Rapid Peptide Reagent Isolation in a Disposable 

Microfluidic Cartridge 

by Dimitra N. Stratis-Cullum, Joshua M. Kogot, 

and Paul M. Pellegrino 

ARL-TR-5357 September 2010 

Approved for public release; distribution unlimited.
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Army Research Laboratory 

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ARL-TR-5357 September 2010 

Rapid Peptide Reagent Isolation in a Disposable 

Microfluidic Cartridge 

Dimitra N. Stratis-Cullum, Joshua M. Kogot, 

and Paul M. Pellegrino

Sensors and Electron Devices Directorate, ARL 

Approved for public release; distribution unlimited. 
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Rapid Peptide Reagent Isolation in a Disposable Microfluidic Cartridge 

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14. ABSTRACT 

In this collaborative development project, the challenges in affinity reagent isolation were addressed by combining two 

seemingly disjoint technologies—biosynthetic display libraries and microfluidic systems. To isolate optimal ligands from these

libraries, our project developed an automated magnetic bacterial cell sorting system, termed micromagnetic cell sorter (MMS), 

to screen microbial libraries with unprecedented throughput using a disposable microfluidic cartridge. We report on the sorting

system performance in rare cell and ultra-rare cell recovery (populations less than 0.001%), and compare the results to the 

standard manual method, magnetic activated cell Sorting (MACS). To demonstrate the system effectiveness in reagent 

isolation through sorting against an anthrax toxin target, protective antigen was performed and comparable results to standard 

screening methods were obtained. In addition, the good recovery performance through MMS selection yielded a consensus 

sequence among 24 unique binders. 

15. SUBJECT TERMS

Reagents, peptide, affinity, microfluidics, protective antigen, cell sorting, bioassay, m olecular recognition elements, ICB 

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Dimitra N. Stratis-Cullum 

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iii

Contents 

List of Figures iv 

List of Tables v 

Foreward vi 

Acknowledgments vii 

1. Introduction 1 

2. Experimental 6 

2.1 Micromagnetic Cell Sorter (MMS) System 6 

2.2 Sorting Procedures and Sample Preparation 8 

3. Results and Discussion 10 

4. Conclusion 14 

5. Future Work 15 

6. References 17 

List of Symbols, Abbreviations, and Acronyms 20 

Distribution List 21 
iv 

List of Figures 

Figure 1. Estimated average time required for completion of ligand/affinity reagent 

development using existing approaches. Phage display assumes 3–5 rounds of selection 

by panning. .................................................................................................................................1

Figure 2. Schematic diagram of CPX bacterial library scaffold. ....................................................3

Figure 3. Schematic description of peptide library screening using Cynvenio’s 

micromagnetic sorter (not to scale). ...........................................................................................5

Figure 4.  Photos of the micromagnetic sorting (MMS) platform. (a) MMS disposable 

cartridge with front side showing sample luer interfaces and backside showing separation 

region, on-cartridge valves and fluid path ways; (b) automated MMS instrument with an 

volumetric control module to precise fluid injection speeds and volumes, so that the 

separation results were highly repeatable independent of sample and buffer viscosity, or 

manufacturing variations in the disposable cartridge; (c) Stepper motors are implemented 

to actuate injectors with micro-switches for injector location sensing; (d) Off-shelf 

syringes are used as injectors for volumetric sample injection. .................................................7

Figure 5. FACS analysis of the fraction of target-binding clones in the enriched population 

after incubation with fluorescently labeled PA protein target over the course of three 

rounds of selection. Following one round of MMS, 0.7% (net) of the population exhibit 

PA binding peptides. Following two rounds of MMS, 56.5% (net) of the population 

exhibit target-binding peptides. 65.1% (net) of the population exhibit target-binding 

peptides after three rounds MMS selection. ............................................................................11

Figure 6. Peptide sequences of clones selected by MMS system for binding to protective 

antigen, revealing a six residual consensus sequence of WXCFTC among 24 individual 

clones. ......................................................................................................................................12

Figure 7. FACS analysis of three selected single clones labeled with biotinylated protective 

antigen. The negative control (top row image) was run with secondary label alone 

(SAPE); the test samples (bottom row image) were labeled with 150nM PA followed by 

SAPE. All of the tested samples show binding to PA as evidence by the increased 

positive signal (increased population in gate). .........................................................................13

 
v 

List of Tables 

Table 1. Desired features and importance in ligand isolation. ........................................................3

Table 2. Rare-cell recovery results as % recovered for the comparison of the MMS and 

manual magnetic sorter (MACS). Although both MMS and MACS show the capability 

of rare-cell recovery to 10–8 cells, the most reliable level for rare-cell recovery in a single 

magnet sort according to the large variance is 10–6 for the MMS and 10–5 for the MACS, 

as determined by the increased variance next cell recovery sample. The average standard 

deviation across all samples in the 4 independent trials for the MMS is 8.6% compared to 

