Finding Denue Virus: One Particle at a time

Yuexing (Zee) Zhang
Biosciences Division, ANL

Ultra-sensitive detection of infectious viral pathogens and/or disease biomarkers requires not only single molecule level sensitivities, but also effective strategies to screen bulk samples to “hunt down” target species at very low concentrations.  We address these challenges by the combination of fluorescence cross correlation spectroscopy (FCCS) and innovative microfluidic structures that enable bulk sample screening.

We focus on the detection of dengue viral pathogens in aqueous sample.  A monoclonal antibody was used to specifically recognize and bind to the dengue virus.  By labeling the dengue with red fluorescent dye and the anti-dengue antibody with green fluorescent dye, we were able to identify dengue/anti-dengue binding events by monitoring the FCCS cross correlation amplitude (Fig.1).

To achieve bulk sample screening, we constructed a home made “microchannel-linked microchamber array” structure using soft lithography methods involving SU-8 photoresist and PDMS.  Aqueous bulk sample was compartmentalized and confined inside microchambers, each in the volume of ~25 femtoliters, for sequential FCCS screening (Fig.2).  Dengue/anti-dengue binding was successfully detected inside individual microchambers (Fig.3).

Fig.1

(A) FCCS measurement for the Dengue/anti-Dengue mixture in bulk solution.  The blue trace is a representative curve (cross correlation amplitude vs. delay time) for the Dengue/anti-Dengue mixture.  In comparison, the black trace shows the FCCS profile for the Dengue/anti-M13 mixture.  The amplitude is close to zero.  (B) Photon counting traces of the Dengue/anti-Dengue mixture.  The green trace is the AF-488 dyed anti-Dengue (left-side tick labels), and the red trace is the AF-647 dyed Dengue viral particles (right-side tick labels).  The arrows indicate the overlapping spikes of the two traces.  (C) Autocorrelation profiles of the AF-647 dyed Dengue (red curve; tick labels at right) and AF-488 dyed anti-Dengue (green curve; tick labels at left).  Autocorrelation of a dilute AF-488 dyed anti-Dengue solution (unbound) is presented as the black dotted line.

Fig.2

Microfabricated SU-8 mold and PDMS microfluidic structure. (A) Two-layer SU-8 mold on a silicon wafer, defined by photolithography. Image was taken with an optical microscope (50× objective). Upper right: an overview of the entire 128×128 microchamber array, with microchannel patterns at both sides.  (B) A closer look of the same SU-8 mold under SEM (top view; 3600× magnification).  The bright round dots are 5 μm-tall cylinders used to define individual microchambers.  The darker stripes are 0.7 μm-thick lines to create the interconnecting microchannels.  The contrast reflects the difference in depth.  (C) A molded PDMS microstructure sealed onto a cover glass, immediately after oxygen plasma surface treatment.  Fluorescent dye (AF-488) solution was introduced into the fluidic structure and filled the entire microchamber array.  The fluorescence image was taken by an optical microscope with an epi-fluorescence setup (40× objective).  The microchambers are shown as bright dots.  The interconnecting channels are shallower and darker.  No leakage occurred.

Fig.3

(Main panel) FCCS curves taken inside 3 different microchambers (5 μm in diameter).  Chambers A (blue trace) and B (red trace) showed positive cross correlation amplitude, reflecting the presence of Dengue/anti-Dengue complex.  Chamber C (black) showed a cross correlation amplitude close to 0, suggesting there is few (if any) Dengue/anti-Dengue association detected.  The insert (magenta) showed a cross correlation curve taken from a smaller microchamber (2.5 μm in diameter).  Strong oscillations at delay time \tau~1-70 ms were present.  The oscillation was attributed to the interference of stray excitation lasers.

Reference

Y. Zhang, J. T. Bahns, Q. Jin, R. Divan, and L. Chen, Analytical Biochemistry, in press (2006).