to electrode and QCM surfaces. Atom transfer radical polymerization (ATRP) is used to grow brushes on either gold or silicon oxide surfaces. An ATRP initiator containing oligo(ethylene glycol) prevents non-specific adsorption of bio-molecules on the sensor surface.

Monoclonal antibodies specific for H5 are prepared in milligram amounts from cloned, secreting cell lines. Binding activity of purified antibodies is confirmed in hemagglutination inhibition and ELISA assays using killed H5N1 X PR8 viruses.

Preparation of H5 proteins from avian influenza virus is based on the full-length H5 gene, expressed in Tetrahymena. Other cell lines expressing several alternative versions of the recombinant H5 protein are being constructed to streamline protein purification and provide alternatives for conjugation to polymer brushes.

Summary
Antibodies produced by the immune system are often the first signs of infectious diseases. Because antibodies recognize a vast repertoire of antigenic groups with exquisite specificity they are also valuable reagents for medical therapeutics, vaccine development, and research. The important need for detection of antibodies in diverse media is currently limited by sensitivity and specificity. We are constructing an electrochemical/gravimetric device to reach new detection limits, based on intrinsic catalytic activities of antibodies. Polymer brushes, modified with selected antigenic groups and other agents, provide the interface on the surface of the detection device, such that antibodies bind specifically and the ACWOP activity can be measured sensitively. The device is initially being developed and optimized with a model antibody system that can then be generalized to antibodies of high medical importance. We are currently adapting the device for detection of antibodies specific for avian influenza viruses (H5N1) that pose pandemic threat. The technological developments will be readily adaptable to detection of other antibodies and also to other biosensors on the micro- and nanoscales.

Accomplishments

  • Measured ACWOP for anti-H5 and other antibodies at levels of >20 mole equiv/mole Ab
  • Quantified polymer brushes for specific binding of anti-DNP
  • Refined design and measurement for electrochemical detection of H202
  • Enhanced production of recombinant H5 proteins and specific anti-H5 monoclonal antibodies

Fig.1: ACWOP: Polyclonal sheep, H5 antibodies or Cholera Toxin B subunit(10 uM) in PBS (pH 7.4) were irradiated with UV light in the presence of 100 uM tris (2,2'-bipyridyl) ruthenium (II) as a photosensitizer. Hydrogen peroxide generation was quantified using Amplex Red.

 

Fig.2: Schematic depiction of the production and indirect detection of H2O2. Horseradish peroxidase (HRP) and Amplex Red are used as enzyme and electrochemical mediator, respectively. Peroxy radicals formed by the activity of HRP on the peroxide irreversibly oxidize Amplex Red to resorufin which is subsequently quantified electrochemically by its reduction to dihydroresorufin [Adapted from Lyon et al Anal Chem 2006]

Fig.3: Quantification of anti-DNP IgG antibody binding specifically to DNP-functionalized PAA brush on a QCM electrode.

Dm = -Df.Cf = 354 ng/cm2

[Total mass change = +112.2 ng]

Surface conc. on Ab = 4.32 x 10-13 mol/cm2

Conc. of DNP on PAA = 2.4 x 10-11 mol/cm2

 

This material is based upon work supported in part by the STC Program of the National Science Foundation under Agreement No. ECS-9876771. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

 Home    Program    Research Areas    Partners    Education    Members    Facilities