Label-Free Detection of Bacillus anthracis Spore Uptake in

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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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Analytical Chemistry

Label-free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements Colin G. Heberta‡ Sean Hartb, Tomasz A. Leskic, and Alex Terraya§ , Qin Lua* a

Naval Research Laboratory, Chemistry Division, Bio/Analytical Chemistry Section, Code 6112 4555 Overlook Ave. S.W. Washington, DC 20375 USA b

c

LumaCyte, LLC. 1145 River Road, Suite 16, Charlottesville, VA 22901 USA

Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Code 6910 4555 Overlook Ave. S.W. Washington, DC 20375 USA

*Corresponding Author: Qin Lu: [email protected] ‡Present address: LumaCyte, LLC. 1145 River Road, Suite 16, Charlottesville, VA 22901 USA §

Present address: MRIGlobal, 65 West Watkins Mill Road, Gaithersburg, MD 20878 USA

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Abstract

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importance with respect to both anthrax disease progression, spore detection for biodefense, as well as

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understanding cell clearance in general. While most detection systems rely on specific molecules, such as

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nucleic acids or proteins, and fluorescent labels to identify the target(s) of interest, label-free methods

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probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with

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a particular biological event. Optical chromatography is a label free technique that uses the balance

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between optical and fluidic drag forces within a microfluidic channel to determine the optical force on

Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant

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cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage

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cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores,

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the exposure was detected in as little as one hour without the use of antibodies or fluorescent labels of any

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kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both

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with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical

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force arises independent of phagocytosis. These results demonstrate the capability of optical

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chromatography to detect subtle biological differences in a rapid and sensitive manner, and suggest future

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potential in a range of applications, including the detection of biological threat agents for biodefense, and

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pathogens for the prevention of sepsis and other diseases.

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Keywords: optical chromatography, laser, microfluidic, Bacillus anthracis, macrophage

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Introduction

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environmental insults, macrophages have the potential to act as a sensor indicating the onset of

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an infection or exposure event. For this reason, the interaction between macrophages and

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various pathogens, including Francisella tularensis,1 Salmonella enterica serovar

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Typhimurium,2 Staphylococcus aureus,3 and Bacillus anthracis spores,4 has been the subject of a

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number of studies. In the case of B. anthracis spores in particular, studies have predicted that

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macrophage-mediated transport plays an important role in the spread of vegetative cells into the

As one of the body’s first lines of defense against inhalational anthrax and other

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bloodstream and throughout the rest of the body.5,6,7 However, most tools for the detection of

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spores or bacteria inside macrophages rely on fluorescent labeling of the bacteria through the use

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of an antibody or other labeled molecule. While powerful in their specificity, these labels are

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limited in several ways. In the case of fluorescent labeling of bacteria or spores, an appropriate

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target antibody or fluorescent molecule must exist that can selectively bind the target of interest.

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These labels can be expensive, labile (requiring special storage and transport), and limited to a

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particular bacteria or target protein. Even using antibodies which are exquisitely sensitive, non-

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specific interactions can still be problematic.8,9 Furthermore, if the target is changed in some

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way, the antibody or label may no longer be able to bind, diminishing if not eliminating detection

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capability. Finally, the labeling procedure is typically considered labor-intensive.

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In contrast, label-free detection and characterization methods seek to surmount these

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obstacles by utilizing the intrinsic differences between individual or populations of cells.

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Through the exposure of cells to one or more forces, including optical,10 magnetic,11

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mechanical,12 and electrical,13,14 these differences can be exploited to allow the separation or

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characterization of these cells. When considering the possibility of using macrophages as the

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basis for a sensitive detection system, either in vivo or in vitro, the ability to detect a wide range

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of infectious threats would be highly desirable. Label-free methods facilitate this approach as it

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may be possible to detect the response of a small but significant number of host cells, and/or a

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general shift in the cell population that reflects an exposure event, without relying on a molecule

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or condition specific to a certain pathogen. Once a population shift or change has been detected,

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cells showing a shift or change can be collected and subjected to more specific confirmatory

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analyses in order to facilitate diagnosis or treatment.

