Advances in aptamers-based lateral flow assays

Accepted Manuscript Advances in aptamers-based lateral flow assays Miriam Jauset-Rubio, Mohammad S. El-Shahawi, Abdulaziz S. Bashammakh, Abdulrahman O. Alyoubi, Ciara K. O´Sullivan PII:

S0165-9936(17)30261-3

DOI:

10.1016/j.trac.2017.10.010

Reference:

TRAC 15028

To appear in:

Trends in Analytical Chemistry

Received Date: 16 July 2017 Revised Date:

12 October 2017

Accepted Date: 12 October 2017

Please cite this article as: M. Jauset-Rubio, M.S. El-Shahawi, A.S. Bashammakh, A.O. Alyoubi, C.K. O ´Sullivan, Advances in aptamers-based lateral flow assays, Trends in Analytical Chemistry (2017), doi: 10.1016/j.trac.2017.10.010. 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 proof before it is published in its final 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.

ACCEPTED MANUSCRIPT

ADVANCES IN APTAMERS-BASED LATERAL FLOW ASSAYS Miriam Jauset-Rubio a* Mohammad S. El-Shahawi b, Abdulaziz S. Bashammakh b, Abdulrahman O. Alyoubi b and Ciara K. O´Sullivan a,c* a

To whom correspondence may be addressed, email: [email protected]; [email protected]

SC

*

RI PT

Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira I Virgili, 43007 Tarragona, Spain b Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Kingdom of Saudi Arabia. c Institució Catalana de Recerca I Estudis Avancats, Passeig Lluís Companys 23, 08010 Barcelona, Spain

M AN U

Abstract

TE D

The use of lateral flow assays exploiting antibodies is well established in different fields due to their advantages, which include low cost, ease of production and rapid response, with the only required end-user intervention being sample addition. In recent years, aptamer-based lateral flow assays are garnering increasing interest offering a highly cost-effective and more flexible alternative to antibodies. In this review, an overview of the aptamer-based lateral flow assays developed to date is provided, highlighting the advantages of using aptamers and their ability to be incorporated into formats not possible with antibodies.

Keywords

AC C

EP

Aptamers; Lateral flow aptamer assays; Point-of-care; Point-of-need; Sandwich aptamer assay; Competitive aptamer assay

1. Introduction

Aptamers are nucleic acids that can bind to a wide range of diverse targets, from small molecules to proteins and even cells [1–7]. Aptamers offer several advantages over their antibody counterparts, including their flexibility to adapt to different assay formats. Once aptamers are selected they can be synthesised with high reproducibility and purity and are chemically stable, being able to recover their native conformation following denaturation. Furthermore, aptamers can be selected against specific regions of targets [8,9], which is sometimes difficult for antibodies, since the animalimmune system is inherently generated towards specific epitopes on target molecules. Aptamers possess excellent selectivity and affinity toward their targets, binding with dissociation constants (KD) ranging from picomolar to nanomolar [10,11]. Small variations in the target molecule can disrupt aptamer binding, as exemplified by the P á g i n a 1 | 30

ACCEPTED MANUSCRIPT aptamers for theophylline and L-arginine, which can discriminate closely related chemical structures by factors as high as four orders of magnitude [12,13].

RI PT

Due to their specific ability to undergo conformational changes upon target binding, their high stability, specificity and low cost, they have become increasingly important as recognition elements for analytical tools, including electrochemical and fluorescent biosensors [14–21], colorimetric assays [22,23], surface plasmon resonance assays [24–26] and amplification techniques [27–29]. However, the majority of these methods require trained personnel, expensive instrumentation, and are often laboratory based, limiting their use at point-of-need and point-of-care settings.