21.8% for the MACS. ..............................................................................................................14
vi 

Foreward 

The U.S. Army Research Laboratory (ARL) was the government partner on a three-year, 6.2 

Research Project funded through the Army university-affiliated research center (UARC), the 

Institute for Collaborative Biotechnologies (University of California, Santa Barbara under grant 

DAAD19-03-D-0004). This ICB 6.2 program enables transition of ICB 6.1 technologies, and in 

this project two 6.1 technologies (peptide display technology, and microfluidic cell sorting) 

produced an affinity reagent isolation platform. The purpose of this report is to highlight the 

Army evaluation of the affinity sorter technology produced during this program, and to provide 

recommendations for future work related to this area. 
vii 

Acknowledgments 

This work was supported by the U.S. Army, through the Institute for Collaborative 

Biotechnologies (ICB) at the University of California, Santa Barbara under grant DAAD19-03-

D-0004. We wish to acknowledge the industrial partners Cynvenio Biosystems LLC and 

CytomX LLC (subcontract KK6125), and academic partners at the University of California 

Santa Barbara to include the following researchers: S. Moore, J. Rice, P. Bessette, B. 

Valdovinos, N. Stagliano, Y. T. Zhang, P. Pagano, D. Chang-Yen, M. Turewicz, A. deFusco, P. 

Daugherty, H. T. Soh, A. J. Heeger, J. Ballew, and J. Adams. We thank Ms. Rebecca Brown of 

the Edgewood Chemical Biological Center (ECBC) for her supporting discussions during this 

work that has proven critical to the ongoing transition of this effort. We would also like to 

extend our appreciation to Oak Ridge Associated Universities for the postdoctoral fellowship 

supporting J. Kogot, and to the Sensors and Electron Devices Directorate (SEDD) of the U.S. 

Army Research Laboratory (ARL) in Adelphi, MD, for their support. 

 
viii 

INTENTIONALLY LEFT BLANK. 
1 

1. Introduction 

There are two main challenges in creating a high speed affinity ligand isolation technology 

against unknown/un-catalogued pathogens: (1) creation of a large (high diversity) and robust 

cell based library, and (2) creation of an ultrahigh throughput, disposable screening system. The 

approach developed in this work aims to address these challenges by combining two innovative 

technologies whose roots originate to the Institute for Collaborative Biotechnology’s (ICB) 6.1 

research program. In this report, we report on the sorting system performance in rare cell and 

ultra-rare cell recovery, and demonstrate the system effectiveness in reagent isolation through 

sorting against an anthrax toxin target, protective antigen. 

Methodologies to develop highly specific affinity reagents for diagnostics, sensing, and therapy 

are critical to the U.S. Army mission in both the medical and nonmedical applications. With the 

development of hybridoma monoclonal antibody technology in 1975 by César Milstein and 

Georges Köhler (1), affinity reagents quickly became a cornerstone of diagnostic and sensing 

technology. Subsequently, a powerful new approach to more quickly generate affinity reagents, 

termed (bacterio) phage display technology, was developed by George Smith in 1985 (2). Phage 

display technology further transformed biotechnology by allowing researchers to generate tailormade affinity reagents substantially faster than hybridoma technology and in as little as two 

weeks (figure 1). 

Figure 1. Estimated average time required for completion of ligand/affinity reagent 

development using existing approaches. Phage display assumes 3–5 rounds of 

selection by panning. 

Despite recent advances in molecular recognition element alternatives, technology transfer to the 

Army has been challenging, and the current state-of-the-art continues to be limited by the high 

cost, limited availability, and often inadequate biochemical properties of affinity reagents for 
2 

many different applications. Given the enormous number of pathogens and toxins of concern to 

the Army, and their constant evolution, the demand for new reagents far exceeds the rate at 

which they can be produced. Consequently, methodologies to identify and produce ligands with 

high affinity and specificity to particular target molecules quickly, efficiently, reproducibly, and 

inexpensively are urgently needed. 

Bacterial display technology offers an alternate strategy for generating tailor-made affinity 

ligands in a short time period (3–4). In this method, cellular machinery is used to generate 

billions of diverse polypeptide molecules that can be screened with high throughput methods to 

identify unique polypeptide sequences for a desired target (4). Currently, a number of systems, 

including messenger ribonucleic acid (mRNA) and ribosome display (5), eukaryotic virus 

display (6–7), and bacterial and yeast surface display (3, 8), are used to rapidly generate affinity 

reagents that can be used for diagnostics, proteomics, and therapeutic applications (9–10). 

Bacterial cell surface display is advantageous because the use of bacterial cells simplifies the 

polypeptide selection method and enables fast screening of potential recognition elements using 

fluorescence activated cell-sorting (FACS). Furthermore, polypeptide affinity reagents could 

offer more stable alternatives to antibody technology, enabling more rugged application in the 

field (e.g., enhanced thermal stability). 