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The use of optical force for manipulating and interrogating cells and microparticles has become a well-established field of study with applications in the chemical, biological, and

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medical fields.10, 15,16,17,18,19,20 Optical chromatography is a technique which relies on the balance

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of optical and fluidic forces in a microchannel.21,22 In contrast to other optical force techniques

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such as optical tweezers or optical trapping that generally use a highly focused laser beam,

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optical chromatography employs a mildly focused beam. As particles encounter the beam, they

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experience optical pressure that results from photons imparting a fraction of their momentum as

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they either scatter at the surface or refract through a particle. The magnitude of this force

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depends on the size, refractive index, shape, morphology and composition of the particle,21,23

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with higher refractive index particles experiencing a greater force. In an optical chromatography

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system, force differentials will form the basis for characterization and separation. Thus, it is

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important to determine and understand the optical force exerted on cells and how these optical

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forces are influenced by cellular changes linked to specific events.

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In this study, we present data on the optical force changes associated with the engulfment

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of both microparticles and B. anthracis spores by macrophages, and the response of

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macrophages to B. anthracis spores after phagocytosis was inhibited by cytochalasin D, a cell-


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permeable and potent inhibitor of actin polymerization. In both cases, the optical force of

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individual macrophages increased when compared to unexposed macrophages.

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Materials and Methods

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Microchip construction The microfluidic flowcell consists of five plates of fused silica and is similar in

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construction to flowcells used in previous work.24 Fluid connectors (Idex Health Sciences, Oak

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Harbor, WA) were screwed into a custom plastic holder that aligns the fluidic connections with

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the ports on the microfluidic chip.

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Fluidic system The fluidic control system utilizes five pressurized control vessels filled with

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approximately 1mL of liquid, generally PBS buffer (pH 7.4, Sigma, St. Loius, MO, USA), which

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occupies about 2/3 of the vessel volume. By controlling the pressure above the liquid in each

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vessel, pulse-free, stable and reproducible fluid flow can be achieved.25 The multi-reservoir

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design isolates the flow system, increasing the stability and the flexibility to control the direction

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of flow. Flow direction and flow rate were precisely measured to a resolution of ±1.5 nl/min

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using a calibrated commercial liquid mass flow meter (Sensirion Inc., Westlake Village, CA,

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USA).

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Optics and imaging

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The laser used in this study is a continuous wave (CW) 1064 nm ytterbium fiber laser

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(IPG Photonics, Oxford, MA, USA) controlled by custom software written in Labview. The

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laser is focused into the microfluidic system by a 0.5 inch diameter plano-convex 100 mm focal

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length lens. Unless otherwise noted, the laser power in all situations was 3W. The microfluidic



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flowcell is mounted on a 5 axis positioner (New Focus, San Jose, CA, USA) and the entire

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aligned optical and fluidic system can be visualized using a 10x objective and lens tube system

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(Infinity Photo-Optical, Boulder, CO, USA) connected to a CCD camera (Basler, Inc. Exton, PA,

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UA) illuminated by a PL-800 fiber optic light (Edmund Optics, Barrinton, NJ, USA). The

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camera/objective and illuminator were mounted independently of the laser, each on separate x-y-

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z translational platforms from Thorlabs (Newton, NJ, USA). Data collection and analysis were

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performed using StreamPix Version 5 (Norpix, Inc., Quebec, Canada).

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Macrophage cell culture

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RAW264.7 cells (ATCC# TIB-71) were maintained per the manufacturer’s instructions

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in Dulbecco’s modified Eagle’s medium (ATCC, Manassas, VA, USA) supplemented with 10%

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fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Cells were grown at 37° C in a humidified

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incubator supplemented with 5% CO2.

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B. anthracis spore preparation

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B. anthracis Sterne (nonpathogenic, vaccine strains) 34F2 was obtained from Colorado

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Serum Co. (Denver, CO), and 7702 containing pRP1028 plasmid that produces Red Fluorescent

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Protein (RFP) was provided by Dr. Molly Hughes (University of Virginia, Charlottesville, VA).

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Spores were prepared as previously described.26 The details are given in the Supporting

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Information.

