Author’s Accepted Manuscript Replacing Antibodies With Aptamers In Lateral Flow Immunoassay Ailiang Chen, Shuming Yang

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To appear in: Biosensors and Bioelectronic Received date: 3 March 2015 Revised date: 12 April 2015 Accepted date: 13 April 2015 Cite this article as: Ailiang Chen and Shuming Yang, Replacing Antibodies With Aptamers In Lateral Flow Immunoassay, Biosensors and Bioelectronic, This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Replacing antibodies with aptamers in lateral flow immunoassay

Ailiang Chena,b,, Shuming Yanga,b

aInstitute of Quality Standards and Testing Technology for Agro-products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing, 100081, China bKey Laboratory of Agri-food Quality and Safety, Ministry of Agriculture, Beijing, 100081, China Abstract: Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential enrichment (SELEX) with high sensitivity and specificity. Aptamers show remarkable advantages over antibodies due to the nucleic acid nature and target-induced structure-switching properties and are widely used to design various fluorescent, electrochemical, or colorimetric biosensors. However, the practical applications of aptamer-based sensing and diagnostics are still lagging behind those of antibody-based tests. Lateral flow immunoassay (LFIA) represents a well established and appropriate technology among rapid assays because of its low cost and user-friendliness. The antibody-based platform is utilized to detect numerous targets, but it is always hampered by the antibody preparation time, antibody stability, and effect of modification on the antibody. Seeking alternatives to antibodies is an area of active research and is of tremendous importance. Aptamers are receiving increasing attention in lateral flow applications because of a number of important potential performance advantages. We speculate that aptamer-based LFIA may be one of the first platforms for

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commercial use of aptamer-based diagnosis. This review first gives an introduction to aptamer including the selection process SELEX with its focus on aptamer advantages over antibodies, and then depicts LFIA with its focus on aptamer opportunities in LFIA over antibodies. Furthermore, we summarize the recent advances in the development of aptamer-based lateral flow biosensing assays with the aim to provide a general guide for the design of aptamer-based lateral flow biosensing assays. Key words: Aptamer; Antibody; Lateral flow; Immunochromatography; Strip test

Contents 1. Aptamer ................................................................................................................................. 3 1.1. Aptamer preparation by SELEX ...................................................................................... 3 1.2. Aptamer applications ..................................................................................................... 5 1.3. Comparison of aptamers with antibodies ...................................................................... 7 2. Lateral flow immunoassays ................................................................................................... 9 2.1. Principles of lateral flow assays ...................................................................................... 9 2.2. Advances in lateral flow immunoassays....................................................................... 11 3. Antibodies' limitations and aptamers' opportunities in LFIA .............................................. 13 4. Designing aptamer-based LFIA ............................................................................................ 20 4.1. Sandwich format .......................................................................................................... 21 4.2. Competitive (or inhibition) format ............................................................................... 25 4.3. Signal amplification for aptamer hybridization based LFIAs ........................................ 29 4.4. Other formats of LFAA .................................................................................................. 31 5. Future Prospect ................................................................................................................... 34 Acknowledgements ................................................................................................................. 35 References ............................................................................................................................... 35 Abbreviations: OTA: ochratoxin A; GNP: gold nanoparticle; QD: quantum dot; LF: lateral flow; LFIA: lateral flow immunoassay; cDNA: complementary single-stranded DNA; NC: nitrocellulose membrane; SELEX:


systematic evolution of ligands exponential enrichment; AFB1: aflatoxin B1, LFAA: lateral flow aptamer assay; PCR: polymerase chain reaction; POCT: point-of-care testing; DNA: deoxyribose nucleic acid; ELISA: enzyme-linked immunosorbent assay; ICA: immunochromatographic assay. LMW: low-molecular-weight.

1. Aptamer 1.1. Aptamer preparation by SELEX

Aptamers are short single-stranded DNA or RNA (often DNA) ligands that can adopt specific three-dimensional conformations to combine with target analytes with high specificity and affinity in a similar way to antibodies (Ellington and Szostak 1990; Tuerk and Gold 1990). Aptamers usually vary in length from 10 to 100 bases, and their typical structural motifs include stems, internal loops, purine-rich bulges, hairpin structures, hairpins, pseudoknots, kissing complexes, and G-quadruplex structures. Aptamers can bind to a wide variety of targets after undergoing adaptive conformational changes and three-dimensional folding. They can be selected from a combinatorial DNA library by in vitro selection termed systematic evolution of ligands exponential enrichment (SELEX) (Gopinath 2007; Zhu et al. 2014). Generally, the DNA library is synthesized as a large pool (1014-1018) of single-stranded oligonucleotides with 20-70 bp random sequences in the middle and constant primer binding sites at both ends. The SELEX process comprises the following steps (Fig. 1): incubation of the interest target with the random DNA library; isolation of the DNA that binds to the target; DNA amplification by PCR; preparation of single-stranded DNAs for the next-round library; repeat of steps 1–4 for 5 to 15 cycles involving interval counter screening with non-target substances to remove nonspecific ssDNAs; finally cloning and sequencing of the enriched library. 3

Fig. 1. Typical aptamer selection (SELEX) process.

This is easier said than done. Many researchers found it inefficient to obtain a good aptamer with high specificity and sensitivity, especially for small-molecule targets that have few sites for aptamer binding or immobilization before SELEX. Such inefficiency is attributed to limited sequence diversity in the library, PCR bias (Hoon et al. 2011; Tsuji et al. 2009), and lack of methods for monitoring the SELEX process (Schutze et al. 2010). Over the past 25 years, the aptamer selection performance has been improved with many refinements and modifications, which make the selection highly efficient, very rapid, cost- and labor-effective, highly stable, and specific. The milestones in technology innovation of SELEX includes Cell-SELEX (Daniels et al. 2003), CE-SELEX (Mendonsa and Bowser 2004), NON-SELEX (Berezovski et al. 2006), MonoLEX (Nitsche et al. 2007), M-SELEX (Lou et al. 2009), X-aptamer (He et al. 2012), SEL-Seq (Bawazer et 4