M AN U

SC

Lateral flow assays (LFA) are a paper-based platform for the detection of targets that has garnered considerable interest due to its potential to provide results in a matter of minutes, with the only required end-user intervention being sample addition. Due to the low development costs and ease of production, LFAs are well established for pointof-need applications, and have been widely used in diverse fields including biomedicine, quality control, food safety, as well as environmental health [30]. In addition, they can be applied to a range of biological samples including urine [31], saliva [32], sweat [33], serum [34], plasma and blood [35]. The technical basis for LFAs was derived from the latex agglutination assay reported in 1956 by Plotz and Singer [36], but it was not until the early 1980s when a LFA was commercially launched by Unipath, with the first product being the urine-based pregnancy test [37], and since then, hundreds of lateral flow immunoassays have been reported and commercialised.

AC C

EP

TE D

LFA is based on affinity interactions, where a visible line is formed on a test strip when a liquid sample containing a particular analyte of interest is applied. It is composed of different parts (sample pad, conjugate pad, nitrocellulose membrane and wicking or absorbent pad) assembled on a plastic backing pad, which provides mechanical support. The sample pad, transports the sample to other components of the lateral flow strip via wicking. The conjugate pad contains labelled biorecognition molecules, which are released upon contact with the liquid sample as it traverses the membrane by capillary action. Test and control lines are drawn over the nitrocellulose membrane, which is critical in determining the sensitivity of the LFA and should provide optimal and stable binding of capture probes. Finally, the absorbent pad provides the capillary based driving force, which maintains the flow rate of the liquid sample over the membrane and prevents back flow [38] (Figure 1).

Figure 1: Generic representation of lateral flow assays P á g i n a 2 | 30

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

Various lateral flow assay formats have been reported exploiting aptamers as the bioaffinity agent, with the majority being based on either sandwich or competitive assays (Table 1). A sandwich assay is the preferred assay for larger molecules or when there are multiple epitopes in the target and when dual aptamers for the target exist. The competitive format is preferred for low molecular weight targets or targets with a single specific epitope, although it can also be used for larger molecules, and is particularly useful for targets against which only one aptamer exists. In the sandwich format, a labelled reporter molecule reacts with the analyte of interest, and this complex is then captured on the test line by an immobilised capture molecule. The excess of unbound labelled reporter molecules is then captured at the control line. In the competitive format, the target analyte competes with the same target immobilised on the test line, for binding to the labelled reporter molecule, and different approaches have been developed for the control line [39]. Reporter labels used include gold nanoparticles (AuNP), latex spheres and quantum dots (QD). It should be highlighted that the assay specificity will be dependent on the inherent specificity of the aptamer(s) exploited in the assay and this specificity is inferred during the selection process via the inclusion of counter and negative selection steps.

TE D

In the following sections, an overview of the aptamer based lateral flow assays reported to date is presented. Sandwich LFAs exploiting dual aptamers, a combination of antibodies with aptamers or the use of split aptamers is described. Competitive assays exploiting either the aptamer target / complementary DNA immobilised at the test line, which competes with target introduced in the sample for binding to labelled aptamer are detailed and the issue of the control line discussed. Sophisticated assay formats for ultrasensitive detection are also outlined and the review concludes with a critical analysis of the potential advantages of aptamers over antibodies as well as the current shortcomings of aptamer for their use in LFAs, and future perspectives for aptamer based lateral flow assays.

EP

2. Sandwich aptamer lateral flow assay

AC C

The sandwich assay format is the most commonly used format for testing large analytes, which have multiple binding sites and can exploit dual aptamers, a combination of antibodies and aptamers, or split aptamers. In a typical format, reporter labelled antibody or aptamer is absorbed on the conjugate pad. A primary antibody or aptamer against the target analyte is immobilised on the test line and a secondary antibody or aptamer against the labelled bioconjugate is immobilised on the control line. When the analyte is applied to the sample pad it migrates to the conjugate pad, where the target analyte binds to the labelled bioconjugate and this complex passes across the membrane via capillary movement to the test line where it is captured. The excess of labelled aptamer/antibody will then be captured at the control line. The appearance of both lines, test line and control line is indicative of a positive result, with a single line at the control line representing a negative result, whilst confirming correct assay function.