The development of a bacterial display system suitable for robust reagent discovery has proven 

challenging (11). Several different bacterial display systems have been reported (12–14); the 

broader utility of bacterial display, however, has been limited by technical problems, including 

accessibility on the cell surface and adverse effects on cell growth and viability (11, 15). Several 

groups have demonstrated that peptide libraries can be constructed in E. coli (11) as insertions in 

extracellular proteins such as pili or flagella subunits (13), or as insertions into outer membrane 

proteins (16). Though a few of these systems have provided encouraging results in a few 

different applications (16, 17), none of these bacterial display systems has been demonstrated to 

be suitable for routine isolation of high affinity peptide ligands that bind to arbitrary targets. 

Moreover, the construction of a large, high-complexity peptide library in a format that could 

serve as a single resource for many ligand isolation applications has not been reported. 

Through the 6.1 research component of the ICB, the Daugherty Lab at the University of 

California-Santa Barbara (UCSB) achieved several technical breakthroughs in bacterial display, 

yielding a more robust peptide library methodology. Briefly, this peptide library employs an ecoli bacterial display platform, generated from the extracellular loop of OmpX outer-membrane 

protein (see figure 2). The randomized portion of the library is a15-mer, yielding a greater than 

1010 member library. 
3 

Figure 2. Schematic diagram of CPX bacterial library scaffold. 

The design, construction, and application of the Daugherty display scaffolds have been well 

documented in a number of reports. (4, 18–19) In the work reported herein, this novel peptide 

library was used and combined with high throughput, on-chip cell sorter technology. It is 

important to note, however, that the automated micromagnetic cell sorter (MMS) platform can be 

adapted to other library technology (which was out of the scope of this work). 

In table 1, a series of features of an ideal cell-sorting platform for microbial library screening is 

shown. Ideally, a system which is designed to sort quickly sort through a 1010 member library of 

potential binders must have both high-throughput and high purity, in addition to recovery 

sorting. The simplicity of operation is critical to practical application, and the integration of 

functions could ultimately allow for widespread, automated use. Finally, the ability to have a 

disposable interface would be advantageous to laboratories working with hazardous target 

material(s), to minimize human exposure and contamination. 

Table 1. Desired features and importance in ligand isolation. 

Desired Features Importance in Ligand Isolation 

High-throughput Process libraries of 109–1011 cells in minimal time

High recovery High statistical certainty of collection/low loss 

probability 

High purity sorting Collect target cells without non-target cell 

contamination 

Simple, unattended operation Allow for technology transfer/dissemination 

Automation & scale-up Generate ligands for many different targets 

Integration of functions Integrate clone isolation, sequencing, and 

characterization

Reusable or disposable Process clinical or pathogen containing samples with 

minimal risk of cross-contamination

To isolate the bacterial clones that express peptide sequences with high affinity to the target, the 

conventional approach in this field uses multiple rounds of magnetic separation for preenrichment, followed by FACS sorting (20–23). Although this hybrid approach has proven to be 

effective manual magnetic sorting, is labor-intensive, and the sorting results are known to be 

CPX Bacterial Display 

Bacterial display 

peptide library 

(> 1010 members) 

Target 

protein 

N 

C 

Peptide 

ligand 
4 

operator-dependent (24). Moreover, the high capital and maintenance cost of FACS instruments 

limit its accessibility. In addition, when dealing with infectious pathogens, a biohazard aerosol is 

potentially generated at the nozzle; additional steps need to be taken to reduce this hazard, such 

as adding an aerosol management unit, further increasing cost. 

To address the need for a rapid, safe, efficient, cost effective, and reproducible method for 

peptide affinity ligand selection, our program developed an automated magnetic bacterial cellsorting system, the MMS, equipped with disposable microfluidic cartridges shown in figure 3. 

During the affinity ligand screening process (illustrated in figure 3), the target molecule is 

biotinylated and incubated with the bacterial display library. Cells displaying peptides that bind 

to the target molecule become labeled with biotinylated target. The resulting target-binding cells 

are incubated with streptavidin-coated magnetic beads and captured by the magnetic particles 

through biotin-streptavidin binding (4). After sample preparation, MMS sorting is performed to 

isolate the magnetically-labeled bacterial clones from the rest of the library. The collected cells 

were cultured overnight for processing in the next round of selection. The low cost disposable 

cartridge developed herein aims to mitigate hazard exposure through controlled containment of 

the hazard through selection and disposal, with minimal handling by laboratory personnel. 
5 

Figure 3. Schematic description of peptide library screening using Cynvenio’s micromagnetic sorter 

(not to scale). 