al. 2014), and particle display (Wang et al. 2014; Zhu et al. 2014). Besides, in order to increase the aptamer potency by enhancing nuclease resistance or to increase the target affinity by providing more target recognition functionalities or increasing the diversity of oligonucleotide libraries, various modifications of nucleotides are introduced into the pre- or post-SELEX (also named Mod-SELEX) process, including 2′-amino pyrimidines, 2′-fluoro pyrimidines, 2′-O-methyl nucleotides, 5-modified pyrimidines, as well as locked nucleic acids (Keefe and Cload 2008; Liu and Li 2014; Schmidt et al. 2004; Shigdar et al. 2013; Yang et al. 2011). For example, Macugen® and several other aptamers currently in clinical development are generated using Mod-SELEX with the libraries containing 2′-fluoro-pyrimidines (Esposito et al. 2011; Yang et al. 2011). Although much effort has been made to improve the efficiency of aptamer SELEX, new technologies are highly required to achieve efficient and rapid aptamer selection. High-throughput sequencing, digital PCR, and computational selection of aptamers may continuously introduce improved aptamer selection methods, which are exemplified by recent works of Hoon et al. and Harrison et al. (Harrison et al. 2013; Hoon et al. 2011). There are now also some alternatives for increasing the selection efficiency. Companies such as BasePair Biotechnologies (Houston, TX, USA) and BioAptus (Belo Horizonte, Brazil) have developed methods for rapidly screening aptamer libraries and generating highly purified and high-affinity aptamers with a short turnaround time, greatly decreasing the reagent cost. 1.2. Aptamer applications

To date, various aptamers have been identified for hundreds of targets ranging from small ions (e.g. Zn2+ (Ciesiolka et al. 1995) and K+ (Qin et al. 2010)) and small organic compounds (e.g. organic dyes (Wilson and Szostak 1998), neutral disaccharides (Yang et al. 1998), and antibiotics 5

(Kwon et al. 2014; Song et al. 2012)) to large molecules like glycoproteins (such as CD4 (Zhao et al. 2014)) or even more complex targets (e.g. whole living cells (Cerchia et al. 2009)). For example, ethanolamine (HOCH2CH2NH2) reported by Mann et al. that has only four non-hydrogen atoms and may cause several diseases is possibly the smallest aptamer target discovered so far (Mann et al. 2005). The Ellington laboratory has developed the website, a comprehensive aptamer database that collects and organizes all known information about aptamers. About 60 biotech and pharmaceutical companies are offering aptamer (synthetic antibody) products and services, among which the following companies should be given more attention: AM Biotechnologies, LLC (U.S.), Aptagen, LLC (U.S.), Aptamer Sciences, Inc. (S. Korea), Aptamer Solutions, Ltd. (U.K.), Aptitude Medical Systems, Inc. (U.S.), Base Pair Biotechnologies (U.S.), Bioapter S.L. (Spain), Integrated Biotechnologies, Inc. (U.S.), NeoVentures Biotechnology, Inc. (Canada), NOXXON Pharma AG (Germany), OTC Biotech (U.S.), Regado Biosciences, Inc. (U.S.), and TriLink BioTechnologies (U.S.).

Various aptamer-based bioassays including fluorescent, colorimetric, and electrochemical methods have been developed and extensively adopted for an impressively wide variety of applications in clinical, medical, environmental monitoring, and food analysis (Dong et al. 2014; Santosh and Yadava 2014; Wang and Farokhzad 2014; Wu et al. 2014; Yoshida et al. 2014). Especially, as a small-sized non-immunogenic molecule, the aptamer and its functionalized products have been extensively used for targeted drug delivery, molecular diagnostics, imaging and tracking systems in clinical practice, biosensor systems, and biomarker discovery (Blank and Blind 2005; Sundaram et al. 2013; Zhou and Rossi 2011; Zhou and Rossi 2014). Indeed, the 6

VEGF165 aptamer has been approved as a drug for age-related macular degeneration, and several other aptamers have been tested in clinical trials (Yoshida et al. 2014). In July 2013, the latest comprehensive report entitled "Aptamers Market-Technology Trend Analysis By Applications-Therapeutics, Diagnostics, Biosensors, Drug Discovery, Biomarker Discovery, Research Applications with Market Landscape Analysis -Global Forecasts to 2018" was commercially published by MarketResearch ( The published book described promising aptamers in the clinical trial pipeline and predicted that these synthesized chemical antibodies would soon take precedence over monoclonal antibodies in therapeutics, diagnosis, and imaging. It also claimed that the global aptamer market was valued at $287 million in 2013 and would reach $2.1 billion by 2018.

1.3. Comparison of aptamers with antibodies

Aptamers have been proposed as a potential substitute for conventional antibodies due to their similar recognition properties. Apart from this, as a chemical antibody, nucleic acid aptamer has some superior characteristics such as small size, high stability (dehydrated form), lack of immunogenicity, easy chemical synthesis, adaptive modification, and cell-free evolution (Table 1). For bioanalysis applications, aptamers are suitable for nearly all antibody-based designs, in which both aptamers and antibodies can be used as molecular recognition elements. For example, the ELISA-like sandwich format assay of thrombin has been developed by using two thrombin aptamers that recognize different epitopes (Ikebukuro et al. 2005; Park et al. 2014; Ruslinda et al. 2012; Tennico et al. 2010). Label-free aptamer sensors have also been developed, in which aptamers are immobilized on a support and target-binding signals are detected using optical or 7

electrochemical systems such as fluorescence (Shen et al. 2013b), surface plasmon resonance (Bai et al. 2013a), quartz crystal microbalance (Tombelli et al. 2005), cantilever (Bai et al. 2014a; Lim et al. 2014), or a field effect transistor system (Martinez et al. 2009). These aptamer sensors have advantages such as stability and reusability, although similar assays can also be developed using antibodies. Besides, aptamers show more flexibility in designing different sensing formats because they are composed of nucleic acids with intra- and inter-molecular hybridization, enzymatic replication, as well as easy sequence determination characteristics. Possessing these properties, target-induced structural change based aptamer sensors have been developed for homogeneous assays (Lau and Li 2014; Zheng et al. 2012), polymerase- and/or nuclease-combined aptamer sensors have been developed for ultrasensitive assays (Guo et al. 2014; Li et al. 2014; Wu et al. 2015; Zhang et al. 2014; Zhou et al. 2014), and microarray/next-generation sequencing based aptamer sensors have been developed for multiplexed assays (Sassolas et al. 2011; Sosic et al. 2013). For disease therapeutic and diagnostic applications, aptamers have more access to biological compartments and higher bioavailability than antibodies due to their 20- to 25-fold smaller sizes compared with full-sized monoclonal antibodies (Bruno 2013; Meng et al. 2012; White et al. 2000). Aptamers are chemically synthesized and can be easily linked to a wide range of diagnostic agents or chemotherapeutic agents to improve disease diagnosis and treatment efficacies (Shigdar et al. 2013).