2.1. Sandwich aptamer lateral flow assay using pair of aptamers P á g i n a 3 | 30

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

The use of pairs of aptamers has been reported for several analytes, as depicted in Figure 2. The first report of a pair of aptamers used in a lateral flow assay was in 2009 by Xu et al. [40], using the well-known thrombin dual aptamers, TBA1 (a 15-mer aptamer, which binds to the fibrogen-binding site) and TBA2 (a 29-mer aptamer, which binds to the heparin binding domain). Thiolated TBA1 aptamer with a 20-T linker was chemisorbed onto gold nanoparticles and used as the labelled reporter aptamer. Both the test and the control line were functionalised with streptavidin and biotinylated TBA2 aptamer was immobilised on the test line, and a biotinylated 20-A oligomer (DNA 1 on the control line via streptavidin-biotin interactions. In the presence of thrombin, the complex between the target and TBA1-AuNPs was captured by the TBA2 aptamer immobilised on the test line and a characteristic red band was observed. The excess of bioconjugate (TBA1-AuNPs) was captured at the control line via hybridisation between the DNA1 and the 20-T linker of the TBA1-AuNP bioconjugate, resulting in a second red band. In the absence of thrombin, the red band was only observed at the control line demonstrating that the assay was working properly. The limit of detection achieved was 2.5 nM (300 fmol in 120 µl of sample) using a portable strip reader with 10-minute assay time. This device is often used in lateral flow assays to convert the visual qualitative results to quantitative/semi-quantitative results by measuring the intensity of the test and control lines. In addition, the authors tested the specificity of the assay using other proteins such as IgG, IgM, HSA and casein and negligible results were obtained, demonstrating the specificity of the LFA. Finally, the approach was tested in spiked human plasma samples, achieving a LOD of 0.6 pmol.

Figure 2: Schematic of LFA with dual aptamers

In the same year, Liu et al. [41] reported another lateral flow using a pair of aptamers for the detection of cancer cells. The principle of the assay is the same as described above, using aptamers selected against Ramos cells, and in this case the control line consisted of an oligonucleotide complementary to a part of the AuNP-labelled aptamer. A LOD of 4000 Ramos cells by visual detection and 800 Ramos cells using a portable strip reader was achieved, with an assay time of 15 minutes. The assay was also successfully tested in human blood.

P á g i n a 4 | 30

ACCEPTED MANUSCRIPT A further example of a similar type of sandwich assay using a pair of aptamers was recently reported by Raston et al. [42] for the detection of vaspin. The only difference in this case, was the control line, where a sequence that was fully complementary to the aptamer sequence was immobilised. The results were read and recorded 5 minutes after sample loading, achieving a LOD of 5 nM by naked eye and 0.137 nM and 0.105 nM in buffer and spiked human serum, respectively, using Image J software analysis.

RI PT

Other molecules tested in sandwich assay lateral flows are arboviruses, including the Chikungunya and the Tick-borne encephalitis virus (TBEV) [43]. The only difference in the architecture of the assay is the preparation of bioconjugate, where biotinylated aptamer was bound to streptavidin coated gold nanoparticles. 2.2. Sandwich aptamer lateral flow assay using a combination of antibodies and aptamers

AC C

EP

TE D

M AN U

SC

An alternative type of sandwich assay is based on the use of a combination of antibodies and aptamers that bind to different sites on the target (Figure 3). Whilst the incorporation of antibodies is not ideal as it contradicts the advantages of the use of aptamers in LFAs as they have an inferred cost as well as stability issues. In fact this is one of the true shortcomings of the use of aptamers in LFAs, as despite the fact that sandwich assays are the preferred type of assay, there are very few examples of dual aptamers that can be used in a sandwich format and for the extensive implementation and commercialisation of aptamer based LFA, a concerted effort to select dual aptamers (i.e. pair of aptamers binding to different sites on the target analyte) is required. If successful, aptamers could even be exploited for sandwich assays for the detection of small molecules either via the use of split aptamers, or selecting an aptamer against an aptamer-small molecule complex, highlighting a sandwich format not possible with antibodies.

