Despite the fact that aptamers were first described in 1990, they have not been extensively exploited in one of the most
common bioassay formats – the lateral flow device. Nevertheless, aptamers are receiving increasing attention in lateral flow
applications [1–3] due to a number of important potential performance advantages.
Top 5 Reasons for Choosing
Aptamers in Lateral Flow Assays
Tertiary structure of a DNA aptamer
#1. Aptamers have a virtual unlimited shelf-life when dried on a lateral flow strip.
It is generally accepted that the shelf-life of protein based reagents is limited to a few months refrigerated and only days at
room temperature [4, 5]. While detection Ab or Ab:enzyme conjugates are dried in a high sucrose solution on the conjugate
pad, and the capture Ab is painted as a stripe in the detection zone can help preserve these reagents, the performance of lateral flow devices can quickly degrade under uncontrolled conditions [4]. In contrast, given the very high stability of DNA at high
temperatures and its ability to refold upon hydration, it should be feasible to distribute aptamer-based lateral flow tests to very
low resource settings and highly sub-optimal field conditions with limited loss in performance.
#2. Aptamer “sandwich” pairs can be readily identified by high-throughput screening.
For most lateral flow applications, a “sandwich” format is desired. This allows a capture agent and a detection agent to bind the
analyte on two non-competing epitopes thereby facilitating a readout that is directly proportional to the analyte concentration (in
contrast to a competitive lateral flow assay [6]).
In addition to selection strategies that can facilitate the identification of aptamer pairs [7], at Base Pair, we have developed a
proven proprietary, high-throughput post-screening method to identify complementary aptamers binding separate epitopes on
the same target protein in less than 2 weeks. Customers willing to sign a non-disclosure agreement are welcome to review example data demonstrating the power of this process.
#3. Aptamers can be selected to bind difficult targets under non-blood conditions.
Abs sometimes cannot be developed to small molecules, peptides, or other “difficult” targets which are not detrimental to aptamer selection. Antibody generation is also limited to physiological pH, salt, and background protein conditions. For many
desired assays, however, other binding conditions such as urine, saliva, or environmental samples would be preferred, and it
can be difficult to identify a conventionally-selected antibody that can perform under the desired conditions. Base Pair has selected a number of aptamers to difficult targets that bind under the denaturing conditions of urine or other “non-blood” conditions. For more available information on the development of aptamers to small molecules, please see our publications.
8058 El Rio St., Houston, TX 77054 • basepairbio.com • [email protected]
Top 5 Reasons for Choosing Aptamers in Lateral Flow Assays
#4. Aptamers allow very well-defined, single-site conjugation chemistries.
Unlike antibodies, which are usually post modified through multiple lysine residues (via primary amine), aptamers can be
readily synthesized with a single biotin, thiol, or other reactive groups for well-defined, single-site conjugation. Numerous
groups have reacted aptamers having 3’- or 5’-thiol termini with gold surfaces or gold nanoparticles, an excellent example
in the case of lateral flow was demonstrated by Xu et al. in 2009 in which a sandwich, aptamer lateral flow assay was developed for the target thrombin using two well-studied aptamers [8]. Obviously, synthetic single site conjugation removes
degradation and uncertainty regarding heterogeneous distribution of labels which can complicate the development of quantitative assays. In general, experts in the lateral flow field concur that these assays are moving from qualitative, Yes/No
tests towards quantitative assays with digital readout [9].
#5. Aptamers can be tailored to have very fast on-rates during their selection.
In the lateral flow application, the capture antibody especially will have a very short allowed binding time (typically seconds
or less). All antibodies produced by conventional immunization are “selected” under the physiological conditions of the
blood in the immunized animal and there is no control for the developed kinetics (kon and koff) of the developed Abs. Thus,
polyclonal antibody preparations will represent an ensemble mixture of kinetic behavior, and even monoclonal Abs to be
utilized in lateral flow need to be screened for proper binding behavior. In contrast, aptamers are selected by a purely in
vitro process, very short allowed binding times can be incorporated in the aptamer selection procedure itself thus producing
aptamers with very fast on-rates. We have successfully utilized this strategy at Base Pair to produce aptamers with very
desirable characteristics for lateral flow applications for a number of customers without the need to screen numerous
clones.
References:
1. Wang C, Zhang L, Shen X: Development of a nucleic acid lateral flow strip for detection of hepatitis C virus (HCV) core antigen. Nucleosides Nucleotides Nucleic Acids 2013, 32:59–68.
2. Liu G, Mao X, Phillips JA, Xu H, Tan W, Zeng L: Aptamer-nanoparticle strip biosensor for sensitive detection of cancer cells. Anal.Chem 2009,
81:10013–10018.
3. Liu J, Mazumdar D, Lu Y: A simple and sensitive “dipstick” test in serum based on lateral flow separation of aptamer-linked nanostructures.
Angew. Chem. Int. Ed. Engl 2006, 45:7955–7959.
4. Chiodini PL, Bowers K, Jorgensen P, Barnwell JW, Grady KK, Luchavez J, Moody AH, Cenizal A, Bell D: The heat stability of Plasmodium lactate
dehydrogenase-based and histidine-rich protein 2-based malaria rapid diagnostic tests. Trans. R. Soc. Trop. Med. Hyg.2007, 101:331–337.
5. Lee LG, Nordman ES, Johnson MD, Oldham MF: A Low-Cost, High-Performance System for Fluorescence Lateral Flow Assays. Biosensors 2013,
3:360–373.
6. Lateral flow test. Wikipedia, the free encyclopedia 2013.
7. Gong Q, Wang J, Ahmad KM, Csordas A, Zhou J, Nie J, Stewart R, Thomson JA, Rossi JJ, Soh HT: Selection Strategy to Generate Aptamer Pairs that
Bind to Distinct Sites on Protein Targets. Anal Chem 2012, 84:5365–5371.
8. Xu H, Mao X, Zeng Q, Wang S, Kawde A-N, Liu G: Aptamer-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for
Protein Analysis. Analytical Chemistry 2009, 81:669–675.
9. Jackson GW: Rapid Discovery of Aptamer Affinity Reagents and Their Use in Lateral Flow Devices. In Lateral Flow – The Next Generation Practicalities
of developing and manufacturing quantitative assays June 25-27, 2013 in Irvine CA. 2013.
8058 El Rio St., Houston, TX 77054 • basepairbio.com • [email protected]