The target protein (e.g., protective antigen) is biotinylated and labeled with streptavidin-coated 

superparamagnetic beads; thus, bacterial cells displaying peptides that have affinity to target 

protein are captured on the superparamagnetic beads, allowing continuous-flow separation by 

Cynvenio MMS. The design also allows non-binding bacterial cells flow out as waste. The 

binding population is eluted from the MMS cartridge, and can either be amplified by overnight 

culture growth for a further round of labeling and sorting, or plated on solid media to isolate 

single clones for sequence determination. An example of the typical results of DNA sequencing, 

translation, sequence alignment, and consensus determination is shown for clarity. 
6 

Experiments were conducted on the MMS system to determine the rare cell and ultra-rare cell 

recovery (populations less than 0.001%) (25–26) capability of the instrument compared to 

manual MACS. Rare-cell isolation has potential in medicine for cell identification, such as 

cancer cell isolation and population enrichment (27–29), and could have potential military and 

national security applications for isolation of potential pathogens. 

As a further demonstration of the MMS system’s effectiveness for binder isolation, protective 

antigen (PA) of an anthrax toxin (Bacillus anthracis) was chosen as a target. The CytomX 

Therapeutics eCPX bacterial display library, expressing ~3 x 1010 discreet random peptides, was 

screened for affinity reagents, which bound to protective antigen. 

2. Experimental 

2.1 Micromagnetic Cell Sorter (MMS) System 

The MMS is an automated magnetic separation system consisting of a disposable microfluidic 

cartridge (figure 4a) and a companion instrument (figure 4b). The disposable cartridges are 

made of injection-molded polypropylene (Pinnacle Polymers PP 5135C). The 200-μm deep 

fluidic channels are defined by two injected parts, which are laser-welded (California Lasers, 

Simi Valley, CA), and a portion is heat-staked with a hydrophobic membrane for bubble removal 

(Pall Co, Ann Arbor, MI). The trapping region was designed to accommodate up to 1 × 109

 of 

1 μm trapped magnetic beads and process up to 1 × 1011 bacterial cells. Female luer fittings on 

the top of the cartridge allow for a leak-proof interface between the cartridge and disposable 

syringes (Becton Dickinson, San José, CA). The luer fittings on the cartridge are designed to 

hold a reservoir array for pneumatically driven applications, as well as the injector inputs. There 

are a total of four luer ports required for two sample injectors (1 or 5 mL volume), one 

running/wash buffer injector (up to 10 mL) and one elution buffer injector (up to 3 mL volume). 

Strategically designed micro-channels allow for full automation of magnetic separation on the 

cartridge. To accomplish this, five pneumatically actuated pinch valves are located on the 

underside of the cartridge, allowing for the redirection of flow. These valve membranes require 

a force of ~15 lb/in2

 to seal and are robust enough to be actuated multiple times. 
7 

Figure 4. Photos of the micromagnetic sorting (MMS) platform. (a) MMS disposable cartridge with front side 

showing sample luer interfaces and backside showing separation region, on-cartridge valves and 

fluid path ways; (b) automated MMS instrument with an volumetric control module to precise fluid 

injection speeds and volumes, so that the separation results were highly repeatable independent of 

sample and buffer viscosity, or manufacturing variations in the disposable cartridge; (c) Stepper 

motors are implemented to actuate injectors with micro-switches for injector location sensing; 

(d) Off-shelf syringes are used as injectors for volumetric sample injection. 

The instrument uses a cRIO controller with LabVIEW script (National Instruments, Austin, TX), 

outfitted with standard digital and analog in/out modules for control of the internal components. 

Flow rates within the cartridge are controlled by four stepper motors (figure 4c) and controller 

boards (Haydon and Anaheim Automation, respectively), which physically push on the injectors 

(figure 4d). These motors are fitted with micro-switches (Panasonic ECG, Secaucus, NJ) that 

allow for the automatic calculation of input volume. Valves on the cartridge are actuated using 

pneumatically controlled air cylinders (SMC Corp, Noblesville, IN) and a DC diaphragm pump 
8 

(Thomas provided by Nor Cal Controls, San José, CA). There are 70 custom neodymium-iron 

boron magnets, which are position-controlled by another Haydon stepper motor. The magnets 

are distributed equally among top and bottom portions of a magnetic rack, which sandwich the 

disposable cartridge. A single motor, in conjunction with a spring, allows for both horizontal 

and vertical movement of magnets. This facilitates horizontal movement required for trapping 

and elution, and vertical movement capable of agitating the sample within the cartridge. 

Software control is provided using a LabVIEW interface. Push-button applications have been 

created for bacterial library sorting. Advanced users can generate custom sorting routines, which 

allow full access to all the operation parameters with minimal training. Direct control of flow 

rates, wash stringencies, and positive/negative selection criteria enables the end-user to specify 

the magnetic bead and protocol of choice, and optimize it for applications beyond bacterial 

library sorting (i.e., cell culture, flow cytometry, toxicology studies, etc.). 