Figure 3: Schematic of combination of antibody and aptamer LFA

One example is the detection of salivary α-amylase (sAA). In this case, AMYm1 aptamer was modified with biotin and linked to streptavidin-AuNPs (aptamer-BiotinSA-AuNP). On the test line anti-sAA antibody was immobilised and sAA protein was immobilised on the control line. In the presence of sAA, the complex between the target and aptamer-Biotin-SA-AuNP was captured by the antibody immobilised on the P á g i n a 5 | 30

ACCEPTED MANUSCRIPT test line and the excess of bioconjugate (aptamer-Biotin-SA-AuNP) was captured on the control line, resulting in two red bands. The colour of the test line intensified as the concentration of sAA increased. In the absence of target, the bioconjugate was captured on the control line and only one band was observed. In addition, the strip perfomance was demonstrated with 0.1% (v/v) human saliva [44]. 2.3. Sandwich aptamer lateral flow assay using split aptamer

EP

TE D

M AN U

SC

RI PT

Finally, Zhu et al. [45] have pioneered the possibility to use the sandwich assay format for the detection of small molecules using split aptamer fragments, which could find extremely wide application. This strategy consists of two DNA probes that only assemble in the presence of the target ATP. One thiolated split aptamer (aptamer part 1) was chemisorbed on AuNPs and the other split aptamer was biotinylated (aptamer part 2) and immobilised on the nitrocellulose membrane by streptavidin-biotin interactions. DNA probe complementary to the aptamer part 1 – AuNP bioconjugate was immobilised (DNA1) on the control line (Figure 4). In the presence of ATP, a complex between aptamer part 1-AuNP/ATP/biotinylated aptamer part 2 was formed on the test line giving a red band. Excess aptamer part 1-AuNP was captured on the control line by hybridisation with DNA1. This assay was tested in urine samples, achieving a LOD of 0.5 µM and high specificity against UTP, CTP and GTP was observed.

AC C

Figure 4: Schematic representation of split aptamer LFA

3. Competitive lateral flow aptamer assay

Competitive assays or inhibition assays are usually used for low molecular weight compounds where only one aptatope may be available, or, alternatively, when only one aptamer is available. Two different formats of competitive assay have been reported based on different capture molecules on the test line. One approach is based on competition between the target analyte in solution to be tested and the same target analyte immobilised on the test line (Figure 5a), whilst in the other approach, DNA partially complementary to the aptamer-AuNP conjugate is immobilised on the test line and competes against the target analyte to bind to the gold nanoparticle labelled aptamer (Figure 5b). In both cases, P á g i n a 6 | 30

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

the sample containing the target analyte is applied to the sample pad and binds to the labelled aptamer adsorbed on the conjugate pad. This complex traverses the membrane to the test line, where competition between the target analyte and the capture molecule immobilised on the test line takes place. In the case of competitive LFAs, a decreasing intensity of the band at the test line with increasing concentration of the target analyte in the sample is observed. Various solutions have been proposed for the control line including the inclusion of additional bases to the aptamer for subsequent hybridisation with the complementary to these bases immobilised at the control line. However, these additional bases may impede aptamer folding and binding. Alternatively, an additional labelled DNA sequence is co-adsorbed on the conjugate pad with the labelled aptamer, and an oligo complementary to this additional DNA sequence is immobilised at the control line. Whilst this control can indicate correct wicking of this additional labelled DNA, it does not demonstrate that the labelled aptamer is functional or that it correctly wicked to the test line, and furthermore adds additional cost and complexity.

