Affinity Probe Capillary Electrophoresis Evaluation of 

Aptamer Binding to Campylobacter jejuni Bacteria 

by Dimitra N. Stratis-Cullum, Sun McMasters, and Paul M. Pellegrino 

ARL-TR-5015 November 2009 

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Army Research Laboratory 

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ARL-TR-5015 November 2009 

Affinity Probe Capillary Electrophoresis Evaluation of 

Aptamer Binding to Campylobacter jejuni Bacteria 

Dimitra N. Stratis-Cullum, Sun McMasters, and Paul M. Pellegrino 

Sensors and Electron Devices Directorate, ARL 

Approved for public release; distribution unlimited. 
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4. TITLE AND SUBTITLE 

Affinity Probe Capillary Electrophoresis Evaluation of Aptamer Binding to 

Campylobacter jejuni Bacteria 

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6. AUTHOR(S) 

Dimitra N. Stratis-Cullum, Sun McMasters, and Paul M. Pellegrino 

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14. ABSTRACT 

In this work, we developed a capillary electrophoresis immunoassay to evaluate aptamer binding to bacterial cells. This work 

is the first known demonstration of aptamer affinity probe capillary electrophoresis to a whole cell target. The aptamer probes 

investigated were developed against a Campylobacter jejuni bacterial cell target, and we evaluated binding relative to other 

common food-borne pathogens. Specific binding affinity was evidenced by a pronounced mobility shift and peak broadening 

with increasing bacteria concentration. Little to no mobility shift was observed for food-borne pathogens, Salmonella 

typhirium and Escherichia coli, even when increasing concentrations 10-fold over target. These results suggest that affinity 

probe capillary electrophoresis could be useful for qualitative screening of aptamer candidates to bacterial cell targets. 

15. SUBJECT TERMS

Bioassay, aptamer, affinity probes, capillary electrophoresis, food pathogen, Campylobacter jejuni

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OF ABSTRACT 

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19a. NAME OF RESPONSIBLE PERSON

Dimitra N. Stratis-Cullum 

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Unclassified 

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(301) 394-0794 

Standard Form 298 (Rev. 8/98) 

Prescribed by ANSI Std. Z39.18
iii 

Contents 

List of Figures iv

Acknowledgments v

1. Inroduction 1

2. Experimental 2

2.1 Materials ..........................................................................................................................2

2.2 Capillary Electrophoresis System and Separation Protocols ..........................................3

3. Results and Discussion 5

4. Conclusion 8

5. References 9

List of Symbols, Abbreviations, and Acronyms 13

Distribution List 14 
iv 

List of Figures 

Figure 1. Schematic diagram of the capillary electrophoretic analysis of aptamer:bacterial 

cell complexes using laser-induced fluorescence detection. ......................................................4

Figure 2. Simplified schematic diagram illustrating free target and aptamer probe migration 

along with the anticipated complex migration zone highlighted in red. ...................................5

Figure 3. Reference electropherogram of the FITC-labeled, DNA aptamer measured under 

the experiment separation conditions, 45 mM tris-borate, 1 mM EDTA buffer solution at 

pH 8. ...........................................................................................................................................6

Figure 4. Electropherograms of fluorescein-labeled aptamer at a fixed concentration of 80 

nM with C. jejuni concentrations with no target (black), 6.4 × 106

 cells/mL (red), 13 × 

106 cells/mL (pink), and 16 × 106 cells/mL (pink) in 45 mM tris-boric acid-EDTA with 

0.05 % SDS at pH 8.3 under identical electrophoretic conditions. ............................................7

Figure 5. Electrophoretic analysis of solutions containing 80 nM aptamer with (a) no cells, 

(b) 4.8 107 cells/mL of C. jejuni, (c) 4.8 107 cells/mL Salmonella typhimurium and, (d) 

4.8 107 Escherichia coli O157:H7. Fluorescence intensity of each electropherogram is 

normalized to that of free DNA aptamer for comparison. ........................................................8
v 