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Particle and spore uptake by macrophage cells

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Macrophage cells were seeded onto 24 well plates such that a near-confluent (~95%)

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monolayer was present after 2 days of incubation. Just prior to the addition of microparticles or

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spores, several wells were harvested and counted in order to accurately determine the

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multiplicity of infection (MOI) for the experiment. Subsequently, the medium was aspirated


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from both control and experimental wells and replaced with fresh medium. Microparticles or

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spores (MOI of 10 or 100) were then added to the experimental wells at the appropriate

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concentration. After the designated incubation time (3 h for microparticles or 1 h for spores),

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cells were washed three times with PBS (pH 7.4) to remove any externally bound particles or

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spores. Cells were detached from the well plates using trypsin and resuspended in medium prior

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to analysis. During binding experiments, cells were incubated with fresh medium containing

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cytochalasin D (10 µM) for 1 hour prior to the addition of spores. Spores were then added to this

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medium directly prior to 1 hour incubation time. Microparticles used include 1.0 µm fluorescent

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polystyrene (PS) particles (Polysciences, Warrington, PA, USA) and 2.0 µm silica (Si) particles

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(Bangs Labs, Fishers, IN, USA). Particles per cell were determined using fluorescence (or in the

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case of Si, brightfield) microscopy and visually counting the number of particles within each

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cell. In the case of fluorescent spores, confocal microscopy was used to visualize the spores both

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inside and outside the cells.

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Optical chromatography system calibration

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Each day before cell measurements were made, the optical chromatography system was

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calibrated using a NIST traceable 1.8 micron PS particle (Polysciences, Warrington, PA, USA).

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The laser alignment and the beam’s focal point inside the microchannel were calibrated by

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trapping a representative number (N > 10) of particles at the same location against a fixed fluid

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flow rate. This calibration ensured that a constant optical force was applied to the particle at a

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predetermined location, countering a predetermined hydrodynamic dragging force and helping

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minimize any drift in the trapping flow rate that might occur as a result of changes in the

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environmental conditions within the lab.

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Results and Discussion 7 ACS Paragon Plus Environment


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Optical force effects from macrophage - microparticle interaction Because the size and refractive index of microparticles are well defined, they can serve as

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an initial analog for bacillus spores or bacteria, as well as preliminary samples to test the

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sensitivity of the system for detecting optical force changes that result from engulfed particles.

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Two types of particles were used, PS and Si. PS particles have a refractive index of ~1.57 at

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1064 nm,27 which is similar to that of B. anthracis (1.52828). Si particles have a lower refractive

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index (~1.45), which is closer to that of bacterial and eukaryotic cells. Flow rates for trapping

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unexposed RAW 264.7 macrophage cells were used as the reference. Following particle

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engulfment, the flow rates required to trap macrophage cells at the same location against a

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calibrated laser beam were measured. Cell sample (500,000 cells/ml, 200 µL) was loaded into a sample injection tube which

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was then connected to the optical chromatography system. As individual cells entered the flow

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cell, the laser shutter was opened, trapping cells within the beam against the fluidic flow inside

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the channel. Once cells were trapped within the beam, the flow rate was adjusted in order to

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balance the cells at a predetermined location (approximately 3/5 of the distance down the

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channel and 265 µm from the focal point of the laser), at which point the flow rate was recorded.

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When a cell is trapped, the net force acting on it is zero. Thus, the drag force and the optical

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forces acting in opposite directions along the axis of the beam are equal (FOptical = FDrag).

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Assuming laminar flow and a spherical cell, the drag force can be calculated using Stokes’ law:

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FDrag = 6πηαν

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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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fluid flow velocity (m/s).29 Given that the fluid flow velocity is directly proportional to the drag


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force, it is also directly proportional to the optical force and can, therefore, be used to compare

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individual and populations of cells.

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A comparison between the trapping flow rate of unexposed macrophage cells and cells

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incubated with microparticles is shown in Figure 1A. Unexposed cells have an average trapping

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flow rate of 245 ± 26 nL/min. In comparison, cells incubated with 1.0 µm PS microparticles

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experience the highest optical force, with an average trapping flow rate of 431± 91 nL/min.

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Though less pronounced, cells incubated with 2.0 µm Si microparticles also had a significant (p

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< 0.01) increase in optical force when compared to cells alone, with average trapping flow rate of

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294 ± 34 nL/min. Cells exposed to 1.0 µm PS microparticles have the broadest histogram, with

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the trapping flow rates of these cells ranging from 305 nL/min to 610 nL/min (Figure 1B). In

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contrast, unexposed cells have a narrow distribution around the population average, with no cells

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trapped at flow rate > 325 nL/min. As shown in Figure 2A, nearly all (97%) exposed cells have

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engulfed at least one particle, with the number of particles per cell ranging from 0 to 24. On

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average, the particles represent just ~ 0.6 percent of the volume of the cell (assuming an average

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cell size of 10 microns), but result in a 75 percent increase in optical force, demonstrating that

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the uptake of even a small number of particles of higher refractive index dramatically alter the

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optical force exerted on a macrophage cell. With the highest number of particles engulfed (24),

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the total volume of 24 particles only accounts for 2.4 percent of the cell volume, but the highest

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flow rate measured (610 nL/min) represents a nearly 150 percent increase in the optical force.

