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Article
Label-free Detection of Bacillus anthracis Spore Uptake in
Macrophage Cells Using Analytical Optical Force Measurements
Colin G Hebert, Sean Jeffrey Hart, Tomasz A. Leski, Alex Terray, and Qin Lu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01983 • Publication Date (Web): 06 Sep 2017
Downloaded from http://pubs.acs.org on September 11, 2017

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Page 1 of 29 Analytical Chemistry

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4 1 Label-free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells
5 2 Using Analytical Optical Force Measurements
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7 4 Colin G. Heberta‡ Sean Hartb, Tomasz A. Leskic, and Alex Terraya§ , Qin Lua*
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a
10 6 Naval Research Laboratory, Chemistry Division, Bio/Analytical Chemistry Section, Code 6112
11 7 4555 Overlook Ave. S.W. Washington, DC 20375 USA
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9 LumaCyte, LLC. 1145 River Road, Suite 16, Charlottesville, VA 22901 USA
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16 11 Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Code 6910
17 12 4555 Overlook Ave. S.W. Washington, DC 20375 USA
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23 17 *Corresponding Author: Qin Lu: qin.lu@nrl.navy.mil
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25 19 ‡Present address: LumaCyte, LLC. 1145 River Road, Suite 16, Charlottesville, VA 22901 USA
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20 Present address: MRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20878 USA
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1 Abstract
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6 3 Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant
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8 4 importance with respect to both anthrax disease progression, spore detection for biodefense, as well as
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10 5 understanding cell clearance in general. While most detection systems rely on specific molecules, such as
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12 6 nucleic acids or proteins, and fluorescent labels to identify the target(s) of interest, label-free methods
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14 7 probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with
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17 8 a particular biological event. Optical chromatography is a label free technique that uses the balance
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19 9 between optical and fluidic drag forces within a microfluidic channel to determine the optical force on
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21 10 cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage
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23 11 cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores,
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25 12 the exposure was detected in as little as one hour without the use of antibodies or fluorescent labels of any
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27 13 kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both
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30 14 with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical
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32 15 force arises independent of phagocytosis. These results demonstrate the capability of optical
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34 16 chromatography to detect subtle biological differences in a rapid and sensitive manner, and suggest future
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36 17 potential in a range of applications, including the detection of biological threat agents for biodefense, and
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38 18 pathogens for the prevention of sepsis and other diseases.
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50 23 Keywords: optical chromatography, laser, microfluidic, Bacillus anthracis, macrophage
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1 Introduction
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6 3 As one of the body’s first lines of defense against inhalational anthrax and other
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8 4 environmental insults, macrophages have the potential to act as a sensor indicating the onset of
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5 an infection or exposure event. For this reason, the interaction between macrophages and
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13 6 various pathogens, including Francisella tularensis,1 Salmonella enterica serovar
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15 7 Typhimurium,2 Staphylococcus aureus,3 and Bacillus anthracis spores,4 has been the subject of a
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18 8 number of studies. In the case of B. anthracis spores in particular, studies have predicted that
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20 9 macrophage-mediated transport plays an important role in the spread of vegetative cells into the
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22 10 bloodstream and throughout the rest of the body.5,6,7 However, most tools for the detection of
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25 11 spores or bacteria inside macrophages rely on fluorescent labeling of the bacteria through the use
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27 12 of an antibody or other labeled molecule. While powerful in their specificity, these labels are
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13 limited in several ways. In the case of fluorescent labeling of bacteria or spores, an appropriate
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32 14 target antibody or fluorescent molecule must exist that can selectively bind the target of interest.
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34 15 These labels can be expensive, labile (requiring special storage and transport), and limited to a
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37 16 particular bacteria or target protein. Even using antibodies which are exquisitely sensitive, non-
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39 17 specific interactions can still be problematic.8,9 Furthermore, if the target is changed in some
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41 18 way, the antibody or label may no longer be able to bind, diminishing if not eliminating detection
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44 19 capability. Finally, the labeling procedure is typically considered labor-intensive.
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46 20 In contrast, label-free detection and characterization methods seek to surmount these
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48 21 obstacles by utilizing the intrinsic differences between individual or populations of cells.