2.2 Sorting Procedures and Sample Preparation 

Display Library 

A bacterial display library (Cytomx Therapeutics eCPX library) that contained approximately 3 

× 1010 members was screened for clones that display PA binding peptides The random library is 

first grown in 500 mL LB media containing 25 μg/mL chloramphenicol (LB-Cm25) to an 

OD600nm of approximately 0.6 (Eppendorf Biophotometer; Eppendorf, Hamburg, Germany). At 

this point in exponential growth phase, the cells were induced by the addition of arabinose to a 

final concentration of 0.04% (w/v); the enhanced circularly permuted OmpX (eCPX) gene 

expressing the library peptides was under the control of an arabinose inducible promoter (30). 

The cells were shaken at 37 °C for an additional 45 min, after which the OD600nm was again 

measured, and, using the assumption that an OD600nm of 1.0 relates to a bacterial concentration of 

1 x 109 cfu/mL, approximately 2 x 1011 cells were pelleted by centrifugation at 3000 g for 

20 min. 

SA-binder depletion 

The bacterial pellet was re-suspended in 1.5mL of PBSB (PBS buffer plus 0.5% BSA) 

containing 1 × 109

 paramagnetic beads (Invitrogen DynabeadsMyOneStreptavidin C-1; 

Invitrogen, Carlsbad, CA). The cell suspension was incubated at 4 oC for 45 min, with rotation 

to allow depletion of streptavidin binders from the library prior to selections. To remove these 

beads and any cells bound to them, the sample was loaded onto an MMS cartridge and separated 

at a sample flow rate of 50 mL/hr and buffer flow rate of 10 mL/hr. The MMS cartridge 

captured the unwanted bead bound cells and allowed collection of the depleted library ready for 

enrichment. For the SA binder depletion using a benchtop magnetic bead separator (manual 

MACS), the bacterial cell pellet with 1 × 109

 paramagnetic beads was pelleted using a magnet 

next to the tube. The magnetic separation was performed for 5 min to allow the bead pellet to 

form. The sample was washed and aspirated with 5 × 1 mL PBS washes, and resuspended in 

1 mL PBSB for PA binder enrichment. 
9 

PA-binder enrichment 

The SA-binder depleted library was centrifuged at 3000 g for 20 min, resuspended in 1 mL 

PBSB buffer containing 600 nM biotinylated protective antigen (List Biological Laboratories, 

Inc; Campbell, CA), and incubated at 4 oC for 45 min. Cells were centrifuged as previously 

described and re-suspended in 1 mL PBSB buffer with 1 × 109 pre-washed magnetic beads. 

After 45 min at 4 oC with rotation, the cell-beads suspension was loaded into an MMS cartridge 

(or separated by manual MACS using the same methods as SA binder depletion). Bacterial cells 

bound to PA were trapped on cartridge and then eluted into a collection vessel. A second round 

of sorting was performed, following the same protocol as the first; however, the assay parameters 

were adjusted to account for the smaller starting population and to increase the selection pressure 

in the second round. Therefore, we used 1 x 108

 cells in 50 μL of 300 nM PA and 1 x 108

magnetic beads. Cells were incubated static on ice for all labeling steps. Also, 1 μM biotin was 

added in the washing buffer to compete with any remaining streptavidin binders (peptides which 

bind to streptavidin typically have a much lower affinity than biotin). In the third round of MMS 

sorting, cells were labeled with 150 nM biotinylated PA, and then labeled with 1 x 106

 magnetic 

beads in 50 μL of PBSB. After each round of magnetic separation, the bead-bound enriched 

library was added to LB-Cm25 media, supplemented with 0.2% glucose to inhibit expression of 

the eCPX gene and, therefore, prevent growth bias. The cultures were then grown overnight at 

37 oC with shaking. 

Analysis of PA binder enrichment by FACS 

To quantify the library enrichment of potential PA binders, we performed FACS analysis (BD 

FACSAria; BD Biosciences, Franklin Lakes, NJ) using biotinylated PA (EZ-Link Sulfo-NHS 

biotinylation kit; Thermo Scientific, Rockford, IL) labeled with alternating fluorescent secondary 

labels—streptavidin, R-phycoerythrin conjugate (SAPE; Invitrogen, Carlsbad, CA), anti-biotinphycoerythrin (Miltenyi Biotec; Bergisch Gladbach, Germany), and Neutravidin, Rphycoerythrin conjugate (NAPE; Invitrogen, Carlsbad, CA)—similar to previously published 

procedures (4, 12). Following each round of PA selection, the arabinose-induced cell population 

was incubated with 100 nM biotin-PA solution for 45 min. The sample was centrifuged at 

3000 g for 10 min to remove unbound biotin-PA, and was resuspended in a 25 L solution of 

PBSB with secondary label concentration of 5 g/mL and incubated for 45 min at 4 oC. The 

sample was centrifuged and resuspended in 1 mL ice-cold BD FACSFlow (BD Biosciences, 

Franlin Lakes, NJ) sheath immediately prior to FACS analysis. Cells labeled with SAPE exhibit 

increased red fluorescence and are easily distinguishable by flow cytometry. 