Figure 5: Schematic representation of competition with (a) immobilised target and (b) immobilised oligonucleotide with partial complementarity to aptamer sequence

3.1. Competition between the target analyte in solution and the target analyte immobilised on the membrane Jauset-Rubio et al. reported the detection of the anaphylactic allergen β-conglutin, based on competition between target analyte in solution and target immobilised on the membrane [46]. Thiolated aptamer was chemisorbed on in-house prepared AuNPs using an optimised surface chemistry (aptamer-AuNP). β-conglutin and a full complementary aptamer sequence (DNA1) were immobilised on the test line and P á g i n a 7 | 30

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

control line, respectively. With increasing concentrations of β-conglutin in solution, less aptamer was free to bind to the immobilised β-conglutin on the membrane. As a result, there was a decrease in the intensity of the test line and at concentrations greater than 10 µM no colour was observed at the test line. The authors reported on an interesting approach for the control line, exploiting the inherent properties of the nucleic acid nature of aptamers, using a full-length 94-mer oligonucleotide, completely complementary to the β-conglutin aptamer. Following competition at the test line, either the unbound aptamer-AuNP bioconjugate, or the complex of target β-conglutin (aptamer-AuNP/β-conglutin), or a combination of both, are wicked to the control line. The aptamer has much greater affinity for its complementary DNA than for its cognate aptameric target, and thus was effectively displaced from the complex, hybridising to DNA1 at the control line. In the absence of β-conglutin, the conjugate mainly bound to the β-conglutin immobilised on the membrane and there was a small excess that migrates to the control line. In addition, the authors tested the specificity of the assay against two non-specific proteins (streptavidin and bovine serum albumin) demonstrating high specificity. The LOD was calculated using a Smartphone camera followed by analysis using Image J software being 55 pM or 1.1 fmol. The entire assay was completed in just 5 minutes. 3.2. Competition between the target in solution and a complementary DNA probe

TE D

Wang et al. designed a fluorescent strip for Ochratoxin A (OTA) using a Quantum-dot (QD) labelled aptamer with a poly A linker. OTA competed with DNA probe (DNA2) immobilised on the test line for binding to the aptamer-labelled with QDs. The unbound labelled aptamer wicked to the control line, where a polyT probe (DNA1) was immobilised and recognised the poly A linker added to the aptamer sequence. In the absence of OTA, the QD-labelled aptamer bound to the DNA2 on the test line and to DNA1 on the control line [47]. The LOD achieved was 4.7 nM (1.9 ng/ml; 0.75 fmol in 100 µl, as reported by the authors), which is comparable to ELISA and fluorescence polarisation immunoassay methods [15], but inherently less time consuming.

AC C

EP

In 2011, the same group reported a similar assay [48], but in this case used a AuNPlabelled aptamer reporter bioconjugate. The LOD was improved to 2.48 nM (1 ng/ml) for visual detection and 0.45 nM (0.18 ng/ml) with an optical strip reader. In addition, the assay was applied to analysis of spiked red wine samples. No cross-reactivity with other mycotoxins (zearaleone, fumosin B1, deoxynivalenol and microcystin-LR) was observed. Recently, Zhou et al. [49] reported a similar lateral flow assay for the detection of OTA in Astragalus membranaceus. The LOD achieved was 2.48 nM (1 ng/ml) and they demonstrated good reproducibility of the assay, and applied it to 9 different real samples contaminated with A. membranaceus. Aptamer based lateral flow assays have also been reported for another mycotoxin, Aflatoxin B1 (AFB1). The format for the detection of this target was a competition between the target (AFB1) and Cy5-modified DNA probe to bind to biotinylated aptamer. Streptavidin and anti-Cy5 antibody were immobilised on the test and control line, respectively. The aptamer, and different dilutions of AFB1 and Cy5-modified DNA P á g i n a 8 | 30