Table 1 Comparison of aptamers with antibodies Characteristics

Applicable target Selection Production

Aptamer Any targets from ions to whole cells, including non-immunogenic or toxic targets In vitro selection under a variety of conditions

Antibody Common proteins or haptens. Difficult for non-immunogenic or toxic targets

Limited to physiologic conditions by animal immunization Efficient with chemical Time-consuming and costly 8

synthesis with a low cost Batch activity Uniform Redox-insensitive. Difficult to aggregate due to lack of large Stability hydrophobic cores. Tolerant of (or able to recover at) pH and temperature. Long shelf life. Tolerant of transportation without Shelf life any special cooling requirements. In a low nanomolar to Affinity picomolar range Detection Better range Easily modified with other Modification active groups in scale with a low cost Good reusability through a Reusability reversible conformation switch Oriented Easy through various immobilization modifications All molecular recognition element based approaches Nucleic acid amplification based Sensing design ultrasensitive assays Target-induced structural change based homogeneous assays immunogenicity and In vivo Low high bioavailability due application to a small size

Varied Redox-sensitive. Easy to aggregate. Sensitive to (or unable to recover at) pH and temperature.

Short shelf life. Requiring a continuous cold chain. In a low nanomolar to picomolar range Good Difficult and expensive to be modified Poor reusability due to irreversible conformation changes Difficult through protein A/G All molecular recognition element based approaches / / High immunogenicity and low bioavailability due to a large size

2. Lateral flow immunoassays 2.1. Principles of lateral flow assays

To provide patient-centered healthcare for more people or cut the healthcare cost, the use of point-of-care testing (POCT) has constantly increased over the past 40 years, aiming at delivering cost-effective healthcare to patients near their homes. As the dominant technologies in this review, lateral flow immunoassays (LFIAs), also known as immunochromatographic assays (ICAs) or strip tests (Dzantiev et al. 2014), are currently used for qualitative, semi-quantitative, and to some extent quantitative monitoring in resource-limited or non-laboratory environments.


The LFIA biosensing platform mainly comprises the sample pad, conjugated pad, test pad, absorbent pad, and backing pad (Fig. 2). The absorbent pad provides the driving force based on the capillary effect, and the backing pad provides a certain mechanical support for the device. The test pad, which is generally composed of nitrocellulose (NC) membrane, provides a platform for both reaction and detection. Capturing molecules (e.g., antibodies) can be deposited on the NC membrane to form a test zone and a control zone by electrostatic interaction, hydrogen bonds, and/or hydrophobic forces. The conjugated pad is generally composed of glass fiber, on which recognition labels are preloaded and kept stable during their entire shelf life period. The sample pad also commonly consists of glass fiber, which is used to filter specific sample components and can also change the pH value of a sample and release analytes with high efficiency. Every two adjacent components overlap each other by a small part for coordination of the fluid flow. When an assay is performed, a small volume of a sample is applied onto the sample pad, migrates along the conjugated pad, and then carries conjugated particles to the test pad. Analytes in the given sample interact with recognition labels as well as specific biological components, which are then bound at the test line and control line.

Two formats, i.e., sandwich and competitive (or inhibition) formats, are available for LFIAs (Wong and Tse 2009). In the

sandwich format, a recognition label reacts with an analyte of interest (if exists) to form a label–analyte complex, and then the

complex is captured in the test zone via the interaction between the analyte and the corresponding capturing molecules. The

excess free recognition labels exceeding the test zone are then captured by another type of capturing molecules to form a control

line. In the competitive format, a recognition label reacts with capturing molecules deposited in both the test zone and the control

zone. In this manner, the analyte competes for the binding sites with the capturing molecules on the test zone, leading to


non-aggregation of recognition labels in the test zone. In previous studies, both sandwich and competitive format assays were

used for qualitative and quantitative detection of proteins and nucleic acids. In general, sandwich format assays are performed

when there are multiple epitopes in the target analytes, while competitive format assays are preferred when the target analytes

exhibit a low molecular weight or have a single specific antigen.

LFIA incorporates the advantages of homogeneous and heterogeneous analytical methods by combining the speed of homogeneous immunoassay with the separation of reacted and unreacted compounds in a variety of heterogeneous methods. Another advantage of the immunochromatography is that the fluid flow through the carrier (e.g., sorbent and membrane) enables the separation of reacted products from unreacted products without any additional precipitation or washing procedure.

Fig. 2. Typical lateral flow test strip configuration (sandwich format).

2.2. Advances in lateral flow immunoassays

Lateral-flow tests can be conducted with a low-cost, lightweight, and portable tool, but a growing demand for higher sensitivity is challenging the existing formats of this method. For example, cardiac markers, biothreats, and single cell detection require sensitivities that cannot be achieved by the current assays. To address this problem, lateral-flow tests incorporate novel materials in labeling approaches. LFIAs can use different labels such as carbon nanoparticles (Posthuma-Trumpie et al. 2009), latex beads (Bonenberger and Doumanas 2006; Linares et al. 11