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51 22 Through the exposure of cells to one or more forces, including optical,10 magnetic,11
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53 23 mechanical,12 and electrical,13,14 these differences can be exploited to allow the separation or
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24 characterization of these cells. When considering the possibility of using macrophages as the
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1 basis for a sensitive detection system, either in vivo or in vitro, the ability to detect a wide range
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6 2 of infectious threats would be highly desirable. Label-free methods facilitate this approach as it
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8 3 may be possible to detect the response of a small but significant number of host cells, and/or a
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4 general shift in the cell population that reflects an exposure event, without relying on a molecule
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13 5 or condition specific to a certain pathogen. Once a population shift or change has been detected,
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15 6 cells showing a shift or change can be collected and subjected to more specific confirmatory
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18 7 analyses in order to facilitate diagnosis or treatment.
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20 8 The use of optical force for manipulating and interrogating cells and microparticles has
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22 9 become a well-established field of study with applications in the chemical, biological, and
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25 10 medical fields.10, 15,16,17,18,19,20 Optical chromatography is a technique which relies on the balance
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27 11 of optical and fluidic forces in a microchannel.21,22 In contrast to other optical force techniques
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12 such as optical tweezers or optical trapping that generally use a highly focused laser beam,
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32 13 optical chromatography employs a mildly focused beam. As particles encounter the beam, they
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34 14 experience optical pressure that results from photons imparting a fraction of their momentum as
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37 15 they either scatter at the surface or refract through a particle. The magnitude of this force
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39 16 depends on the size, refractive index, shape, morphology and composition of the particle,21,23
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41 17 with higher refractive index particles experiencing a greater force. In an optical chromatography
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44 18 system, force differentials will form the basis for characterization and separation. Thus, it is
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46 19 important to determine and understand the optical force exerted on cells and how these optical
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48 20 forces are influenced by cellular changes linked to specific events.
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51 21 In this study, we present data on the optical force changes associated with the engulfment
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53 22 of both microparticles and B. anthracis spores by macrophages, and the response of
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23 macrophages to B. anthracis spores after phagocytosis was inhibited by cytochalasin D, a cell-
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1 permeable and potent inhibitor of actin polymerization. In both cases, the optical force of
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6 2 individual macrophages increased when compared to unexposed macrophages.
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4 Materials and Methods
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13 5 Microchip construction
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15 6 The microfluidic flowcell consists of five plates of fused silica and is similar in
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18 7 construction to flowcells used in previous work.24 Fluid connectors (Idex Health Sciences, Oak
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20 8 Harbor, WA) were screwed into a custom plastic holder that aligns the fluidic connections with
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22 9 the ports on the microfluidic chip.
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25 10 Fluidic system
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27 11 The fluidic control system utilizes five pressurized control vessels filled with
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12 approximately 1mL of liquid, generally PBS buffer (pH 7.4, Sigma, St. Loius, MO, USA), which
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32 13 occupies about 2/3 of the vessel volume. By controlling the pressure above the liquid in each
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34 14 vessel, pulse-free, stable and reproducible fluid flow can be achieved.25 The multi-reservoir
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37 15 design isolates the flow system, increasing the stability and the flexibility to control the direction
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39 16 of flow. Flow direction and flow rate were precisely measured to a resolution of ±1.5 nl/min
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41 17 using a calibrated commercial liquid mass flow meter (Sensirion Inc., Westlake Village, CA,
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44 18 USA).
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46 19 Optics and imaging
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48 20 The laser used in this study is a continuous wave (CW) 1064 nm ytterbium fiber laser
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51 21 (IPG Photonics, Oxford, MA, USA) controlled by custom software written in Labview. The
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53 22 laser is focused into the microfluidic system by a 0.5 inch diameter plano-convex 100 mm focal
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23 length lens. Unless otherwise noted, the laser power in all situations was 3W. The microfluidic
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1 flowcell is mounted on a 5 axis positioner (New Focus, San Jose, CA, USA) and the entire
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6 2 aligned optical and fluidic system can be visualized using a 10x objective and lens tube system
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8 3 (Infinity Photo-Optical, Boulder, CO, USA) connected to a CCD camera (Basler, Inc. Exton, PA,
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4 UA) illuminated by a PL-800 fiber optic light (Edmund Optics, Barrinton, NJ, USA). The
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13 5 camera/objective and illuminator were mounted independently of the laser, each on separate x-y-
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15 6 z translational platforms from Thorlabs (Newton, NJ, USA). Data collection and analysis were
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18 7 performed using StreamPix Version 5 (Norpix, Inc., Quebec, Canada).