Ultra-Rare Cell Recovery 

To measure the rare and ultra-rare cell recovery of the MMS, cells not expressing surface display 

peptides (negative control) were doped with a known quantity of cells expressing a known PA 

binding sequence. A 1 × 10–3 or 0.1% PA binder (1 L PA binding bacteria in 1mL on negative 

control bacteria) sample was diluted in negative control library to create samples ranging from 
10 

1 × 10–3 to 1 × 10–8, or 0.0000001% PA binding cells. The samples were analyzed by FACS 

before and after MMS (or MACS) sorting to determine the ultra-rare cell recovery capability of 

each technique. 

3. Results and Discussion 

Three key parameters are used to evaluate cell sorting which is required for binder isolation. The 

first is throughput, which measures how many cells can be sorted per second. As discussed 

previously, the MMS platform is designed for high throughput screening since it is capable of 

screening a bacterial library containing 3 × 1010 members in 15 min. The second is purity, (the 

fraction of collected cells which actually bind the target), and the third is recovery (the fraction 

of binders collected relative to the total number of binders in the naïve library). 

To evaluate the purity of the isolated fractions, we performed FACS analyses of PA binder 

populations in the positive sorting sample and the negative control sample. Figure 5 shows the 

FACS analysis results of the fraction of target-binding clones in the enriched population after 

incubation with fluorescently labeled PA protein target. The intensity of red fluorescence (xaxis) represents the level of binding on the cell surface, which may be due to either a high 

expression or a high affinity for the target. To assess recovery, serial dilutions of the collected 

bacterial populations were grown for 14 to 18 h on LB-Cm25 plates at 37 oC. The serial dilutions 

enable a calculation of resultant library diversity compared to the original eCPX library diversity 

of (3 x 1010) and provide verification that enrichment has occurred after each round of selection. 
11 

Figure 5. FACS analysis of the fraction of target-binding clones in the enriched population after incubation with 

fluorescently labeled PA protein target over the course of three rounds of selection. Following one round 

of MMS, 0.7% (net) of the population exhibit PA binding peptides. Following two rounds of MMS, 

56.5% (net) of the population exhibit target-binding peptides. 65.1% (net) of the population exhibit 

target-binding peptides after three rounds MMS selection. 

FACS analysis shows that after one round of MMS selection, the frequency of cells capable of 

binding to PA reached 0.7%; the second and third rounds further enriched the population to 

56.1% and 65.5%, respectively, with MMS selection (figure 5). Individual clones were picked at 

random from the positive populations and sequenced (Genewiz, South Plainfield, NJ). 

Sequences were analyzed and aligned using the Vector NTI software suit (Invitrogen, Carlsbad, 

CA). Sequencing of 24 clones obtained after three rounds of sorting yielded 15 clones 

displaying a consensus motif WXCFTC. After repeating the selection process, a total of 24 

distinct peptide sequences showed the WXCFTC consensus (figure 6). This consensus was the 

same as the best binder found with the conventional MACS/FACS approach SM545 

(GSFYDSILFYCMTCR). Ten randomly selected clones, expressing peptides with the 

consensus sequences, all show binding to 150 nM PA as measured by flow cytometry; results for 

three of the clones are shown in figure 7. 
12 

Figure 6. Peptide sequences of clones selected by MMS system for binding to protective 

antigen, revealing a six residual consensus sequence of WXCFTC among 24 

individual clones. 
13 

Figure 7. FACS analysis of three selected single clones labeled with biotinylated protective antigen. The 

negative control (top row image) was run with secondary label alone (SAPE); the test samples (bottom 

row image) were labeled with 150nM PA followed by SAPE. All of the tested samples show binding to 

PA as evidence by the increased positive signal (increased population in gate). 

Both MMS and MACS show the capability of ultra-rare cell recovery of 3.00% for the MMS and 

5.68% for the MACS at 1 x 10–8 cells (table 2), which represents a greater rare cell population 

than any of the presort populations measured. The MMS is more consistent than the MACS at 

recovering all ultra-rare and rare cell populations, given the lower standard deviation for 

recovery for nearly all the samples tested (except at 1 x 10–7, which has a lower standard 

deviation with the MACS). Overall, the MMS proved to have less variance between rare cell 

recovery experiments than the MACS, given that the average standard deviation for the MMS 

was only 8.6%, compared to 21.8% for MACS for all rare cell populations between 0.1% and 

0.0000001%. 
14 

Table 2. Rare-cell recovery results as % recovered for the comparison of the MMS and manual magnetic sorter 

(MACS). Although both MMS and MACS show the capability of rare-cell recovery to 10–8 cells, the 

most reliable level for rare-cell recovery in a single magnet sort according to the large variance is 10–6 for 

the MMS and 10–5 for the MACS, as determined by the increased variance next cell recovery sample. The 

average standard deviation across all samples in the 4 independent trials for the MMS is 8.6% compared 

to 21.8% for the MACS. 