2012), selenium (Lou et al. 1993), colloidal carbon (Lonnberg and Carlsson 2001; Posthuma-Trumpie et al. 2008), dye-loaded liposomes (Ho et al. 2008; Pyo and Yoo 2012), and europium chelate-loaded silica nanoparticles (Xia et al. 2009). The latest labels include quantum dots (Berlina et al. 2013; Ren et al. 2014) and upconverting phosphors (Corstjens et al. 2014; Fat et al. 2012; Hampl et al. 2001; Huang et al. 2009). Among all these labels, gold nanoparticles are the most widely used and are still popular in LFIA. The most sought-after property of the gold label is its ability to give color while allowing a constant flow of the test solution through NC membrane. Gold which is inert possesses optical properties, and it is easy to visualize by simple and inexpensive laboratory preparation, stable in liquid or dried form, and easy to conjugate with biological materials. Because all of these, gold is the most preferred label for LFIAs. There has been an industry-wide move to integrate a reader system into a point-of-care (POC) immunoassay system, for which a large proportion is based on the use of fluorescence for reasons largely of sensitivity, availability, and cost. To achieve high sensitivity, non-visual labels such as fluorescent particles can be expected to generate sensitivity 1-2 logs higher than typical colloidal gold or colored visual labels. The direct use of fluorescent labeling can also overcome the low efficiency of particle release from the conjugate pads, which is a major contributor to the standard coefficient of variation in the signal strength of LFIA. The progress in detection/quantification technologies, readout devices, and manufacturing techniques increases the reproducibility and sensitivity of lateral-flow assays while maintaining the ultimate attractiveness of rapid tests: sample-to-answer in a single step (Ge et al. 2014). With technologies established to synthesize reagents and relatively inexpensive equipment required to fabricate test strips (dispensers, cutters, and laminators), to produce 12

immunochromatographic kits is relatively easy in various applications such as veterinary medicines, food and beverage manufacturing, pharmaceutical, medical biologics, personal care product manufacturing, environmental remediation, and water utilities. Lateral flow immunoassays have been used to detect the levels of various hormones in blood and serum (Jeong et al. 2013; Tripathi et al. 2012) and heavy metals like Hg, Ar, and Pb in the environment (Chai et al. 2010; Knecht and Sethi 2009). The assays are also used for rapid onsite plant pathogen detection (Braun-Kiewnick et al. 2011; Zhao et al. 2011), toxicity and adulterant detection in both raw and cooked food substances (Bai et al. 2013b; Blazkova et al. 2009; Dzantiev et al. 2014; Lai 2014; Singh et al. 2015), detection of industrial water toxicity of different contaminants (Concejero et al. 2001; Hua et al. 2012; Khreich et al. 2010), onsite detection of various plant viruses, and detection of many human infectious diseases like syphilis (Yang et al. 2013), malaria (Gillet et al. 2010), and tuberculosis (Denkinger et al. 2013; Mdluli et al. 2014). Over the past 30 years, lateral flow technologies have been developing. According to Rosen and O’Farrell (2011), more than 100 companies in the world are carrying out a wide range of tests with the total market value of about US$3360 in 2010. With a compounded annual growth rate (CAGR) of 7%, the global market is expected to reach $4675 million by 2015.

3. Antibodies' limitations and aptamers' opportunities in LFIA Many efforts have been made for lateral flow immunoassays to improve the performance of existing technologies including better sensitivity, reproducibility, quantification, and multiplexing capability. With continuously improved materials, reagents, approaches, manufacturing equipment, and manufacturing processes, LFIAs have evolved into a true laboratory-based system 13

when a whole new generation of facilitative technologies is introduced. LFIAs utilize the lateral flow strip as a component of a highly specific platform, with advanced facilitative technologies such as readers, specifically designed sample handling devices, and cartridge approaches with on-board functionality, which is essentially creating laboratory analyzers with the lateral flow format at their hearts. Even though many advances have been made, most of the recent LFIAs that have been extensively applied are based on antibodies used as affinity reagents. However, the utilization of antibodies may encounter some drawbacks to their production, stability, and modification, and therefore other alternative candidates are being searched for. As listed in Table 1, aptamers show some inherent advantages over antibodies in developing rapid assay kits. The antibody limitations (■) and aptamer opportunities (●) in developing LFIAs were listed below.

Most LFIAs require a controlled temperature for storage and suffer from a short shelf life of about 6-24 months for optimal stability. Certain assays are completely intolerant of higher temperatures. This is largely a function for the stability of antibodies used in the system. This temperature control requirement means a high cost on the supply chain and may also prevent tests in high-temperature field environments. This is a major issue in diagnostic applications in the developing world or in, for example, biowarfare, agricultural, or veterinary testing applications in the developed world.

Given the very high stability of DNA at high temperatures and its ability to refold upon hydration, it should be feasible to apply aptamer-based lateral flow tests to very low resource settings and highly sub-optimal field conditions with minimum performance loss. 14

For most lateral flow applications, a "sandwich" format is desired, which allows capture and detection agents to bind to 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). However, it is time- and effort-consuming, and it is sometimes difficult to screen an antibody pair bound to the same target at different sites, especially for a low-weight molecule, which has only one epitope, i.e., one antibody available. In this case, developing a "sandwich" format LFIA is impossible for these analytes.

Compared with the antibody preparation, the strategies for selecting aptamers based on in vitro SELEX from a more than 1014 library facilitate identification of aptamer pairs (Gong et al. 2012). Besides, studies proved that one aptamer could be split into two pieces, which could simultaneously bind to the same target such as thrombin, ATP, theophylline, 17 beta-estradiol, and cocaine (Bai et al. 2014b; Chen et al. 2012a; Chen and Zeng 2013; Cheng et al. 2013; He et al. 2013; Liu et al. 2014; Wu et al. 2010; Zuo et al. 2009). That means even if there is only one aptamer sequence, a "sandwich" biosensor is possible, though such a sandwich format LFAA based on split aptamers has not yet been reported.

High affinity and good specificity are the most important issues to consider during antibody screening. Also, antibody resistance to rough chemical conditions is a key issue to be considered when the antibody works with organic extracts or different samples. Antibody generation is restricted by physiological pH, salt, and background protein conditions as well. For many desired assays, however, other binding conditions such as urine, saliva, or 15

environmental samples would be preferred, and it is difficult to identify a conventionally selected antibody under the desired conditions. Anyway, protein-based antibodies show high sensitivity to denaturing agents, which limit their applications or require complicated sample pretreatment. 

In this respect, nucleic acid based aptamers show more resistance to different chemical buffers such as large ranges of pH, ion strength, and organic reagents. Even if aptamers are denatured, they can refold again, in most cases, after denaturing conditions are removed. They can also bind to their targets under the denaturing conditions, such as urine or other "non-blood" conditions.

