(698d) A Viscosity-Based Measurement System for Pathogen Detection | AIChE

(698d) A Viscosity-Based Measurement System for Pathogen Detection


Clayton, K. N. - Presenter, Purdue University
Moehling, T., Purdue University
Berglund, G. D., Purdue University
Lee, D. H., Purdue University
Witten, A. J., Purdue University
Wereley, S. T., Purdue University
Linnes, J. C., Purdue University
Kinzer-Ursem, T. L., Purdue University

There is a great need to develop
rapid, sensitive, and accurate infectious disease diagnostics. Current gold
standard pathogen detection often involves fluorescence-based DNA amplification
readings or antibody-based assays to confirm disease presence in either patient
or environmental samples. Here we introduce an alternative method, particle
diffusometry, where the presence of amplified DNA is detected by measuring
change in solution viscosity. The presence of V. cholerae, S. aureus,
and K. pneumoniae with specialized oligonucleotide primers allows for
amplification using loop-mediated isothermal amplification (LAMP). This
amplification increases the length of polynucleotides in the solution, which in
turn makes the fluid sample more viscous. We suspend 200 nm particles in a
fluid sample and image their Brownian motion with fluorescence microscopy. In
the presence of amplified pathogen DNA, these particles exhibit slower Brownian
motion, indicating a change in the particle diffusion coefficient, and in turn the
solution viscosity. Particle diffusometry calculates the diffusion
coefficient of the particles by correlating successive particle images (at
time Dt) to one another
(cross-correlation, sc) and the particle image
with itself (autocorrelation, sa) at a magnification (M):

From the diffusion coefficient, the viscosity (η) of
the solution is calculated by the Stokes-Einstein equation, where k is
the Boltzmann’s constant, T is absolute temperature, and a is the
hydrodynamic radius of the 200 nm particle.

particle diffusometry we successfully determine the presence of amplified V. cholerae
DNA in blinded studies involving (A) V. cholerae DNA with oligos that
underwent LAMP, and the following controls: (B) no V. cholerae DNA but
with oligos that underwent LAMP, (C) V. cholerae DNA with oligos that
did not undergo LAMP, and (D) no V. cholerae DNA but with oligos that
did not undergo LAMP. Particle diffusometry measurements are also comparable to
qPCR (fluorescence signal). We compare both techniques using a concentration
range of pure DNA that is equivalent to what is found in samples containing 1-10,000
cells/mL. Further, we determine the change in viscosity of DNA amplification in
the presence of whole cells for V. cholerae, K. pneumoniae, and S.
. Finally, we can measure the presence of DNA amplified V.
cells in environmental pond water sources, indicating the
feasibility of particle diffusometry for the point of care. From these studies,
we find that particle diffusometry has a lower limit of detection of 100
cells/mL, a relevant concentration for the detection of V. cholerae in
water sources. We will also discuss our current work on the implementation
of particle diffusometry onto a smartphone-based platform as a quantitative
point of care pathogen detection device.