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20 8 Macrophage cell culture
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22 9 RAW264.7 cells (ATCC# TIB-71) were maintained per the manufacturer’s instructions
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25 10 in Dulbecco’s modified Eagle’s medium (ATCC, Manassas, VA, USA) supplemented with 10%
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27 11 fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Cells were grown at 37° C in a humidified
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32 13 B. anthracis spore preparation
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34 14 B. anthracis Sterne (nonpathogenic, vaccine strains) 34F2 was obtained from Colorado
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37 15 Serum Co. (Denver, CO), and 7702 containing pRP1028 plasmid that produces Red Fluorescent
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39 16 Protein (RFP) was provided by Dr. Molly Hughes (University of Virginia, Charlottesville, VA).
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41 17 Spores were prepared as previously described.26 The details are given in the Supporting
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44 18 Information.
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46 19 Particle and spore uptake by macrophage cells
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48 20 Macrophage cells were seeded onto 24 well plates such that a near-confluent (~95%)
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51 21 monolayer was present after 2 days of incubation. Just prior to the addition of microparticles or
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23 multiplicity of infection (MOI) for the experiment. Subsequently, the medium was aspirated
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1 from both control and experimental wells and replaced with fresh medium. Microparticles or
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6 2 spores (MOI of 10 or 100) were then added to the experimental wells at the appropriate
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8 3 concentration. After the designated incubation time (3 h for microparticles or 1 h for spores),
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4 cells were washed three times with PBS (pH 7.4) to remove any externally bound particles or
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13 5 spores. Cells were detached from the well plates using trypsin and resuspended in medium prior
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15 6 to analysis. During binding experiments, cells were incubated with fresh medium containing
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18 7 cytochalasin D (10 µM) for 1 hour prior to the addition of spores. Spores were then added to this
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20 8 medium directly prior to 1 hour incubation time. Microparticles used include 1.0 µm fluorescent
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23 9 polystyrene (PS) particles (Polysciences, Warrington, PA, USA) and 2.0 µm silica (Si) particles
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25 10 (Bangs Labs, Fishers, IN, USA). Particles per cell were determined using fluorescence (or in the
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28 11 case of Si, brightfield) microscopy and visually counting the number of particles within each
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30 12 cell. In the case of fluorescent spores, confocal microscopy was used to visualize the spores both
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35 14 Optical chromatography system calibration
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37 15 Each day before cell measurements were made, the optical chromatography system was
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39 16 calibrated using a NIST traceable 1.8 micron PS particle (Polysciences, Warrington, PA, USA).
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42 17 The laser alignment and the beam’s focal point inside the microchannel were calibrated by
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44 18 trapping a representative number (N > 10) of particles at the same location against a fixed fluid
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19 flow rate. This calibration ensured that a constant optical force was applied to the particle at a
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57 24 Results and Discussion
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1 Optical force effects from macrophage - microparticle interaction
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6 2 Because the size and refractive index of microparticles are well defined, they can serve as
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8 3 an initial analog for bacillus spores or bacteria, as well as preliminary samples to test the
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4 sensitivity of the system for detecting optical force changes that result from engulfed particles.
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13 5 Two types of particles were used, PS and Si. PS particles have a refractive index of ~1.57 at
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15 6 1064 nm,27 which is similar to that of B. anthracis (1.52828). Si particles have a lower refractive
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18 7 index (~1.45), which is closer to that of bacterial and eukaryotic cells. Flow rates for trapping
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20 8 unexposed RAW 264.7 macrophage cells were used as the reference. Following particle
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22 9 engulfment, the flow rates required to trap macrophage cells at the same location against a
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25 10 calibrated laser beam were measured.
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27 11 Cell sample (500,000 cells/ml, 200 µL) was loaded into a sample injection tube which
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12 was then connected to the optical chromatography system. As individual cells entered the flow
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32 13 cell, the laser shutter was opened, trapping cells within the beam against the fluidic flow inside
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34 14 the channel. Once cells were trapped within the beam, the flow rate was adjusted in order to
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37 15 balance the cells at a predetermined location (approximately 3/5 of the distance down the
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39 16 channel and 265 µm from the focal point of the laser), at which point the flow rate was recorded.
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17 When a cell is trapped, the net force acting on it is zero. Thus, the drag force and the optical
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44 18 forces acting in opposite directions along the axis of the beam are equal (FOptical = FDrag).