Acknowledgments 

We wish to thank Dr. Andy Senecal from the U.S. Army Soldier & Biological Chemical 

Command from Natick Soldier Center for providing us with C. jejuni-aptamer sequences 

(Contract# DAAD16-00-C-09221) (36). We would also like to extend our appreciation to Oak 

Ridge Associated Universities for the postdoctoral fellowship, and to the Sensors and Electron 

Devices Directorate of the U.S. Army Research Laboratory (ARL) in Adelphi, MD for their 

support. 
vi 

INTENTIONALLY LEFT BLANK. 
1 

1. Inroduction 

Many applications rely on biological recognition as a means of achieving specificity for a 

particular target species (1–5). Although antibodies are most commonly employed, aptamers are 

another class of affinity probe and they possess a number of advantages over traditional 

bioreceptors (4, 6–9). Aptamers consist of a short, single-stranded ribonucleic acid (RNA) or 

deoxyribonucleic acid (DNA) oligonucleotides that provide three-dimensional (3D) 

conformational dependent binding capabilities towards a variety of target types via noncovalent 

interactions (6, 9–11). The molecular basis for these high binding affinities is due to an intricate, 

at least partial, encapsulation of the target species with a variety of interactions within the 

aptamer-target complex, including stacking, shape complementarity, electrostatic interactions, 

and hydrogen bonding (12). Unlike antibodies, aptamers can readily be mass-produced through 

standard oligonucleotide synthesis techniques (once identified) without the use of an animal host. 

Other distinct advantages over antibody affinity probes include a greater stability, robustness, 

and the ability to be easily chemically modified during the production process to allow for a 

variety of transduction schemes (9, 13). Aptamers exhibiting specific binding affinity have been 

developed for a wide range of targets, including small organic molecules, amino acids, 

oligosaccharides, and proteins, with limited reports of whole cells and tissues (1, 7, 13–24). 

However, there are very few reports of aptamers developed against whole-cell targets, making 

the aptamers under investigation here of particular interest, as they were developed against C. 

jejuni bacterial cells. 

There are a number of recent reports on the development of capillary electrophoresis (CE)-based 

methods for the investigation of aptamer affinity probe interactions (4, 8, 25–34), as well as for 

facilitating the development of aptamers through CE-enhanced sorting of aptamer libraries (8, 

34, 35). These methods can be divided into two main categories—noncompetitive and 

competitive (30, 31). Both typically employ laser-induced fluorescence detection. Some of the 

primary advantages to using affinity probe capillary electrophoresis methods over other 

techniques include: 1) only small sample quantities needed for analysis; 2) the sample does not 

necessarily need to be pure, provided that the complex formation is distinguishable, (3) radiolabeling is not required; 4) the analysis is readily automated; and 5) the interactions can be 

investigated in free solution without requiring immobilization of the target or affinity probe, 

which can lead to interferences in binding performance (31). 

In the noncompetitive affinity probe CE format, the probe is fluorescently labeled and incubated 

with the target species, and affinity interactions are observed through a mobility shift between 

the free probe and the target probe complex. Although the initial sample is in equilibrium, the 

separation can be performed under non-equilibrium conditions, even with low affinity aptamer 

probes. By contrast, the competitive format—a known quantity of labeled antigen or target 
2 

species—is added to the background electrolyte. In this case, a peak associated with the free 

target antigen increases when unlabeled target is introduced, while the peak associated with the 

complex decreases due to competition for binding sites. 

