Probing the Limits of Aptamer Affinity with a
Microfluidic SELEX Platform
Kareem M. Ahmad1, Seung Soo Oh2, Seon Kim3, Forrest M. McClellen4, Yi Xiao2*, H. Tom Soh1,2,5*
1Interdepartmental Program in Biomolecular Science and Engineering, University of California Santa Barbara, Santa Barbara, California, United States of America,
2Materials Department, University of California Santa Barbara, Santa Barbara, California, United States of America, 3Department of Electrical and Computer Engineering,
University of California Santa Barbara, Santa Barbara, California, United States of America, 4Chemistry and Biochemistry Department, University of California Santa
Barbara, Santa Barbara, California, United States of America, 5Department of Mechanical Engineering, University of California Santa Barbara, Santa Barbara, California,
United States of America
Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity
reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their
selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for
molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high
performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high
affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with
different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer
sequences that bind to platelet derived growth factor B (PDGF-BB; pI=9.3) and thrombin (pI=8.3) with respective
dissociation constants (Kd) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these
targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI=5.3) with a Kdof 3.1 nM.
Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further
explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection
protocol, and report an inverse correlation between protein charge and aptamer Kd.
Citation: Ahmad KM, Oh SS, Kim S, McClellen FM, Xiao Y, et al. (2011) Probing the Limits of Aptamer Affinity with a Microfluidic SELEX Platform. PLoS ONE 6(11):
Editor: Maxim Antopolsky, University of Helsinki, Finland
Received March 25, 2011; Accepted October 10, 2011; Published November 14, 2011
Copyright: ? 2011 Ahmad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was funded by Armed Forces Institute for Regenerative Medicine Sub-Award 0010745, and National Institutes of Health NIH R01EB009764
NIH U01HL099773-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (YX); firstname.lastname@example.org (HTS)
Aptamers ,  are nucleic acid-based affinity reagents
selected through an in vitro process that can bind to a range of
molecular targets, including small molecules, proteins, and cells
. Aptamers offer a number of advantageous features, including
low cost, high thermal stability and the capacity for chemical
synthesis and modification, making them a promising alternative
to antibodies and other protein-based reagents . However, due
to the iterative nature of the aptamer selection process (systematic
evolution of ligands by exponential enrichment; SELEX), the
generation of aptamers requires a significant investment of time,
labor and resources , and selections often fail to yield molecules
with affinities suitable for molecular diagnostics or therapeutics.
Thus, a selection technique that can efficiently and reproducibly
generate high performance aptamers is urgently needed.
Towards this end, our group recently reported a microfluidics-
based approach for the rapid generation of DNA aptamers.
Microfluidic SELEX (M-SELEX) exerts stringent selection
pressures by employing minimal amounts of target molecules
and continuous washing to isolate high affinity aptamers in fewer
rounds of selection compared to conventional methods , ,
. To extend this method, in this work, we report a universal M-
SELEX process that can isolate high affinity aptamers for a range
of protein targets with varying isoelectric points (pIs) within three
selection rounds. Using a common selection protocol, we have
discovered new aptamer sequences that bind to PDGF-BB  and
thrombin ,  with higher affinities than those previously
published. We also report a new aptamer that binds apolipopro-
tein E3 ,  with low nanomolar affinity. Based on the results
obtained from these selections, we make the observation that
target protein pI appears to be inversely related to the Kdof the
selected aptamers. To further explore this relationship, we
performed microfluidic selections with PDGF-BB under varying
pH conditions to alter its charge state, and report differences in the
affinities of the resulting aptamer pools.
Microfluidic selection of aptamers
We performed DNA aptamer selections for three separate
protein targets under the same selection conditions. For every
round of selection, we used the micro-magnetic separation (MMS)
device  to efficiently separate non-specifically-bound DNA from
target-bound aptamers. For each target, we performed three
rounds of selection with increasing stringency (Fig. 1). This
stringency was controlled in two ways. First, we used a low
concentration of target protein during incubation and decreased
PLoS ONE | www.plosone.org1 November 2011 | Volume 6 | Issue 11 | e27051
thrombin and ApoE. The electrostatic potentials are mapped to
the van der Waals surfaces of these three proteins. The bottom set
of views show the proteins rotated 180u about the vertical axis.
These three maps suggest a structural basis for the relationship
between charge and binding affinity. Reflecting its high net
positive charge (z=+15), a substantial portion of the PDGF-BB
homodimer surface has a highly positive surface potential. These
regions present sizable areas for possible interactions with the
highly negatively charged DNA aptamers, and were identified as
the actual binding sites for a previously-isolated PDGF-AB
aptamer through cross-linking
(z=+2.6) contains two somewhat smaller distinct regions of
positive surface potential, and these were also identified as the
binding sites of two previously-published thrombin aptamers ,
. The surface potential of ApoE (z=24.3) is more
heterogeneous, reducing the area available for aptamer binding.
These data support the view that aptamers often preferentially
bind to positively charged surface patches, as has been observed in
crystal structures of aptamers bound to proteins , , ,
, , .
Electrostatic potential surfaces of PDGF-BB,
(MMS) device and pump configuration. The organization
of the fluidic connections between the pumps and MMS chip are
shown. Syringe pumps (not shown) were used to infuse buffer
Schematic of micro-magnetic separation
from each selected round 3 pool.
Selected sequences. 60-mer random regions cloned
We synthesized these aptamer sequences with a 59-FAM
fluorophore and assayed their binding affinity using the bead-
Sequences of previously published aptamers.
This list was compiled from PubMed searches and includes both
RNA and DNA aptamers against protein targets. Where not
provided in the reference, isoelectric points were determined by
UniProt (www.uniprot.org) and ExPASy (www.expasy.org).
A list of 75 previously published aptamers.
Microfabrication was carried out in the Nanofabrication Facility at UCSB.
We thank Jinpeng ‘‘JP’’ Wang for assistance with the lambda exonuclease
digestion and Qiang ‘‘Jackson’’ Gong for assistance with the radiolabeling
and filter binding assay. We also thank Herb Miller and Les Wilson for use
of the scintillation counter and Kalju Khan for assistance with the
isoelectric focusing experiments. The critical reading of and contributions
to the manuscript by Janet Kayfetz are appreciated.
Conceived and designed the experiments: KMA YX HTS. Performed the
experiments: KMA FMM YX. Analyzed the data: KMA YX HTS.
Contributed reagents/materials/analysis tools: KMA SSO SK. Wrote the
paper: KMA SSO YX HTS.
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