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46 19 Assuming laminar flow and a spherical cell, the drag force can be calculated using Stokes’ law:
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51 21 where η is the fluid’s dynamic viscosity (N s/m2), α is the radius of the cell (m), and v is the
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54 22 fluid flow velocity (m/s).29 Given that the fluid flow velocity is directly proportional to the drag
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1 force, it is also directly proportional to the optical force and can, therefore, be used to compare
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6 2 individual and populations of cells.
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8 3 A comparison between the trapping flow rate of unexposed macrophage cells and cells
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4 incubated with microparticles is shown in Figure 1A. Unexposed cells have an average trapping
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13 5 flow rate of 245 ± 26 nL/min. In comparison, cells incubated with 1.0 µm PS microparticles
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6 experience the highest optical force, with an average trapping flow rate of 431± 91 nL/min.
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18 7 Though less pronounced, cells incubated with 2.0 µm Si microparticles also had a significant (p
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20 8 < 0.01) increase in optical force when compared to cells alone, with average trapping flow rate of
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23 9 294 ± 34 nL/min. Cells exposed to 1.0 µm PS microparticles have the broadest histogram, with
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25 10 the trapping flow rates of these cells ranging from 305 nL/min to 610 nL/min (Figure 1B). In
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30 12 trapped at flow rate > 325 nL/min. As shown in Figure 2A, nearly all (97%) exposed cells have
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32 13 engulfed at least one particle, with the number of particles per cell ranging from 0 to 24. On
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35 14 average, the particles represent just ~ 0.6 percent of the volume of the cell (assuming an average
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37 15 cell size of 10 microns), but result in a 75 percent increase in optical force, demonstrating that
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39 16 the uptake of even a small number of particles of higher refractive index dramatically alter the
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42 17 optical force exerted on a macrophage cell. With the highest number of particles engulfed (24),
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44 18 the total volume of 24 particles only accounts for 2.4 percent of the cell volume, but the highest
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19 flow rate measured (610 nL/min) represents a nearly 150 percent increase in the optical force.
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49 20 Cells exposed to 2.0 µm Si microparticles show a similar distribution (Figure 2B), but a lower
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51 21 overall number of particle uptake per cell.
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56 23 a macrophage cell after its engulfment of the microparticles, the effective refractive index of
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1 exposed macrophage cells containing PS or Si microparticles can be estimated using Maxwell
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6 2 Garnett Approximation30. It incorporates the refractive index of the macrophage cell (assumed to
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8 3 be 1.38431) and of the particle (PS: 1.57, Si: 1.45), and the volume fraction calculated from the
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4 number of particles per cell. Table 1 gives the average trapping flow rate, particles per cell, force
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13 5 experienced by and the refractive indices of the unexposed cell population and those exposed to
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15 6 PS and Si microparticles. Particles per cell were determined using microscopy and the force was
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18 7 calculated using the Stokes’ law, assuming a spherical shape for the cells. There is minimal
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20 8 difference between unexposed and exposed cells in terms of average effective refractive indices
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22 9 using the average number of particles per cell, presumably due to the low volume fraction of
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25 10 engulfed microparticles, 0.6% for 1.0 µm PS and 1.8% for 2.0 µm Si. At the highest number of
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27 11 uptake, the volume fractions reached 2.4 % for 1.0 µm PS and 8.8% for 2.0 µm Si, but the
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30 12 increase in average effective refractive index in both cases is still negligible (~ 0.001).