The overall rapid assay performance is affected by a number of variables including the antibody quality. This also includes obvious parameters such as the antigen specificity, affinity (as defined by the dissociation constant), and particular binding rate constant for the LFIA. Produced from different animals, polyclonal antibodies are potentially subject to batch-to-batch variations to some degree (Bordeaux et al. 2010; Marx 2013). Even comparable monoclonal antibodies are potentially affected by the decreased production or "drift" and alterations in amino acid composition over long periods (Bordeaux et al. 2010; Marx 2013) because they form in hybridoma cell lines, which may experience changed culture conditions or mutations over time, leading to varied antibody output or composition. That means before each batch of applications with newly prepared antibodies, re-evaluation is required to test the sensitivity, cross-reactivity, stability as well as resistance to sample buffer conditions. It is time- and effort-consuming to perform these screening assays, which 16

involve initial ELISA and subsequent LFIA. Since standardized antibodies defined by their sequences and produced as recombinant proteins have been proposed recently to save millions of dollars and dramatically improve reproducibility (Bradbury and Plückthun 2015), however, it is in any case a long way to meet the expectations. 

In this sense, an aptamer can be replicated by simple chemical synthesis with very high fidelity once a useful DNA sequence of the aptamer is known. All these aptamers show the same quality and are easily quantified. No re-evaluation is needed before a new batch of aptamers comes into use.

The lateral-flow format is the only ubiquitous and universally applicable platform that can be utilized for simple, qualitative, and low-cost POC applications, and it has the capability to be functionalized for highly sensitive, fully quantified, and multiplexed assays. Only low cost is not enough to achieve market penetration in diagnostics, however, the benefits of low-cost and multiplexed diagnostics will be realized upon reaching communities, where they are needed the most. Since high-quality antibodies are limited, the antibodies are highly priced as a kind of rare resources when there is a bulging demand. Except antibodies, all the other materials including the colloid gold and various membranes could be mass produced with a low cost. Now, the antibody has become the bottleneck to reduce the cost of LFIA.

Due to different production ways of aptamers and antibodies, nucleic acid aptamers show great cost advantages over antibodies since aptamers are produced by chemical synthesis while antibodies are produced from animals or cell cultures. Furthermore, because aptamers could readily refold after denaturing conditions are removed, possible 17

regeneration of an aptamer-immobilized biosensor will also reduce the cost (McKeague et al. 2010).

The LFIA performance is affected by the method of reagent immobilization. Oriented immobilization of antibodies (e.g., using protein A or G or species-specific antibodies) can improve the LFIA sensitivity. However, the quality of the antibody involved in the reaction is limited because the maximum amount of the antibody bound to gold particles or immobilized on NC membrane depends on the particle surface area or the membrane adsorption amount, respectively. Worse yet, it is generally impossible to orient the binding of antibodies or to attach to the colloidal gold or NC membrane, indicating that only fewer antibodies could be really functional in the assay.

On one hand, aptamers are much smaller than antibodies and more aptamers can be immobilized on the same GNP or NC membrane surface. On the other hand, aptamers have known sequence information and are easily modified at each end to achieve oriented binding, such as the thiol modification for linking to GNP and biotin-streptavidin modification for immobilization on the NC membrane. The oriented immobilization further increases the number of available aptamers.

The great advantages of LFIA make it attractive to various applications from biomedical diagnosis to food safety detection or environmental analysis. However, many low-molecular-weight (LMW) compounds and metal ions lack their own immunogenicity, and it is difficult to obtain high-affinity antibodies against them even by immunizing animals 18

with their conjugates with polymeric carriers, such as proteins. Also, antibodies cannot be developed into highly poisonous substances through immunization of animals. Therefore, there are many metabolites, drugs, heavy metals, pesticides, and chemical contaminants, which are desperately short of fast sensitive screening methods. 

Nearly any of the substances can be used as targets for aptamer selection in unlimited conditions in vitro and a high-affinity aptamer is more easily selected for a LMW target than an antibody.

Antibodies are usually post-modified through multiple lysine residues (via primary amine), and the heterogeneous distribution of labels can complicate the development of quantitative assays and cause some uncertainty and antibody degradation.

Aptamers can be readily synthesized with a single biotin, thiol, or other reactive group for well-defined single-site conjugation. Obviously, synthetic single-site conjugation facilitates the development of quantitative assays.

In addition to the aptamer opportunities in LFIA mentioned above, an additional advantage of using aptamers is the option of hybridization with complementary DNAs and the deconstruction of hybridization once aptamers meet a target molecule (Chen et al. 2012b). Another advantage is the regeneration by heating or other methods. These advantages make aptamers appealing in the development of low-cost, reusable, and robust analytical methods.


4. Designing aptamer-based LFIA Aptamers have recently attracted considerable attention in diagnostic applications due to their inherent advantages over antibodies. The use of aptamers in the lateral flow technology as an alternative to antibodies is under investigation at the time of writing. Similar to LFIA, there are also two formats, i.e., sandwich and competitive (or inhibition) formats, for lateral flow aptamer assay (LFAA). Beside this, there are also some other aptamer-based LF methods with distinctive nucleic acid characters. This section summarizes recent advances in this field and attempts to provide general guidance on how to design such assays (Table 2).

Table 2 Developed lateral flow aptamer assay (LFAA) Target



Test Zone

Control Zone

Limit Detection

Thrombi n

Sandwic h

Primary aptamer-G NP

Streptavidin-biotincapture aptamer

Streptavidin-biotin -cDNA to primary aptamer

2.5 nM

Ochrato xin A

Competiti Aptamer-G ve NP

Ochrato xin A

Competi tive

Aptamer-Q D

E. coli

Sandwic h

Aptamer-G NP, Aptamer-Q D

Aflatoxi n B1

Competi tive

Cy5-modifi ed DNA probes and biotin-apta mer


(Xu et al. 2009 a) Streptavidin-biotin- Streptavidin-biotin 1 ng mL-1 (Wan cDNA -PolyT 0.18 ng mL-1 g et (with a al. reader) 2011 b) Streptavidin-biotin- Streptavidin-biotin-PolyT 1.9 ng mL-1 (Wan cDNA g et al. 2011 a) Amino end-labeled Anti-digoxigenin 3,000-6,000 E. (Bru capture aptamers antibody coli cells, no 300-600 E. 2014 coli cells (with ) a reader) Streptavidin Anti-cy5 antibody 0.1 ng/ml (Shi m et al. 2014 ) 20