German et al., first demonstrated the potential of aptamers as probes for use in affinity probe 

capillary electrophoresis using a fluorescently labeled aptamer targeting immunoglobulin E (IgE) 

(28). Prior to injection, the IgE analyte was incubated with the aptamer probe and capillary 

electrophoretic analysis yielded two primary electrophoretic zones—one corresponding to the 

free aptamer and the other corresponding to the aptamer IgE complex. In another study, a 

similar mobility shift of the bound complex from the free aptamer was observed using a 

thrombin aptamer to detect thrombin protein. A unique ability of aptamer-based affinity 

capillary electrophoresis to investigate conformational binding dependence of the thrombin 

aptamer (intramolecular G-quadruplex form) with the thrombin protein target has also been 

demonstrated (30). In general, most reports employing affinity probe CE using aptamers have 

been mostly applied to the investigation or detection of various protein analytes. Recently, 

Krylov and coworkers proposed a new method termed Nonequilibrium Capillary Electrophoresis 

of Equilibrium Mixtures (NECEEM) that allows the use of low affinity aptamers as affinity 

probes for quantitative protein analysis (27). In another more recent report, aptamer-based 

affinity probe CE was used to detect sub-nanomolar concentrations of the protein Ricin, 

illustrating the potential application to the detection of biowarfare agents (35). It is important to 

note that, to date, there are no known reports by other research groups on the characterization of 

aptamers targeting whole cells using capillary electrophoresis. The work reported here is the 

first known development of a CE-based immunoassay that evaluates aptamer binding to bacterial 

cells. We report on the method developed and characterization results of the relative binding 

affinity of an aptamers developed against a Campylobacter jejuni bacterial cell target, relative to 

several other common food-borne pathogens, including the vegetative forms of Escherichia coli

O157:H7 and Salmonella typhimurium. 

2. Experimental 

2.1 Materials 

The aptamer sequence used in these studies was derived from a systematic evolution of ligands 

by exponential enrichment under a U.S. Army small business innovation research (SBIR) 

contract through Natick Soldier Center (36). In the report, aptamers were selected against heatkilled Campylobacter jejuni cells (Kirkegaard & Perry Laboratories, catalog # 50-92-93) from a 

DNA library containing 19 random nucleotides, denoted as N, with a sequence of 5’-

TCATCCGTCACACCTGCTCT-N19-GGTGGTGTTGGCTCCCGTAT-3’ in vitro (30). No 

specific surface antigen was targeted, as the entire heat-killed organism was used as the selection 

target. The selection resulted in a handful of aptamers, which were synthesized and high 
3 

performance liquid chromatography (HPLC)-purified from Integrated DNA Technologies 

(www.idtdna.com). A 6-carboxyfluorescein (6-FAM) was included at the 5’ end of DNA 

sequences with an additional thymine base, which was inserted adjacent to the label in order to 

avoid static fluorescence quenching. Sequences were analyzed initially through cell staining and 

enzyme linked immunosorbent assay(ELISA), and the best candidate (5’-

TACCGGCGCTTATTCCTGCT-3) was selected for the affinity probe CE investigations here. 

The concentration of the sequence was determined using the provided molar absorption 

coefficients (260nm) of 173,100 M cm–1.

 Desired stock solutions were freshly prepared with trisborate ethylenediaminetetraacetic acid (EDTA) buffer solutions. 

Lyophilized, heat-killed food pathogen samples were all obtained from Kirkegaard & Perry 

Laboratories (Gaithersburg, MD) at the following catalogue concentrations: Campylobacter 

jejuni cells (4.8 × 108 cells/mL, 2%); Escherichia coli O157:H7 (3 × 109 Cells/mL, 2%); and 

Salmonella typhimurium (5 × 109 cells/mL). It is important to note that all three organisms were 

grown and prepared under identical conditions. Once we received them, the organisms were 

resuspended in tris-borate buffer solutions and stored at –4 °C in single-use aliquots. Working 

solutions were prepared by diluting the stock solution to the desired concentration with a 

working buffer prior to use. 

A buffer solution containing either 89 mM tris-borate/2 mM EDTA or 45 mM tris-borate/1 mM 

EDTA with 0.05% SDS at pH 8.3 was used for all sample preparations and as a background 

electrolyte. All solutions were prepared with 18 mega-ohm polished water from a Barnstead 

Easypure reservoir feed, compact ultrapure water system. A 20x concentrated wash solution 

containing 0.002 M imidazole buffered saline with 0.02% Tween 20 was purchased from 

Kirkegaard & Perry Laboratories (Gaithersburg MD). All solutions used were filtered through a 

Nalgene analytical 0.2 μm cellulose nitrate membrane filter to remove any debris, and then were 

degassed by sonication for 2 min prior to use. All other internal standard chemicals, including 

Fluorescein and Coumarin 334, were purchased from Aldrich and used as received. 