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32 13 Since we cannot attribute the increase in optical force solely to a change in refractive
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14 index brought about by the physical presence of the particles, there must be other changes in the
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37 15 exposed cells due to their interactions with the microparticles that affect the optical force. With
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39 16 regards to the phagocytosis of PS and Si microparticles by macrophages, some suggest that the
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42 17 uptake of these particles is mediated by a macrophage receptor with collagenous structure
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44 18 (MARCO), one of the scavenge receptors on the surface of macrophages.32,33 Others hypothesize
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46 19 that the uptake of PS or Si microparticles is via a nonspecific mechanism involving surface
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49 20 charge interaction. Furthermore, it is believed that the phagocytosis of non-opsonized particles is
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51 21 likely the interplay of microtubules, microtubule associated proteins, and actin nucleation
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22 factors.34 Despite the complexity associated with different phagocytic mechanisms, a number of
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1 on the surface of the phagocyte with ligands on the surface of the particle. This leads to the
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6 2 polymerization of actin at the site of ingestion, and the internalization of the particle via an actin-
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4 phagocytosis of and biological response to the PS and Si microparticles.36, 37
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13 5 Optical force effects from macrophage - B. anthracis Sterne strain spore interaction
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15 6 Following the success achieved in sensitively measuring the change in optical force
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18 7 associated with the engulfment of microparticles by macrophage cells, experiments were directed
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20 8 towards the assessment of any change in optical force that results from the interaction of
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22 9 macrophage cells with more pertinent bioparticles, B. anthracis Sterne strain spores. Cell
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25 10 populations were analyzed in the same manner, except that the incubation time was decreased
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27 11 from three hours down to one hour. Results shown in Figure 3 compare the trapping flow rate of
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32 13 average trapping flow rate (Figure 3A) increased from 250 ± 26 nL/min for unexposed cells to
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34 14 278 ± 37 nL/min and 375 ± 107 nL/min for cells incubated with spores at an MOI of 10 and 100,
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37 15 respectively. A significant (p < 0.01, t-test) increase in the average trapping flow rate, thus the
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39 16 optical force, was measured in cells incubated with spores at both MOIs. The relatively large
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41 17 standard deviation of the MOI 100 population is a result of the high degree of heterogeneity in
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44 18 the population, likely due to a wide distribution of the number of spores taken up by each cell.
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46 19 This heterogeneity between cells can be seen when looking at the histogram data for each cell
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48 20 population (Figure 3B). When compared to unexposed cells, the MOI 10 population has a slight
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51 21 broadening of the peak in the direction of higher optical force, as reflected by a greater
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53 22 percentage of cells trapped at a flow rate > 300 nL/min. At MOI 100, the histogram reflects not
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1 between cells in the population. In fact, 50% of the cells were trapped at a flow rate > 350
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6 2 nL/min, stretching all the way up to 867 nL/min. This variability at MOI 100 is not unexpected,
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8 3 as previous studies using fluorescent labels have shown that the number of spore uptake at MOI
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4 100 can range from 2 to 22, with an average of 9.9 ± 4.6 spores per cell. In contrast, the number
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13 5 of spores per cell at MOI 10 ranges from 0 to 5, with an average of 0.9 ± 1.2 spores per cell.4
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15 6 Thus, the increase in the variability and magnitude of the optical force when moving from MOI
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18 7 10 to MOI 100 observed in this study correlates well with previously published reports.4
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20 8 However, it is important to note that in contrast to previous studies, no antibodies or fluorescent
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22 9 labels were required to measure the difference in optical forces and to distinguish the unexposed
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25 10 cells from the exposed cells in this study.
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27 11 The size of Bacillus anthracis spores has been measured using a transmission electron
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12 microscope26 to have a width of 0.71 µm and a length of 1.28 µm. The refractive index of the
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32 13 spore has been determined to be 1.528.28 Using the Maxwell Garnett Approximation30 and
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34 14 taking the highest number of uptake, 22, to calculate the volume fraction of spores inside the
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37 15 cell, the change in refractive index is, once again, negligible. Thus, as in the case of
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39 16 microparticle phagocytosis, the increase in optical force experienced by the macrophage cells
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17 exposed to spores cannot be attributed solely to the change in refractive index brought about by
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44 18 the physical presence of the spores.
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46 19 In addition to increased optical force, cells exposed to spores often displayed increased
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20 laser scatter. Figure 4 compares an unexposed cell trapped in the beam at 267 nL/min (Figure
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51 21 4A) to an exposed cell at MOI 100 trapped at 867 nL/min (Figure 4B). The latter image displays
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53 22 such a large degree of scattered light that it is difficult to even view the actual cell. Interestingly,
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56 23 our results contrast with side scatter measurements from a traditional flow cytometer which
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1 reported little to no difference between untreated cells and those exposed to B. anthracis Sterne
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6 2 spores at similar MOIs4. Each cell is also shown after it is released by closing the shutter to
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8 3 block the laser beam (Figures 4C and 4D, respectively). The unexposed cell can clearly be seen,
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4 while the exposed cell at MOI 100 is blurred as a result of a much higher trapping flow rate and,
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13 5 thus, the speed at which it exits the trap. It is important to note that there is little to no size
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15 6 difference between the unexposed and exposed cells, suggesting that the increase in optical force
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18 7 is not driven predominantly by the size change, but rather other factors such as cell membrane
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20 8 characteristics, or changes in the cytoskeleton as a result of spore engulfment.