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Cells exposed to 2.0 µm Si microparticles show a similar distribution (Figure 2B), but a lower

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overall number of particle uptake per cell.

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To assess the impact from the change in bulk refractive index on optical force exerted on a macrophage cell after its engulfment of the microparticles, the effective refractive index of



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exposed macrophage cells containing PS or Si microparticles can be estimated using Maxwell

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Garnett Approximation30. It incorporates the refractive index of the macrophage cell (assumed to

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be 1.38431) and of the particle (PS: 1.57, Si: 1.45), and the volume fraction calculated from the

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number of particles per cell. Table 1 gives the average trapping flow rate, particles per cell, force

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experienced by and the refractive indices of the unexposed cell population and those exposed to

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PS and Si microparticles. Particles per cell were determined using microscopy and the force was

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calculated using the Stokes’ law, assuming a spherical shape for the cells. There is minimal

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difference between unexposed and exposed cells in terms of average effective refractive indices

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using the average number of particles per cell, presumably due to the low volume fraction of

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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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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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increase in average effective refractive index in both cases is still negligible (~ 0.001).

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Since we cannot attribute the increase in optical force solely to a change in refractive

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index brought about by the physical presence of the particles, there must be other changes in the

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exposed cells due to their interactions with the microparticles that affect the optical force. With

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regards to the phagocytosis of PS and Si microparticles by macrophages, some suggest that the

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uptake of these particles is mediated by a macrophage receptor with collagenous structure

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(MARCO), one of the scavenge receptors on the surface of macrophages.32,33 Others hypothesize

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that the uptake of PS or Si microparticles is via a nonspecific mechanism involving surface

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charge interaction. Furthermore, it is believed that the phagocytosis of non-opsonized particles is

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likely the interplay of microtubules, microtubule associated proteins, and actin nucleation

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factors.34 Despite the complexity associated with different phagocytic mechanisms, a number of

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shared features follow: particle internalization is initiated by the interaction of specific receptors


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on the surface of the phagocyte with ligands on the surface of the particle. This leads to the

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polymerization of actin at the site of ingestion, and the internalization of the particle via an actin-

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based mechanism.35 Evidence exists that the actin polymerization is essential for the

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phagocytosis of and biological response to the PS and Si microparticles.36, 37

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Optical force effects from macrophage - B. anthracis Sterne strain spore interaction

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Following the success achieved in sensitively measuring the change in optical force

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associated with the engulfment of microparticles by macrophage cells, experiments were directed

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towards the assessment of any change in optical force that results from the interaction of

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macrophage cells with more pertinent bioparticles, B. anthracis Sterne strain spores. Cell

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populations were analyzed in the same manner, except that the incubation time was decreased

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from three hours down to one hour. Results shown in Figure 3 compare the trapping flow rate of

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unexposed cells with cells incubated with B. anthracis spores at an MOI of 10 or 100. The

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average trapping flow rate (Figure 3A) increased from 250 ± 26 nL/min for unexposed cells to

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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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respectively. A significant (p < 0.01, t-test) increase in the average trapping flow rate, thus the

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optical force, was measured in cells incubated with spores at both MOIs. The relatively large

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standard deviation of the MOI 100 population is a result of the high degree of heterogeneity in

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the population, likely due to a wide distribution of the number of spores taken up by each cell.