HCV core antigen

Competi tive

Aptamer-G NP


Streptavidin-biotin -PolyT

100 pg/mL; 10 pg/mL (with a reader)

Ramos cells

Sandwic h

Primary aptamer-G NP

Streptavidin-biotincapture aptamer

Streptavidin-biotin -cDNA to primary aptamer

Thrombi n

Sandwic h

Aptamer-G NP


Streptavidin-biotin -cDNA to primary aptamer

4000 Ramos cells; 800 Ramos cells (with a reader) 0.25 nM

Adenosi ne

Aptamer-G NP aggregates



20 µM


Aptamer-G NP aggregates



20 µM

4.1. Sandwich format

As shown in Fig. 3, Xu (Xu et al. 2009a) and co-workers developed a dry-reagent lateral flow biosensor for visual detection of thrombin according to the conventional sandwich format. Two aptamers were bound to thrombin at two different sites. The thiolated primary aptamer conjugated to gold nanoparticles through the S-Au binding was used as a recognition label and loaded on the conjugated pad. The biotinylated secondary aptamer was used as a capture aptamer immobilized on the test line through streptavidin. When the detection aptamer was dipped into a solution containing thrombin, thrombin would be migrated by capillary action and bind to gold NP-aptamer conjugates. The complexes continued to migrate along the strip and were captured by the secondary aptamer via interaction between the secondary aptamer and 21

(Wan g et al. 2013 ) (Liu et al. 2009 ) (She n et al. 2013 a) (Liu et al. 2006 ) (Liu et al. 2006 )

thrombin. A characteristic red band could be observed because of the accumulation of gold NPs in the test zone (Fig. 3B). In other words, the more target thrombin was in the solution, the stronger intensity was on the test line. The excess gold NP-primary aptamer conjugates passing the test line were captured in the control zone via hybridization events between the control DNA probes (pre-immobilized in the control zone) and the primary aptamer, thus forming a second red band (Fig. 3B, top). In the absence of thrombin, the red band was observed only in the control zone, but not in the test zone (Fig. 3B, bottom). The result was obtained by recording the optical response with a strip reader after 10 min, and the response of the biosensor was linear over the range of 5-100 nM for thrombin with a detection limit of 2.5 nM. This study also compared aptamer-based strips with antibody-based strips, and the result showed that the former exhibited similar sensitivity and even better specificity. The sensor was also capable of detecting human plasma samples.

Fig. 3. Design principle of a sandwich lateral flow sensor for thrombin detection. (Reproduced with permission from ref. (Xu et al. 2009a) Copyright 2009, American Chemical Society.)


Liu et al. developed an aptamer-nanoparticle sandwich lateral flow device using a pair of aptamers capable of specifically binding to Ramos cells (Liu et al. 2009) (Fig. 4). Similar to Xu's work, a thiolated aptamer is immobilized on the GNPs, and a biotinylated aptamer is immobilized in the test zone of NC membrane through streptavidin. Interacting with the aptamer probes of the Au-NP-aptamer conjugates, Ramos cells form Au-NP-aptamer-cell complexes and continue to move along the test strip. Then, a large number of Au-NPs accumulate in the test zone and produce a distinctive red band, which can be used for qualitative (visual) evaluation or quantitative cell detection with a portable strip reader. A biotinylated DNA probe, which is complementary to the aptamers immobilized on the GNPs, is immobilized in the control zone to capture excess Au-NP-aptamers and generate a second red band. Under the optimal conditions, the LFAA was able to detect a minimum of 4000 Ramos cells by visual detection without instrumentation and 800 Ramos cells with a portable strip reader within 15 min.

Fig. 4. Design principle of a sandwich lateral flow sensor for Ramos cell detection. (Reproduced with permission from ref. (Liu et al. 2009) Copyright 2009, American Chemical Society.) 23

In the method mentioned in section 3.1, a DNA probe (complementary to the aptamers immobilized onto the GNPs) was immobilized in the control zone to hybridize with residue aptamers on the GNPs or QDs. However, it is often difficult to obtain a strong and reliable control line since a sample solution can quickly pass the membrane within 10 min, which is not enough for hybridization on the membrane. Wei et al. (Wei et al. 2005) reported that a minimum of 500s was required for hybridizing DNAs on a microarray using a microfluidic device in a liquid solution. To address this problem, Bruno (Bruno 2014) adopted a dual-labeled aptamer approach, with which one end was labeled with biotin binding to streptavidin conjugated on the GNP and the other end was labeled with digoxigenin (Fig. 5). Therefore, the residue aptamer-GNP or QD could be rapidly captured by an anti-digoxigenin antibody immobilized on the membrane and a consistently strong and reliable control line was obtained. In this system, the amino end-labeled capture aptamers were covalently immobilized on the analytical NC membrane by exposure to 254 nm ultraviolet (UV) light for 15 min. The end labeled with the amino group may provide a useful tethering point, from which the residue aptamers can protrude vertically away from the membrane surface and capture bacteria when they pass through the capture zone. The developed sandwich format aptamer-based LF assay shows a visible LOD of ~3,000-6,000 E. coli cells in the buffer by using aptamer-conjugated colloidal gold. The LOD could be reduced to ~300-600 bacterial cells per test by switching to a Qdot 655 aptamer-based LF system.


Fig. 5. Design principle of a sandwich lateral flow sensor for bacterium detection. (Reproduced with permission from ref. (Bruno 2014) Copyright 2014, licensee MDPI, Basel, Switzerland.)

