The new age of Aptamer based Biosensors

Transcription

1 The new age of Aptamer based Biosensors There is an increasing interest in developing new biosensors for diagnostics and basic research applications. In light of the emerging aptamer technology, aptamer-based biosensors (Aptasensors) are expected to be one of the most promising devices in bioassay related applications (Han, Liang, and Zhou., 2010). However given this noted promise, the application of aptamer technology in biosensors remains in an early phase of development. As a result, there remains a need to combine aptamers and their potential and biosensors to tackle the numerous challenges of the currently limited conventional electrochemical bioassays and monitoring diagnostics. The advantages of using aptamers in Biosensors Aptamers have been widely recognised as promising recognition elements for biosensor construction. Unlike monoclonal antibodies (mabs), the traditional affinity reagent used in biosensors, aptamers can be isolated to bind to almost any target of choice, from small molecules to whole cells and microorganisms (Han, Liang, and Zhou, 2010). This markedly broadens the applications of the corresponding biosensors to encompass more targets in medical diagnostics, food and environmental analysis, anti-bioterrorism and much more. Recent examples include the development of an aptasensor for detection of one of the most abundant food contaminating mycotoxins namely Ochratoxin A (Hayat et al., 2013), and optimisation of an aptasensor to detect C-reactive protein, a major cardiac disease biomarker as a diagnostic biosensor (Qureshi et al., 2012). Along with an extended target range, aptamers have gained popularity due to their chemical and physical properties. Due to their small size and versatility, aptamer allow efficient immobilisation at high density which is essential for multiplexing for miniaturised systems (Song et al, 2008). Moreover, aptamers once immobilised, are able to release targets upon washing with different buffers, permitting a test to be potentially reused. Aptamer stability is also a crucial aspect for biosensor development and due to their stable chemical structure negate the need for cold storage.

2 Analogous to immunoassays, aptamer-based bioassays can adopt different assay configurations to produce a clear signal. The ease of aptamer conjugation to signal moieties such as such as electrochemical probes, fluorophores and quenchers is made simple via chemical modification of the aptamer, allowing incorporation of amine or thiol groups which facilitates the development of biosensors. Biosensor design and sensing strategies Aptasensors are well-constructed by a variety of methodologies, including electrochemical biosensors, optical biosensors and mass-sensitive biosensors (Han, Liang, and Zhou., 2010). However, the design strategies of most of these biosensors have some similar elements. These strategies can be divided into four modes: 1. Target-induced structure switching mode (TISS). 2. Sandwich like mode. 3. Target-induced dissociation mode. 4. Competitive replacement mode. Target-induced structure switching mode In TISS, the binding of the targets to the immobilised aptamer causes the aptamers to alter configuration from a flexible to rigid tertiary structure such as G-quaduplex. Such conformational switches would change the relative positions of signal moieties, leading to initiation of a signal. Based on this design, several electrochemical aptasensors have been designed using reporters including methylene blue and ferrocene. Examples of biosensors using TISS include the aptamer beacon (Mir, Jenkins, and Katakis et al., 2008) and the target-responsive electrochemical aptamer switch (TREAS) (Zuo et al., 2007). Another reported example features methylene blue incorporation into the aptamer stemloop structure (Fig. 1). Upon target binding, methylene blue is separated from the surface resulting in a change in signal. This method was able to linearly and selectively detect thrombin with a detection limit of 11 nm (Bang et al., 2005).

3 Figure 1. Design of a signal-off electrochemical aptamer biosensor for thrombin detection (Xiao et al. 2005) Besides electrochemical biosensors, TISS strategy could also be utilised to design other classes of biosensors, including aptamer-based molecule beacons which utilise fluorophores to emit a fluorescence signal. This established method has been proven to be effective in direct detection and quantification of targets in complex biological samples (Han, Liang, and Zhou., 2010). Mass-Sensitive strategy Aptamers are often coupled with biophysical measurement devices to detect a change in mass. Frequently used mass-sensitive techniques include surface plasmon resonance (SPR) (Wang and Zhou, 2008), quartz crystal microbalance (QCM) (Min et al. 2008) and surface acoustic wave device (SAW) (Schlensog, 2004). For example, a Love-wave biosensor array was designed to allow label-free, real-time, and quantitative measurements of protein and nucleic acid binding events by coupling aptamers to the surface of a Love-wave sensor chip. Schlensog and colleagues calibrated the biosensor for human α-thrombin and HIV-1 Rev peptide by binding fluorescently labelled molecules and correlating the mass of the bound molecules to fluorescence intensity in which analyte recognition was specific and Detection limits of approximately 75 pg/cm2 were obtained (Schlensog, 2004). Sandwich Mode Sandwich like assays have gained much popularity due to the popularity of sandwich ELISA in

4 the research and diagnostic environment. In this assay type, an aptamer is immobilized onto a surface as a capture probe and a second aptamer is modified with a label as a signal readout probe. The target can be captured on the surface of electrodes by binding to the capture aptamer and then forming a sandwich complex with the second aptamer. During this process, sandwiched binding complexes bring catalytic labels to the surfaces of electrodes to generate colorimetric, voltammetric, impedimetric, or gravimetric signals for detection (Deng et al. 2014). Some protein targets, such as PDGF-BB and thrombin, have dual binding sites, which permits the recognition by two recognition molecules. As shown in Figure 2, an electrochemical biosensor for PDGF detection was constructed using a sandwich structure. Au-NPs mediated signal amplification has been previously constructed whereby an electrochemical probe added to the assay were directly correlated with the concentration of PDGF (Wang et al, 2009). Figure 2. Schematic for the formation of sandwich structure and Au-NP/electrochemical probe detection of PDGF (Wang et al., 2009). Target-Induced Dissociation/Displacement In Target-Induced Dissociation/Displacement (TID) mode the complementary sequence of aptamers are employed as anchors to localise the aptamers. After incubation with targets,