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This heterogeneity between cells can be seen when looking at the histogram data for each cell

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population (Figure 3B). When compared to unexposed cells, the MOI 10 population has a slight

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broadening of the peak in the direction of higher optical force, as reflected by a greater

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percentage of cells trapped at a flow rate > 300 nL/min. At MOI 100, the histogram reflects not

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only a further increase in trapping flow rate (and thus optical force), but also a wide variability



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between cells in the population. In fact, 50% of the cells were trapped at a flow rate > 350

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nL/min, stretching all the way up to 867 nL/min. This variability at MOI 100 is not unexpected,

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as previous studies using fluorescent labels have shown that the number of spore uptake at MOI

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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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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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Thus, the increase in the variability and magnitude of the optical force when moving from MOI

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10 to MOI 100 observed in this study correlates well with previously published reports.4

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However, it is important to note that in contrast to previous studies, no antibodies or fluorescent

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labels were required to measure the difference in optical forces and to distinguish the unexposed

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cells from the exposed cells in this study.

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The size of Bacillus anthracis spores has been measured using a transmission electron

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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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spore has been determined to be 1.528.28 Using the Maxwell Garnett Approximation30 and

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taking the highest number of uptake, 22, to calculate the volume fraction of spores inside the

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cell, the change in refractive index is, once again, negligible. Thus, as in the case of

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microparticle phagocytosis, the increase in optical force experienced by the macrophage cells

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exposed to spores cannot be attributed solely to the change in refractive index brought about by

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the physical presence of the spores.

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In addition to increased optical force, cells exposed to spores often displayed increased

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laser scatter. Figure 4 compares an unexposed cell trapped in the beam at 267 nL/min (Figure

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4A) to an exposed cell at MOI 100 trapped at 867 nL/min (Figure 4B). The latter image displays

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such a large degree of scattered light that it is difficult to even view the actual cell. Interestingly,

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our results contrast with side scatter measurements from a traditional flow cytometer which


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reported little to no difference between untreated cells and those exposed to B. anthracis Sterne

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spores at similar MOIs4.

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block the laser beam (Figures 4C and 4D, respectively). The unexposed cell can clearly be seen,

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while the exposed cell at MOI 100 is blurred as a result of a much higher trapping flow rate and,

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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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difference between the unexposed and exposed cells, suggesting that the increase in optical force

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is not driven predominantly by the size change, but rather other factors such as cell membrane

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characteristics, or changes in the cytoskeleton as a result of spore engulfment.

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Each cell is also shown after it is released by closing the shutter to

The binding and uptake of Bacillus anthracis spores by macrophage cells is a dynamic

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process and involves multiple receptors and signaling pathways. The recognition of exosporium

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glycoprotein of Bacillus collagen-like protein of anthracis (BclA) by the integrin Mac-1

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receptor, and the binding of rhamnose residues of BclA by the CD-14 co-receptor which induces

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an inside-out signaling pathways involving TLR2 and PI3K, ultimately leads to enhanced Mac-1

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dependent spore internalization.38 The changes in the cell membrane and the cytoskeleton due to

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the binding of spores by the multiple receptors, the activation of multiple signaling pathways, the

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polymerization of actin, and the physical presence of spores inside the macrophages, all of these

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combined, will induce significant changes in cell surface characteristics. Atomic Force

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Microscope investigation on fixed RAW264.7 macrophage cells39 stimulated by

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lipopolysaccharide (LPS) has revealed that the cell surface roughness was increased compared to

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unstimulated ones, presumably due to the aggregation of membrane proteins and the change of

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F-actin structure. Rough surfaces scatter more light than smooth surface, and macrophage cells

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exposed to spores displayed increased laser scatter (Figure 4 B). Even though no AFM images

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are available on macrophage cells exposed to spores, it is reasonable to assume that the exposed



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cells have rougher cell surface than unexposed ones, since the binding and uptake of spores also

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involves the interplay of multiple membrane proteins and the change of actin structure. The

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increased optical force on macrophage cells exposed to spores, therefore, is likely linked to the

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increase in cell surface roughness.

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Optical force effects from macrophage- B. anthracis spore interaction in absence of phagocytosis

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Because an optical force increase was observed following exposure of macrophage cells

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to B. anthracis spores, experiments were conducted to determine to what extent the increase was

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driven by changes to the macrophage cell itself, as opposed to the physical presence of the spores

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inside the cell. To this end, cells were incubated with 10 µM cytochalasin D, an inhibitor of

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actin-dependent uptake pathways such as phagocytosis, prior to incubation with B. anthracis

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spores. As an additional control, the optical force of cells exposed to only cytochalasin D was

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also measured. Results presented in Figure 5A show a slight but statistically significant (p

Label-Free Detection of Bacillus anthracis Spore Uptake in

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