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22 9 The binding and uptake of Bacillus anthracis spores by macrophage cells is a dynamic
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25 10 process and involves multiple receptors and signaling pathways. The recognition of exosporium
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27 11 glycoprotein of Bacillus collagen-like protein of anthracis (BclA) by the integrin Mac-1
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12 receptor, and the binding of rhamnose residues of BclA by the CD-14 co-receptor which induces
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32 13 an inside-out signaling pathways involving TLR2 and PI3K, ultimately leads to enhanced Mac-1
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34 14 dependent spore internalization.38 The changes in the cell membrane and the cytoskeleton due to
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37 15 the binding of spores by the multiple receptors, the activation of multiple signaling pathways, the
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39 16 polymerization of actin, and the physical presence of spores inside the macrophages, all of these
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41 17 combined, will induce significant changes in cell surface characteristics. Atomic Force
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44 18 Microscope investigation on fixed RAW264.7 macrophage cells39 stimulated by
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46 19 lipopolysaccharide (LPS) has revealed that the cell surface roughness was increased compared to
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48 20 unstimulated ones, presumably due to the aggregation of membrane proteins and the change of
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51 21 F-actin structure. Rough surfaces scatter more light than smooth surface, and macrophage cells
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53 22 exposed to spores displayed increased laser scatter (Figure 4 B). Even though no AFM images
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1 cells have rougher cell surface than unexposed ones, since the binding and uptake of spores also
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6 2 involves the interplay of multiple membrane proteins and the change of actin structure. The
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8 3 increased optical force on macrophage cells exposed to spores, therefore, is likely linked to the
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4 increase in cell surface roughness.
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13 5 Optical force effects from macrophage- B. anthracis spore interaction in absence of phagocytosis
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15 6 Because an optical force increase was observed following exposure of macrophage cells
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18 7 to B. anthracis spores, experiments were conducted to determine to what extent the increase was
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20 8 driven by changes to the macrophage cell itself, as opposed to the physical presence of the spores
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9 inside the cell. To this end, cells were incubated with 10 µM cytochalasin D, an inhibitor of
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25 10 actin-dependent uptake pathways such as phagocytosis, prior to incubation with B. anthracis
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27 11 spores. As an additional control, the optical force of cells exposed to only cytochalasin D was
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30 12 also measured. Results presented in Figure 5A show a slight but statistically significant (p <
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32 13 0.01) increase in the average trapping flow rate for cells exposed to cytochalasin D alone, from
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34 14 250 ± 19 nL/min to 277 ± 32 nL/min. It has been reported that cytochalasin D treatment alone
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37 15 can cause disruption and clustering of the actin cytoskeleton of macrophages.40 Therefore, both
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39 16 the optical and drag forces could be affected by changes to the cell membrane and the
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17 cytoskeleton, as mediated by cytochalasin D. When cells are exposed to spores at MOI 100 after
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44 18 incubation with cytochalasin D, there is a larger increase in the average trapping flow rate of
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46 19 cells, which rises to 337 ± 46 nL/min and includes a number of cells with a trapping flow rate
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20 greater than 400 nL/min. A shift, together with a broader distribution of flow rates, can also
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51 21 been seen when examining the population histograms presented in Figure 5B. It is interesting to
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53 22 note that this shift is less than the shift determined in the exposed cells in the absence of
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1 does contribute to an increase in optical force. However, it is perhaps more interesting to note
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6 2 that an increase in optical force can be measured independent of the presence of spores inside the
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8 3 cells, indicating that some other mechanism, perhaps the response of the macrophage itself, is
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4 responsible for the change in its optical properties.
