An Embedded Microretroreflector-based Microfluidic Immunodiagnostic Platform for Sensitive

Pathogen Detection


Balakrishnan Raja
1
, Carmen Pascente
1
, David Shakarisaz
1
, Jennifer Knoop
1
,

Katerina Kourentzi
1
, Steven
Kemper
1
, Paul Ruchhoeft
1
, Juan Olano
2
, Anson Hatch
3
and Richard Willson
1

1. University of Houston, Houston, Texas 2. University of Texas-Medical Branch, Galveston,
Texas 3. Sandia National Laboratory, Livermore, California


Sensitive and rapid pathogen detection, a critical tool in managing infectious diseases, typically relies on
nucleic acid extraction followed by amplification, or labelling with dyes, enzymes or fluors. Signal
detection in these techniques often requires expensive and elaborate instrumentation. This work
introduces embedded, microfabricated linear retroreflectors as bio-sensing surfaces, using micron-sized
magnetic particles as light-blocking labels in a semi-homogeneous format, resulting in a highly sensitive,
automated diagnostic immunoassay.

Several thousand micron-scale linear retroreflectors (dimensions 100 m long 3 m wide 5 m tall)
are fabricated, with precise positioning, on a microfluidic chip using commercially-scalable, inexpensive
photolithographic techniques and then embedded in a transparent, biofunctionalizable poly (methyl
methacrylate) layer. These chips, containing seven parallel microfluidic channels, are assembled on a
LabView-controlled, automated stage, and imaged in real-time using an inexpensive, in-lens illuminated
CMOS camera. Rickettsia conorii, the causative agent of Mediterranean Spotted Fever and an NIAID
Category C priority pathogen, is used as a model pathogen to test the microfluidic assay. Magnetic
microparticles, bearing immuno-captured Rickettsia, are delivered into the microfluidic channels in a
manner that will help them see and bind to Fc-immobilized, polyclonal anti-Rickettsia antibodies on the
polymer surface. These immuno-sandwiches block light and appear as dark spots on the machine-
registered pattern of the originally bright retroreflectors. Following fluidic force discrimination to remove
non-specifically bound particles, an in-house automated difference imaging algorithm then quantifies the
number of captured labels that serves as the readout.




The unique combination of large biosensing areas, optimized surface chemistry and microfluidic
protocols, automated image capture and analysis, and high sensitivity of the difference imaging results in
a rapid, sensitive and automated assay for pathogen detection, with a current limit of detection of the order
of 10
3
R. conorii per mL.


Detection principle of embedded
retroreflectors
SEM image of an array of ten
micro- retroreflectors
CMOS camera images showing darkening by magnetic particles in the
presence of analyte
Absence of Pathogen Presence of Pathogen