Samples were prepared for injection and analysis by capillary electrophoresis in the running 

buffer solutions detailed previously. The specific concentrations used are given in the 

discussion, and ranged from106 to 107 cells/mL. The DNA aptamer concentration was fixed 

throughout the studies at 80 nM. To ensure equilibrium prior to injection, all samples were 

vortexed and incubated at room temperature for 15 min. 

2.2 Capillary Electrophoresis System and Separation Protocols 

All capillary electrophoresis experiments were performed with a Beckman P/ACE MDQ system 

equipped with a 488-nm argon ion laser excitation source. A schematic diagram illustrating the 

CE system and conceptual schematic of the bacteria:aptamer complex separation is shown in 

figure 1. Emission of fluorescent labeled analytes at 520 nm was monitored after separation 

through a bare fused silica capillary tube (50-μm-i.d. with 30 cm effective capillary length and 
4 

40.2 cm total capillary length) purchased from Beckman Coulter. Data were processed using 32 

Karat Software Ver 5.0 HPLC from Beckman Coulter. 

Figure 1. Schematic diagram of the capillary electrophoretic analysis of aptamer:bacterial cell 

complexes using laser-induced fluorescence detection. 

It is important to note that the separation conditions were optimized with thorough rinsing cycles 

between runs to prevent cell adhesion to capillary walls and to yield a reproducible analysis 

(~2% RSD). Conditions were also optimized to allow for easy visualization of affinity probe 

mobility shifts upon binding with a relatively short analysis time. The optimized conditions 

included a new capillary tube pretreated with 1N sodium hydroxide (NaOH) for 10 min, 

followed by rinsing with water for 10 min. The capillary tube was pre-rinsed with background 

electrolyte buffer for 2 min prior to each measurement, with the pressure at 40 psi, and the 

capillary volume was replaced at least 14 times. Prior to injection into the capillary tube, the 

buffer solution containing mixtures of analytes was vortexed and allowed to equilibrate for at 

least 15 min at room temperature. All analytes were injected hydrodynamically for 3 s under 0.3 

psi at the positive end of the capillary tube, and an electric field of 500 V cm–1 was applied under 

normal polarity for separation. This volume corresponds to ca. 2.7 nL according to the Poiseuille 

equation, while the length of the sample plug was 1.36 mm. The background electrophoresis 

buffer, which contained 0.05% sodium dodecyl sulfate (SDS), was replaced after 8–10 

electrophoretic separations to avoid buffer depletion. At the end of each run, the capillary tube 

was washed with 1 N hydrochloric acid (HCl), 1 N NaOH, and 0.1 N NaOH for 1 min, with a 1 

min rinse of polished water in between each treatment. Temperature of the capillary tube and 
5 

samples was regulated to 25 °C at all times. Fluorescein or Coumarin 334 was used as an 

internal standard to correct for changes in the potential field based on the fluorescein 

electrophoretic mobility, while the latter was used to monitor the electroosmotic flow (EOF) of 

the solution. For periodic capillary tube treatments, the tube was rinsed weekly with the wash 

solution and neat ethanol, followed by polished water. This washing cycle was repeated until a 

reproducible (<2% RSD) standard calibration was obtained. 