5 the formed target-aptamer complexes will be released into solution leading to changes of detectable signals. Unlike both TISS and sandwich mode, TID does not rely on a structuredependent assay as demonstrated by Han et al. (2009). TID has also been employed in other biosensor type applications such as surface enhanced resonance Raman scattering (SERS) biosensor (Cho et al., 2008) and colorimetric types assays (Zhao et al., 2007). As aptamers share similar applications to antibodies, numerous detection methods that take advantage of antibodies can be developed into aptamer-based methods. For instance, most immunoassays for small molecules are competitive assays relying on the replacement of surface-bound antibodies by the analyte in solution. This replacement mode could be applied to the aptamer-based assays. This has lead to new design strategies which are classed as competitive replacement modes to be developed. A prime example was the development of a fibre optic microarray biosensor using aptamers as receptors as complementary to the conventional ELISA technique (Lee and Walt, 2000). Aptamer based biosensors using nanoparticles Nanotechnology has recently added a new dimension to the analytical and diagnostics field. Conjugation of aptamers on various nanomaterials has led to highly sensitive and selective aptasensors. Nanomaterials possess a number of attractive properties for biosensor development including size/shape-dependent optical properties, easy tuning of surface properties and catalytic ability and therefore are very useful for signal generation and amplification. Examples of nanomaterials used for biosensors include metallic nanoparticles, quantum dots, silica nanoparticles, and carbon nanotubes (Burda et al., 2005), with interest in aptamer incorporation increasing in popularity. Both gold and magnetic nanoparticles are well characterised and can be used to design aptasensors. Whilst AuNPs are easily conjugated to aptamers via use of a thiol group (Pavlov et al., 2004), magnetic NPs e.g. magnetite have the benefit of easy signal amplification via concentration using magnets and are commonly used for separation experiments. Song and co-workers developed rapid and ultra-sensitive aptasensors for the detection of adenosine monophosphate (AMP) based on dual aptamer conjugated particles system (Song et al., 2009).

6 Carbon nanomaterial based Aptasensors Aside from nanoparticles, other types of nanomaterial have been used for biosensors, including single walled carbon nanotubes (SWCNTs). SWCNTs have many similar properties to nanoparticles, but also benefit from a higher surface area, mechanical strength and thermal and electrical conductivity which are utilised for aptasensor development. Lee and co-workers first demonstrated SWCNT- field effect transistor (FET) biosensor using aptamers in which anti-thrombin aptamers were covalently immobilised to SWCNTs. The SWCNT-FET aptasensor was able to detect thrombin as low as 10nM by measuring conductance (So et al., 2005). Similarly, graphene materials including graphene oxide (GO) are one for the most promising nanomaterials for the applications in biosensors due to their unique electronic, optical and mechanical properties (Yao et al., 2012). The combination of graphene materials and aptamers has supplied high potential to develop novel graphene based aptasensors. As highlighted in Figure 3, Florescence resonance energy transfer (FRET) based aptasensors using GO as a quencher for detection of various targets due its intra-sheet energy and hydrophobic nature (Chen et al., 2012). By addition of target the aptamers are displaced from GO nano-sheets by target induced conformational change of aptamers to folded

7 structure resulting in the increase of florescence signal. This was recently reported by Yi and co-workers and applied not only in vitro analysis but also in vivo molecular probing of ATP (Yi et al., 2014). Figure 3. Carbon nanomaterials-based aptasensors. (a) Aptamer-functionalized SWCNT-FET aptasensor and (b) FRET-based aptasensors using GO as quencher using catalytic recycling of analyte for signal amplification (Kim, Raston, Gu., 2015). Conclusions The innovative surge in developing aptasensors has shown remarkable promise as apatsensors have enough demonstrated their merits and potentials in the bioanalysis field. However and in spite of this excellent progress the market of aptasensors and aptamer based diagnostics is not explored yet. Therefore the development of aptasensors for newly emerged targets of importance presents a golden opportunity to rival traditional immunoassays. Aptamer Group The Aptamer group takes a high-throughput approach using liquid handling robotics to identify aptamers against novel and significant targets to develop sensors for potential clinical/commercial use. We are committed to identify and develop new aptamer based biosensors that truly stand out to a much broader range of research laboratories. Aptamer Group s biomarker discovery, diagnostic and therapeutic divisions aim to conduct further research in raising novel aptamers for your target of interest to help develop biosensors that significantly reduce precious research time and cut cost of development. References: Bamrungsap, S., Chen, T., Shukoor, M.I., Chen, Z., Sefah, K., Chen, Y., Tan, W.H., Pattern Recognition of Cancer Cells Using Aptamer-Conjugated Magnetic Nanoparticles. ACS Nano (5), Bang, GS. Cho, S. Kim, BG. A novel electrochemical detection method for aptamer biosensors. Biosens. Bioelectron. 21 (2005) 863.

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