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13 5 It is possible that spores attached to the outside of cells survived the wash step could have
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15 6 contributed to the increase in optical force. However, previous studies using fluorescently
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18 7 labeled spores have demonstrated that less than 20% of the spores associated with the cells are
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20 8 bound to the outside.4 This data suggests that if the binding of spores to the outside of cells
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22 9 results in an increase in the optical force, it should only play a minor role. To further investigate
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25 10 the number of spores associated with cells that have been treated with cytochalasin D, cells were
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27 11 exposed to spores expressing red fluorescent protein (RFP) in order to visualize the spores both
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12 inside and outside the cells. For both MOI 10 and 100, the number of spores inside and outside
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32 13 each cell was counted for a population of native macrophage cells as well as cells treated with
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34 14 cytochalasin D. Results are presented in Figure 6. In the case of MOI 100 without cytochalasin
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37 15 D, the number of spores associated with the cell was higher and could not be accurately counted,
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39 16 so that population is not shown on the graph. However, the addition of cytochalasin D to the
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41 17 spores before exposing them to a concentration of MOI 100 reduced the number of spores per
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44 18 cell dramatically to 3.3 ± 4.0 spores per cell. In the case of MOI 10, the number of spores per
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46 19 cell dropped from 10.8 ± 10.9 spores per cell to 0.8 ± 1.6 spores per cell upon the addition of
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48 20 cytochalasin D. Notably, the optical force of the cytochalasin D/MOI 100 (trapping flow rate =
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51 21 337 ± 46 nL/min, Figure 5A) population is greater than the MOI 10 native spores (trapping flow
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53 22 rate = 278 ± 37 nL/min, Figure 3A), despite the fact that the average number of spores associated
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1 It has been reported that even in the presence of cytochalsin D, the binding of particles on
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6 2 the surface of macrophages is not completely blocked.40 Therefore, the force increase in the
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8 3 presence of cytochalsin D is likely the outcome of disruption and clustering of the actin
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4 cytoskeleton caused by cytochalsin D. The additional force measured after exposure to spores
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13 5 could be the result of the changes in the cytoplasm due to the activation of multiple signaling
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15 6 pathways by the binding of spores by the receptors. As demonstrated by the minimal number of
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18 7 spores present on the outside of the cells, these spores were likely removed during the washing
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20 8 steps before injecting into the microfluidic flowcell. The detection of optical force changes in
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22 9 macrophage cells exposed to spores, but in the absence of phagocytosis, is an exciting result as it
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25 10 speaks to the potential use of macrophage cells for indirectly sensing exposure to B. anthracis
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27 11 spores. Label free detection of host response may be especially important, given the fact that
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12 spores can remain dormant in the lung for months after initial infection.41,42
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32 13
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34 14 Conclusion
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37 15 Biological detection and analysis systems that rely on specific molecules or labels can be
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39 16 expensive, time consuming to develop, and dependent upon prior knowledge of a specific target.
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41 17 A label-free technology for the detection of macrophage response to various environmental
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44 18 stimuli has been developed which is capable of measuring optical force differences between
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46 19 RAW 264.7 macrophages that have been exposed to B. anthracis spores and unexposed cells.
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48 20 After 1 hour exposure, the average trapping flow rate (and thus optical force) of exposed cells
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51 21 was increased by 11 and 50 percent at MOIs of 10 and 100, respectively, with a number of
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53 22 individual cells having significantly higher flow rates. Interestingly, the optical force
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1 anthracis spore exposure also increased, by a smaller but still significant amount. This result
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6 2 strongly suggests that the disruption and clustering of the actin cytoskeleton caused by
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8 3 cytochalasin D, and the changes in the cytoplasm due to the activation of multiple signaling
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4 pathways through the binding of spores by the receptors can result in a measurable increase in
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13 5 optical force that is separate from the presence of the spores inside the macrophage cell. Further
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15 6 studies will aim at integrating vibrational spectroscopy with the optical chromatography system
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18 7 to develop a label free detection technology with high specificity. It is envisioned that this
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20 8 technology will enable a detailed understanding of how cellular biochemical changes due to the
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22 9 innate immune response by host cells such as macrophages relate to the change in optical force,
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25 10 and allow timely pathogen detection.
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32 14 Acknowledgements
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34 15 A portion of this research was performed while the author (CGH) held a National Research
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37 16 Council Research Associateship Award at the U.S. Naval Research Laboratory. The authors
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39 17 would like to acknowledge funding from the Office of Naval Research and the U.S. Naval
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41 18 Research Laboratory, as well as the Defense Threat Reduction Agency (DTRA) under contract
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44 19 number BA09DET067. They would also like to thank Dr. Molly Hughes, Dr. Ian Glomski, and
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46 20 Ms. Debra Fisher for assistance in obtaining the fluorescent spores, and to thank Dr. Harold D.
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48 21 Ladouceur for helpful discussions.
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7 4 Figure 1. Trapping flow rate values measured after the exposure of various microparticles to
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9 5 RAW 264.7 cells. A). Graph showing the average trapping flow rate of cells alone (41 cells)
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12 6 versus the addition of 1.0 µm PS (25 cells) and 2.0 µm Si microparticles (28 cells). B).