4.2. Competitive (or inhibition) format

Ochratoxin A (OTA) is a type of small molecular mycotoxin with only one aptamer. Wang et al. (Wang et al. 2011b) developed a competitive (or inhibition) lateral flow biosensor based on aptamer-linked gold nanoparticles for visual detection of ochratoxin A (Fig. 6). In this system, the thiol-modified OTA aptamer with a polyA tail was conjugated on the colloidal gold surface. The biotin-modified complementary strand named DNA probe 1 was immobilized through streptavidin binding onto the test zone of the NC membrane. The biotin-modified polyT named DNA probe 2 was immobilized through streptavidin binding onto the control zone of the NC membrane. The aptamer-based strip assay was implemented by the competitive reaction between DNA probe 1 (test line) and the target OTA to combine with aptamers. Once found in the detection solution, OTA would combine with the aptamer-GNP probe, decreasing the amount of aptamer-GNP that could hybridize with DNA probe 1 on the test line and causing the red color intensity to become weaker. In other words, the more target OTA was in the solution, the weaker intensity was on the test line. Regardless of the presence of the target OTA in the detection 25

solution, the aptamer-GNP probe would definitely hybridize with DNA probe 2 on the control line, ensuring the detection validity. The test results indicated that the sensitivity of the aptamer-based test strip was higher than that of the conventional antibody-based strip. The visual LOD of the strip was 1 ng/mL for qualitative detection while the LOD could go down to 0.18 ng/mL for semi-quantitative detection by using a scan reader. Wang et al. (Wang et al. 2011a) also developed a similar fluorescent lateral flow strip for OTA detection by replacing GNPs with quantum dots, with which a LOD of 1.9 ng mL-1 was obtained. The sensitivity by using QDs was slightly lower than that by using GNPs, which may be attributed to other test conditions because the authors said that the QD-aptamer-based fluorescent strips exhibited slightly higher sensitivity than the gold-nanoparticle-labeled strips in the same paper. All the detection results could be achieved within 10-15 min, demonstrating a potential use of the aptamer-based strip in rapid onsite detection.


Fig. 6. Design principle of a competitive lateral flow sensor for OTA detection. (Reproduced with permission from ref. (Wang et al. 2011b) Copyright 2010, Elsevier B.V.)

Using the same principle, Wang et al. (Wang et al. 2013) developed a highly sensitive aptamer-based strip method based on the competitive reaction between DNA probe 1 (test line) and the HCV core antigen to combine with aptamers. The lower LOD of the test strip was calculated to be 10 pg/mL with a scanner and 100 pg/mL with naked eyes. The results indicated that ELISA and strip assay were well agreed with the measured values.

The hybridization deficiency also lies in competitive LFIAs, in which the aptamer-label conjugates 27

unbound to the analyte were hybridized with the cDNA immobilized on the NC membrane. To address this problem, as a modified LFIA, the dipstick assay is also widely accepted, which usually includes two steps, pre-reaction with a target molecule and a receptor (antibody and aptamer) and dipping a dipstick, which involves an absorbent pad and membrane treated with capture reagents that can bind to the receptor. At the pre-reaction step, sufficient hybridization of the aptamer and the cDNA was formed for negative samples whereas the interaction of the aptamer with the target analyte for positive samples was performed in a liquid solution. When a dipstick was dipped into the mixtures, the complexes (target aptamer and hybridized aptamer/cDNA) were trapped by the capture reagents immobilized on the membrane. Using this approach, Shim et al. (Shim et al. 2014) developed a rapid and simple aptamer-based dipstick assay for determination of aflatoxin B1 (AFB1) (Fig. 7). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between the target and the cy5-modified DNA probes. Streptavidin and the anti-cy5 antibody were immobilized as capture reagents on the test and control lines on the dipstick assay membrane. After optimization, the LOD for the dipstick assay was 0.1 ng/ml with AFB1 in the buffer.


Fig. 7. Design principle of a competitive lateral flow sensor for AFB1 detection. (Reproduced with permission from ref. (Shim et al. 2014) Copyright 2014, Elsevier B.V.)

4.3. Signal amplification for aptamer hybridization based LFIAs

High sensitivity is often required for clinical diagnosis, food monitoring, and chemical and biological researches. The sensitivity of the LFIA methods needs to be improved by using aptamers. Shen et al. (Shen et al. 2013a) designed a dual gold nanoparticle conjugate based signal enhancement method in a lateral flow immunoassay, in which more GNPs are captured on the test line, giving a better detection limit. As shown in Fig. 8, the GNPs with a diameter of 30 nm (1st GNP) were modified with DNA1 to form a conjugate (1st GNP-DNA1), and the GNPs with a diameter of 30 nm (2nd GNP) were modified with both DNA2 and the aptamer to form another conjugate (2nd GNP-DNA2/aptamer). The 1st GNP-DNA1 conjugate was designed to bind only to 29

the 2nd GNP-DNA2/aptamer conjugate via hybridization between DNA1 and DNA2, whereas the 2nd GNP-DNA2/aptamer conjugate was also able to combine with the analyte for a sandwich assay. The biotin labeled polyT named DNA3 was immobilized in the control zone to capture excess 1st GNP-DNA1/2nd GNP-DNA2/aptamer complexes via hybridization between DNA3 and polyA tail ended with the aptamer. Using this strategy, the detection limit was improved 30 times and could detect the thrombin concentration in the range of 0.5-120 nM with a detection limit of 0.25 nM.

Fig. 8. Design principle of an aptamer hybridization based lateral flow sensor for thrombin detection. (Reproduced with permission from ref. (Shen et al. 2013a) Copyright 2013, The Canadian Society of Clinical Chemists.)


4.4. Other formats of LFAA

Using aptamer-linked AuNPs, Liu et al. (Liu et al. 2006) developed a simple LFAA for adenosine detection (Fig. 9). In this system, adenosine-responsive nanoparticle aggregates were prepared, which contain two kinds of DNA-functionalized gold nanoparticles (particles 1 and 2 in Fig. 9a) and an aptamer DNA. The detailed DNA sequences, modifications, and linkages are shown in Fig. 9b. Two kinds of thiol-modified DNAs were used to functionalize particle 2, i.e. biotinylated and non-biotinylated. The biotin modification (N) allowed the nanoparticles to be captured by streptavidin. The aptamer-linked nanoparticle aggregates were spotted on the conjugation pad, and streptavidin was applied on the membrane as a thin line (Fig. 9c). If the wicking pad of the device is dipped into a solution, the solution will move up along the device and rehydrate the aggregates. In the absence of adenosine, the rehydrated aggregates will migrate to the bottom of the membrane and stop due to their large sizes (Fig. 9d). In the presence of adenosine, the nanoparticles will be disassembled due to the binding of adenosine by the aptamer (Fig. 9a). The dispersed nanoparticles can then migrate along the membrane and be captured by streptavidin to form a red line (Fig. 9e). Similarly, a cocaine-sensitive device was prepared and applied to human blood serum well (Liu et al. 2006).


Fig. 9. Design principle of a lateral flow aptamer assay for adenosine detection. (Reproduced with permission from ref. (Liu et al. 2006) Copyright 2006, Wiley-VCH.)