3. Results and Discussion 

Based on the previous literature discussion, it was anticipated that the free-bound aptamer probe 

would exhibit a specific electrophoretic mobility in the absence of target and a shifted mobility 

when bound to the target species. Figure 2 illustrates a general migration zone diagram concept, 

where the bound complex should appear in the migration zone highlighted in red between the 

two components. Therefore, in order to demonstrate the ability to perform aptamer and whole 

cell studies using capillary electrophoresis, binding interactions between the aptamer affinity 

probe and C. jejuni were evaluated based on the peak appearance and electrophoretic mobility 

shift of the bound complexes from that of their free forms. The binding specificity of the 

aptamer was then further investigated by examining interactions with other related food-borne 

pathogens under identical conditions. 

Figure 2. Simplified schematic diagram illustrating free target and 

aptamer probe migration along with the anticipated complex 

migration zone highlighted in red. 

The electrophoretic mobilities of the unbound aptamer were first determined using the optimized 

run buffer and conditions: 45 mM tris-borate and 1 mM EDTA with 0.05% SDS solution at pH 

8.3. Figure 3 shows the migration time for the aptamer probe in the absence of a target, which 

was monitored during separation using a fluorescein isothiocyanate (FITC) label and laser-
6 

induced fluorescence detection. Specific apparent mobility was calculated to be 1.8  10–4

 cm

2

V–1s

–1. 

Figure 3. Reference electropherogram of the FITC-labeled, 

DNA aptamer measured under the experiment 

separation conditions, 45 mM tris-borate, 1 mM 

EDTA buffer solution at pH 8. 

To investigate the mobility shift of the bound, aptamer, and Campylobacter jejuni complexes, 

equilibrium mixtures of the aptamer probe (80 nM) with differing concentrations of the 

organisms were analyzed. Shown in figure 3 are electropherogram results obtained from 

equilibrium mixtures of fluorescently labeled aptamer probe with differing concentrations of C. 

jejuni cells. As compared to the free aptamer (shown in black), there was a slight migration shift 

along with tailing when ca. 106

 cell/mL were interacted under identical buffer and separation 

conditions (shown in pink). 

Figure 4 also shows a pronounced mobility shift and broadening of the peak as bacteria titration 

progressed. These peaks clearly eluted faster than those of the unbound aptamers, suggesting 

they originate from aptamer-C. jejuni complexes. Although it was not possible to visualize the 

free bacterial cells in their unmodified form using the fluorescence detection module, the 

direction of mobility shift is consistent with experiments performed during assay optimization in 

preliminary studies, where the approximate mobility was measured to be 2.8  10–4 cm2

 V–1s

–1

under similar experimental conditions (data not shown) by using fluorescent cell staining of the 

bacteria. It is also interesting to note in figure 4 that the tail-end of this peak becomes broad over 

a range of up to 6 min of elution time. This electrophoretic dragging may be attributed to the 

dissociation of the bound complexes since the separation was carried out under non-equilibrium 

conditions. Moreover, this phenomenon may also interplay with the formation of multiple Seq 
7 

1-C. jejuni complexes with differing stoichiometric-binding interactions, generating a 

heterogeneous charge-to-mass ratio of the species. 

Figure 4. Electropherograms of fluorescein-labeled aptamer at a fixed concentration 

of 80 nM with C. jejuni concentrations with no target (black), 6.4 × 106

 cells/mL (red), 

13 × 106

 cells/mL (pink), and 16 × 106 cells/mL (pink) in 45 mM tris-boric 

acid-EDTA with 0.05 % SDS at pH 8.3 under identical electrophoretic conditions. 

In order to investigate the relative specificity of the interaction, the experiments were repeated 

with other food-borne pathogens, Escherichia coli O157:H7 and Salmonella typhimurium. 