Trial 1 Trial 2 Trial 3 Trial 4 Average ± SD

MMS MACS MMS MACS MMS MACS MMS MACS MMS MACS Presort

10–3

(0.1%) 

96.2 0 91.1 11.9 96.9 97.7 97.7 96.9 95.48 ± 

2.98 

68.83 ± 

52.97 

2.83 ± 

1.67 

10–4

(0.01%) 

95.6 94.9 69.2 42.2 90.9 82.3 87.7 76.3 85.85 ± 

11.56 

73.93 ± 

22.53 

1.30 ± 

0.44 

10–5

(0.001%) 

26.2 82.9 39.9 5.2 28.2 25.1 67.6 40.7 40.48 ± 

19.07 

38.48 ± 

32.99 

0.97 ± 

0.29 

10–6 (0.00001%) 

18.2 34.0 15.1 4.8 3.6 9.2 0 4.3 9.23 ± 8.79 13.08 ± 

14.12 

1.03 ± 

0.46 

10–7 (0.000001%) 

2.1 1.6 13.7 5.6 0 8.1 1.1 6.5 4.23 ± 

6.37 

5.45 ± 

2.77 

1.17 ± 

0.49 

10–8 (0.0000001%) 

1.3 1.0 6.9 8.2 3.8 8.6 0 4.9 3.00 ± 

3.04 

5.68 ± 

3.53 

1.10 ± 

0.71 

4. Conclusion 

The U.S. Army Research Laboratory (ARL) was the government partner on a three-year, 6.2 

Research Project funded through the Army university-affiliated research center (UARC), the ICB 

at UCSB under grant DAAD19-03-D-0004). During this program, we transitioned two 6.1 

technologies (peptide display technology, and microfluidic cell sorting) and produced an affinity 

reagent isolation platform. In this report, we highlight the first phase of the Army evaluation of 

the affinity sorter technology. 

Characterization of the MMS system’s performance was achieved by screening against PA from 

Bacillus anthracis. Automated MACS are not currently available for display library screening. 

Therefore, results obtained using the MMS platform were directly compared to those obtained 

using conventional MACS/FACS sorting. In this work, we demonstrated that the automated 

MMS platform is capable of effectively enriching affinity peptides against potential biological 

warfare agents with high throughput. For a typical 1 mL sample volume, MMS requires only 5 

min of user interaction, while manual selection requires more than 20 min. 
15 

With regard to gross throughput per hour, MMS is able to process 5 × 1012 cells/hr (50 mL/hr at 

a cell concentration of 1 x 1011 cells/mL), which is four orders of magnitude higher than that 

achieved using state-of-art FACS instrumentation or a previously reported dielectrophoretic cell 

sorter (31). In addition, for the first time, a PA binder sequence consensus WXCFTC was 

discovered via the high recovery performance of MMS, which results from the MMS sorting 

system’s automation and integrated nature. Furthermore, the sorting protocols described here 

can be easily adapted to select other affinity reagents to targets of interest. 

The current results not only demonstrate the potential of the MMS platform for automated 

reagent discovery, but could lead to a much broader extension to a variety of applications 

requiring rare-cell recovery that were out of the scope of our objectives. For example, the ability 

to consistently recover and isolate a rare cell population from a large negative control population 

provides a useful method for pathogen detection in food and water using this low cost, 

disposable cartridge system. The use of a disposable cartridge permits the analysis of potentially 

hazardous materials with minimal user exposure, and eliminates any concerns for crosscontamination of samples. The MMS sorting platform is also suitable for general magnetic cellsorting, with applications ranging from mammalian cell separation to protein and nucleic acid 

purification. 

5. Future Work 

Although the MMS sorter development shows promise for affinity reagent isolation applications, 

there are a number of areas of ongoing work, as well as recommendations for future research and 

development, that can be made. Our current and future work related to this program includes the 

continued evaluation of binder candidates selected against protective antigen (off scaffold) and 

other targets (on and off scaffold) in terms of binding affinity and specificity. The PA peptide 

binders produced during this work show promise, as the binding affinity on scaffold and in 

preliminary work in peptibody fusions show Kds in the nM range. They exhibited little to no 

cross-reactivity with IgG and other standard protein systems typically used in immunoassay 

chemistries, but extensive cross-reactivity studies are still underway. Also, we are currently 

evaluating Kds of the free, solubilized peptides off scaffold using standard enzyme-linked 

immunoassay methods (ELISA). We plan to follow up with another report detailing the peptide 

analysis of these PA binders. 

We plan to not only extend the sorting to other target analyte materials, but also explore the use 

of alternate library technologies (e.g., yeast library display, aptamers, etc.) with the MMS 

isolation. 

As discussed earlier, a main challenge in affinity reagent isolation from bacterial libraries is 

overcoming the tedious steps involved in manual selection that leads to irreproducible results. 
16 

Although advances are made herein towards automating this application, the final binder 

population selection is expected to be representative of the “family” of binders exhibiting desired 

characteristics. However, statistically, the best binders can be lost through adsorption, etc. 