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14 7 Population histograms showing the distribution of trapping flow rates for individual cells across
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17 8 the same sample populations. Error bars represent the standard deviation across each cell
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19 9 population. (* p < 0.01 when compared to the cells alone).
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21 10 Figure 2. Histograms showing the distribution of internalized microparticles in RAW 264.7 cells
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24 11 exposed to both 1.0 µm PS microparticles (A) and 2.0 µm Si microparticles (B).
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26 12 Figure 3. Trapping flow rate values measured after the exposure of B. anthracis spores to RAW
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29 13 264.7 cells. A). Graph showing the average trapping flow rate of cells alone (61 cells) versus the
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31 14 addition of spores at MOI 10 (29 cells) and MOI 100 (62 cells). B). Population histograms
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33 15 showing the distribution of trapping flow rates for individual cells across the same sample
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36 16 populations. Error bars represent the standard deviation across each cell population. (* p < 0.01
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38 17 when compared to the cells alone).
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18 Figure 4. Microscope images showing two representative RAW 264.7 cells in the optical
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43 19 chromatography system. An unexposed macrophage is shown both trapping at a flow rate of 267
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45 20 nL/min within the laser beam (A) and after it has been released from the beam (C). A cell
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47
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21 exposed to B. anthracis spores at MOI 100 is also shown trapped within the laser beam (B) at a
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50 22 flow rate of 862 nl/min and after release from the beam (D). The vertical bars are drawn on the
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52 23 image to indicate the measurement location. Trapping flow rate is directly proportional to optical
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6 2 264.7 cells treated with cytochalasin D. A). Graph showing the average trapping flow rate of
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8 3 cells alone (57 cells) versus cells treated with 10µM cytochalasin D both with (41 cells) and
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11 4 without (36 cells) the addition of spores at MOI 100. B). Population histograms showing the
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13 5 distribution of trapping flow rates for individual cells across the same sample populations. Error
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6 bars represent the standard deviation across each cell population. (* p < 0.01 when compared to
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18 7 the cells alone and cells + 10µM cytochalasin D).
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20 8
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23 9 Figure 6. Fluorescent spores associated with RAW 264.7 cells treated with cytochalasin D.
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25 10 Cells were exposed to spores producing red fluorescent protein and visualized using confocal
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27 11 microscopy. The number of spores inside and outside each cell was counted for a population of
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30 12 cells. The average and standard deviation are shown for each condition (Ncells = 142, 191, and
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32 13 136, respectively).
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14 Table 1. Average trapping flow rate, particles per cell, optical force, and refractive index for
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37 15 RAW264.7 cell populations alone and exposed to various microparticles. Particles per cell were
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39 16 determined using microscopy and optical force was calculated using the Stokes viscous
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42 17 expression, assuming a spherical shape for the cells.
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44 Condition Trapping Flow Rate (nL/min) Particles Per Cell Optical Force (pN) Refractive Index
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46 Cells (RAW264.7) 245 ± 26 N/A 12.5 ± 1.3 1.384
47 Cells + 1.0 µm PS 431 ± 94 5.8 ± 5.2 22.0 ± 4.8 1.384
48 Cells + 2.0 µm Si 294 ± 36 2.3 ± 2.4 15.0 ± 1.9 1.384
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48 µm PS (25 cells) and 2.0 µm Si microparticles (28 cells). B). Population histograms showing the distribution
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48 spores at MOI 10 (29 cells) and MOI 100 (62 cells). B). Population histograms showing the distribution of
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49 100 is also shown trapped within the laser beam (B) at a flow rate of 862 nl/min and after release from the
50 beam (D). The vertical bars are drawn on the image to indicate the measurement location. Trapping flow
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Figure 5. Trapping flow rate values measured after the exposure of B. anthracis spores to RAW 264.7 cells
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48 versus cells treated with 10 µM cytochalasin D both with (41 cells) and without (36 cells) the addition of
49 spores at MOI 100. B). Population histograms showing the distribution of trapping flow rates for individual
50 cells across the same sample populations. Error bars represent the standard deviation across each cell
51 population. (* p < 0.01 when compared to the cells alone and cells + 10 µM cytochalasin D).
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Figure 6. Fluorescent spores associated with RAW 264.7 cells treated with cytochalasin D. Cells were
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exposed to spores producing red fluorescent protein and visualized using confocal microscopy. The number
48 of spores inside and outside each cell was counted for a population of cells. The average and standard
49 deviation are shown for each condition (Ncells = 142, 191, and 136, respectively).
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