These bacteria were selected to investigate the specificity of aptamer binding due to the fact that 

the preparation of the samples was identical to the C. jejuni material that was used for aptamer 

selection. In this way, observed differences under the same experimental conditions would be 

solely due to the interactions between the aptamer probe and bacteria complexes. Shown in 

figure 5 are the cross reactivity electropherogram results where (a) is the resulting reference 

electropherogram of the aptamer probe in the absence of a target, and (b) shows the pronounced 

mobility shift at a 4.8 x 107

 cell/mL concentration of Salmonella typhimurium. This 

concentration is higher than the concentration range investigated in figure 4 and is indicative of 

the maximum mobility shift observed for the C. jejuni target under this fixed probe 

concentration. Figure 5 (c) and (d) shows the electropherogram results after analysis of the 

aptamer probe interactions with E. coli and S. typhimurium, respectively. These results show 

very little if any cross-reactivity with the other food-born pathogen cell preparations. In 

summary, although not quantitative, the mobility shift is pronounced and easily provides a tool 

for qualitative screening of aptamer binding specificity to whole cell target. 
8 

Figure 5. Electrophoretic analysis of solutions containing 80 nM aptamer 

with (a) no cells, (b) 4.8 107

 cells/mL of C. jejuni, (c) 4.8 107 cells/mL 

Salmonella typhimurium and, (d) 4.8 107 Escherichia coli O157:H7. 

Fluorescence intensity of each electropherogram is normalized to that 

of free DNA aptamer for comparison. 

4. Conclusion 

In this report, a CE-based immunoassay that evaluates aptamer binding to bacterial cells was 

developed. The utility of this approach was demonstrated through binding affinity studies of an 

aptamers-targeting Campylobacter jejuni, relative to other common food-borne pathogens, 

Escherichia coli O157:H7 and Salmonella typhimurium. This is the first known report of an 

aptamer affinity probe, capillary electrophoretic analysis of cell targets. The aptamer probe 

investigated in this work exhibited a pronounced mobility shift upon binding to the 

Campylobacter jejuni target, and minimal shift and broadening to the other two food pathogen 

targets investigated. Although cell targets are much more complex than previously reported, 

aptamer affinity probe CE studies of bacteria, capillary electrophoresis shows potential for 

screening of aptamer probe candidates for binding performance. Moreover, this screening 

method could prove useful for evaluating aptamer binding without requiring immobilization of 

the probe or target on a solid support, which could interfere with conformational and binding 

properties of the probe. Finally, the specific probe investigated exhibit some specificity for the 

selected bacterial target, and warrants further investigation for potential application to food 

pathogen sensing. 
9 

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13 

List of Symbols, Abbreviations, and Acronyms 

CE capillary electrophoresis 

DNA deoxyribonucleic acid 

EDTA ethylenediaminetetraacetic acid 

ELISA enzyme linked immunosorbent assay 

EOF electroosmotic flow 

FITC fluorescein isothiocyanate 

HPLC high performance liquid chromatography 

IgE immunoglobin E 

NaOH sodium hydroxide 

RNA ribonucleic acid 

SBIR small business innovative research 

SDS sodium dodecyl sulfate 
14 

NO. OF 

COPIES ORGANIZATION 

 1 ADMNSTR 

 ELEC DEFNS TECHL INFO CTR 

 ATTN DTIC OCP 

 8725 JOHN J KINGMAN RD STE 0944 

 FT BELVOIR VA 22060-6218 

 1 DARPA 

 ATTN IXO S WELBY 

 3701 N FAIRFAX DR 

 ARLINGTON VA 22203-1714 

 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 AND 

 ENGRG CTR 

 ARMAMENT ENGRG AND 

 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 US ARMY RDECOM 

 NATICK SOLDIER CTR 

 ATTN A SENECAL 

 KANSAS STREET 

 NATICK MA 01760-5020 

 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 

NO. OF 

COPIES ORGANIZATION 

 1 COMMANDER 

 US ARMY RDECOM 

 ATTN AMSRD AMR 

 W C MCCORKLE 

 5400 FOWLER RD 

 REDSTONE ARSENAL AL 35898-5000 

 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 

 18 HCS US ARMY RSRCH LAB 

 2 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 N FELL 

 ATTN RDRL SEE O 

 P PELLEGRINO (2 HCS, 1 CD) 

 ATTN RDRL SEE P GILLESPIE 

 ADELPHI MD 20783-1197 

TOTAL: 31 (1 ELEC, 3, CDS, 27 HCS)