Therefore, additional optimization of the binder population is still needed, such as employing 

traditional soft randomization techniques and re-selection. 

Additional work related to the challenge of understanding molecular recognition includes a new, 

three-year program, beginning in FY10, funded by the Defense Threat Reduction Agency, 

(DTRA) termed “Iterative Modeling of Peptide-Protein Interaction for ‘Smart’ Reagent 

Development.” We have chosen the SM545 PA peptide reported here as a model system and 

aim to ultimately develop a comprehensive modeling toolkit that can be used to predict the best 

(equivalently optimized) smart binders after initial selection. 

Finally, the MMS was very simple to operate; however, the system could benefit greatly from 

further engineering to help with reproducible card placement and full automation. No current 

plans by the industrial partners exist to further develop the platform specifically for bacterial 

library screening, but advances in the fluidics and other cartridge capabilities are underway for 

other commercial applications. 
17 

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20 

List of Symbols, Abbreviations, and Acronyms 

ARL U.S. Army Research Laboratory 

DTRA Defense Threat Reduction Agency 

DNA deoxyribonucleic acid 

ECBC Edgewood Chemical Biological Center 

ELISA enzyme linked immunoassay 

FACS fluorescence activated cell sorting 

ICB Institute for Collaborative Biotechnologies 

IgG immunoglobulin G 

LB lysogeny broth 

LB-Cm25 lysogeny broth with with 25 g/mLchloramphenicol 

MACS magnetic activated cell sorting 

MMS micromagnetic cell sorter 

MRE molecular recognition element 

mRNA messenger ribonucleic acid 

NAPE neutravidin, R-phycoerythrin 

PA protective antigen 

PBSB phosphate buffered saline with 0.1% bovine serum 

SAPE streptavidin, R-phycoerythrin 

UARC university-affiliated research center 

UCSB University of California-Santa Barbara 
21 

NO. OF 

COPIES ORGANIZATION 

1 ADMNSTR 

ELECT DEFNS TECHL INFO CTR 

 ATTN DTIC OCP 

 8725 JOHN J KINGMAN RD STE 0944 

 FT BELVOIR VA 22060-6218 

1 CD OFC OF THE SECY OF DEFNS 

 ATTN ODDRE (R&AT) 

 THE PENTAGON 

 WASHINGTON DC 20301-3080 

1 US ARMY RSRCH DEV AND ENGRG 

 CMND 

 ARMAMENT RSRCH DEV & ENGRG 

 CTR 

 ARMAMENT ENGRG & TECHNLGY 

 CTR 

 ATTN AMSRD AAR AEF T 

 J MATTS 

 BLDG 305 

 ABERDEEN PROVING GROUND MD 

 21005-5001 

1 US ARMY RDECOM 

 EDGEWOOD CHEMICAL 

 BIOLOGICAL CTR 

 ATTN AMSRD ECB RT BI 

 R BROWN 

 5183 BLACKHAWK RD BLDG E3549 

 ABERDEEN PROVING GROUND MD 

 21010-5424 

1 PM TIMS, PROFILER (MMS-P) 

 AN/TMQ-52 

 ATTN B GRIFFIES 

 BUILDING 563 

 FT MONMOUTH NJ 07703 

1 US ARMY INFO SYS ENGRG CMND 

 ATTN AMSEL IE TD A RIVERA 

 FT HUACHUCA AZ 85613-5300 

1 COMMANDER 

 US ARMY RDECOM 

 ATTN AMSRD AMR 

 W C MCCORKLE 

 5400 FOWLER RD 

 REDSTONE ARSENAL AL 35898-5000 

NO. OF 

COPIES ORGANIZATION 

1 US GOVERNMENT PRINT OFF 

 DEPOSITORY RECEIVING SECTION 

 ATTN MAIL STOP IDAD J TATE 

 732 NORTH CAPITOL ST NW 

 WASHINGTON DC 20402 

1 US ARMY RSRCH LAB 

 ATTN RDRL CIM G T LANDFRIED 

 BLDG 4600 

 ABERDEEN PROVING GROUND MD 

 21005-5066 

20 HCS US ARMY RSRCH LAB 

3 CDS ATTN IMNE ALC HRR MAIL & 

 RECORDS MGMT 

 ATTN RDRL CIM L TECHL LIB 

 ATTN RDRL CIM P TECHL PUB 

 ATTN RDRL SEE G WOOD 

 ATTN RDRL SEE O 

 D STRATIS-CULLUM (10 HCS, 1 CD) 

 ATTN RDRL SEE O J KOGOT 

 (3 HCS 1 CD) 

 ATTN RDRL SEE O N FELL 

 ATTN RDRL SEE O P PELLEGRINO 

 (1 HC, 1 CD) 

 ATTN RDRL SEE P GILLESPIE 

 ADELPHI MD 20783-1197 

TOTAL: 32 (27 HCS, 4 CDS, 1 ELECT) 
22 

INTENTIONALLY LEFT BLANK.