It has been demonstrated that some aptamers can be split into two fragments without significant perturbation of their ligand binding abilities (Liu et al. 2010; Xu et al. 2009b; Zhang et al. 2008). Chen et al. (Chen et al. 2012a) split a 15-mer anti-thrombin aptamer and a 27-mer anti-ATP aptamer into two fragments (subunits), respectively. The two subunits of the thrombin aptamer were coupled with the two subunits of the ATP aptamer, respectively, and then two integrated oligonucleotides were obtained. In the absence of the targets, the integrated strands do not interact with each other. In the presence of respective or both targets, however, a tri-component 32

supramolecular aptamer complex is generated on the basis of the target-induced self-assembly of the split aptamer fragments. By appropriate labeling of the aptamer subunit with gold nanoparticles (AuNPs), aptamer subunit−target binding events can be directly visualized with naked eyes based on the strip sensing platform. Based on target-induced self-assembly of split aptamer fragments, Chen et al. firstly developed an LFAA-based two-analyte "OR" and "AND" logic gate for dual detection of thrombin and adenosine (Fig. 10). The simple, easy-to-perform, and cost-effective assay allows portable analysis at ambient temperature. The strip logic system is tolerant to nonspecific interfering agents and can effectively function even in human serum samples. Such logic strips have great promise for applications in intelligent POC and in-field diagnostics.

Fig. 10. Design principle of a lateral flow aptamer assay for protein and small molecule detection. (Reproduced with permission from ref. (Chen et al. 2012a) Copyright 2012, American Chemical Society.)


5. Future Prospect In the past two decades, aptamers has been remarkably developed in analytical chemistry. Aptamers have been shown to be versatile and effective as molecular probes in designing various types of electrochemical (Ho et al. 2012; Liu et al. 2012), fluorescence (Kim et al. 2012; Zhang et al. 2011), chemiluminescence (Freeman et al. 2011), or colorimetric (Zhu et al. 2010) sensing schemes for a broad spectrum of targets with high sensitivity and selectivity, comparable to and sometimes even better than antibody-based assays (Baldrich et al. 2004; Xu et al. 2005; Xu et al. 2009a). Similar to antibodies, aptamers are also a platform technology, making them useful to meet the growing demand for detecting target molecules that have emerged. However, practical applications of aptamer-based sensing and diagnostics, such as home and clinical tests, are still lagging behind antibody-based tests due to two drawbacks. The first disadvantage, common to all aptamer-based sensing methods reported so far, is that the detection still requires professional laboratory-type operations, such as precise transfer of solutions, making it less useful for people who do not have a scientific background. Second, most sensors have low sensitivity for instrument-free observation. For example, to observe a distinct color change with the naked eye, a concentration of 0.5 mM or higher of adenosine is needed for the adenosine sensor, which is approximately 50 times higher than the Kd value (10 mm) of this aptamer (Liu et al. 2006).

The lateral flow technology, as an efficient technique featured rapidity, simplicity, stability, portability, and sensitivity, is one of the most successful platforms for developing bioanalytical assays. Use of aptamers as an alternative of antibodies in gold nanoparticle labeled strip methods has recently achieved satisfactory results. We speculate that this might be one of the first 34

platforms for aptamer-based diagnosis to be commercialized. To achieve this, many issues need to be resolved before aptamers can bring practical impacts. Solving these problems will become a major part of future researches. (1) Sensitivity is a major concern for developing any LFIA. High sensitivity can be achieved by performing signal amplification using polymerases and/or nucleases, longer (up to 200 base) "multivalent" aptamers (Mallikaratchy et al. 2011; McNamara et al. 2008; Zhao et al. 2013), and nanoparticles. (2) The aptamer binding to various membranes and colloidal particles, and long-term stability of DNA conjugates and their flow properties require extensive study and optimization. (3) By using each line of aptamers in the system for an analyte, multiple detection methods should be developed to implement a low-cost and rapid quantitative assay. (4) Despite the common sandwich and competitive format lateral flow, more other formats should be studied based on unique nucleic acid or target induced structure-switching properties of aptamers to improve sensitivity and stability.

Acknowledgements This work was supported by International Science & Technology Cooperation Program of China (2012DFA31140). The authors express their gratitude for the support.

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Figure captions: Fig. 1. Typical aptamer selection (SELEX) process. Fig. 2. Typical lateral flow test strip configuration (sandwich format). Fig. 3. Design principle of a sandwich lateral flow sensor for thrombin detection. (Reproduced with permission from ref. (Xu et al. 2009a) Copyright 2009, American Chemical Society.) Fig. 4. Design principle of a sandwich lateral flow sensor for Ramos cell detection. (Reproduced with permission from ref. (Liu et al. 2009) Copyright 2009, American Chemical Society.) Fig. 5. Design principle of a sandwich lateral flow sensor for bacterium detection. (Reproduced with permission from ref. (Bruno 2014) Copyright 2014, licensee MDPI, Basel, Switzerland.) Fig. 6. Design principle of a competitive lateral flow sensor for OTA detection. (Reproduced with permission from ref. (Wang et al. 2011b) Copyright 2010, Elsevier B.V.) Fig. 7. Design principle of a competitive lateral flow sensor for AFB1 detection. (Reproduced with permission from ref. (Shim et al. 2014) Copyright 2014, Elsevier B.V.) Fig. 8. Design principle of an aptamer hybridization based lateral flow sensor for thrombin detection. (Reproduced with permission from ref. (Shen et al. 2013a) Copyright 2013, The Canadian Society of Clinical Chemists.) Fig. 9. Design principle of a lateral flow aptamer assay for adenosine detection. (Reproduced with permission from ref. (Liu et al. 2006) Copyright 2006, Wiley-VCH.) 40

Fig. 10. Design principle of a lateral flow aptamer assay for protein and small molecule detection. (Reproduced with permission from ref. (Chen et al. 2012a) Copyright 2012, American Chemical Society.)

Highlights: 

Aptamer and lateral flow immunoassay were introduced.

Antibodies' limitations and aptamers' opportunities in LFIA were summarized.

Recent advances in aptamer-based lateral flow assays were reviewed.


Replacing antibodies with aptamers in lateral flow immunoassay.

Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential e...
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