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

probe were sequentially added to the wells of a microtitre plate, then incubated for 20 minutes, and finally dipsticks were placed in each of the wells. In the presence of AFB1, the biotinylated aptamer first bound to the AFB1, preventing hybridisation of the Cy5modified DNA probe with the aptamer. The complex formed (biotin-aptamer-AFB1) wicked to the test line, where it was captured by streptavidin, and the remaining free Cy5-modified DNA probe migrated to the control line where it was captured by the anti-cy5 antibody, with a fluorescent signal observed. In the absence of target, the biotinylated aptamer and the Cy5-DNA probe hybridise on the test line [50] (Figure 6). The LOD of the dipstick assay was 0.32 nM (0.1 ng/ml) of AFB1 in buffer, and 0.3 ng/g of AFB1 in spiked corn samples.

EP

Figure 6: Fluorescent dipstick competition between analyte in solution and DNA probe (Adapted from [50]).

3.3. Signal amplification exploited in lateral flow aptamer assays

AC C

There is an increasing need for the detection of very low concentrations of analytes, which cannot be achieved using the current lateral flow formats[51]. Alternative strategies to improve the sensitivity of lateral flow assays have been reported, and whilst these sophisticated platforms achieve improved detection limits, they do infer added cost and complexity and will need to be simplified and made more costeffective to be truly implemented for use at the point-of-need. These assays are based on the use of different approaches, including the replacement of gold nanoparticles with quantum dots, the use of two types of gold nanoparticles coupled to an enzymatic reaction, which produces more intense bands, thus increasing the sensitivity of the assay. Other approaches exploited the aptamer’s inherent properties such as a conformational change in the aptamer structure upon binding to its target as well as exploiting the nucleic acid nature of aptamers, allowing their amplification via the polymerase chain reaction or isothermal DNA amplification techniques, also improving the limit of detection. P á g i n a 9 | 30

ACCEPTED MANUSCRIPT 3.3.1. Signal amplification using quantum dots

EP

TE D

M AN U

SC

RI PT

The use of AuNP was compared to QDs for the detection of foodborne pathogens [52]. Three different bacterium were tested; Escherichia coli, Listeria monocytogenes and Salmonella enterica. In this LFA, the reporter aptamer was modified on one end with biotin to link to streptavidin modified AuNPs or QDs and on the other end with digoxigenin (AuNP/QD-aptamer1-digoxigenin), which was recognised by an antidigoxigenin antibody immobilised on the control line. On the test line, the capture aptamer (aptamer2) was directly immobilised on the membrane via an amino terminal group using UV treatment. The authors highlighted that the use of a 254 nm UV light could induce structural changes to the aptamers due to induced thymine dimerization and that aptamers that work in other formats may not work or their performance may be affected. In the presence of bacterium, the target was sandwiched on the test line, whilst the digoxigenin of the aptamer was captured on the control line (Figure 7). In the absence of bacterium, the AuNP/QD-labelled aptamer only bound at the control line. The LOD of E.coli was around 3000 live E.coli 8739 cells and 6000 live E.coli O157:H7 cells by visible detection and the LOD was improved ten-fold using QD and UV excitation. This work included an extremely systematic evaluation of various parameters affecting assay performance.

AC C

Figure 7: Schematic representation of signal amplification assay LFA using digoxigenin5’-reporter aptamer (Adapted from [52])

3.3.2. Signal amplification using two types of bioconjugate An alternative approach to enhance the signal was reported by Liu et al. [53], who described a dipstick test for the detection of adenosine and cocaine in serum. In both cases, a linker sequence was added to the aptamer (Aptamer 1). A thiolated oligonucleotide partially complementary to the linker sequence was chemisorbed onto gold nanoparticles (AuNP-DNA1), and a thiolated oligonucleotide complementary to part of the linker sequence and part of the aptamer was also chemisorbed onto gold nanoparticles (AuNP-DNA2). Upon hybridisation of AuNP-DNA1 and AuNP-DNA2 with the aptamer, aggregates were formed due to the proximity of the AuNPs, and these aggregates were spotted and dried on the conjugate pad. Sample was added to the P á g i n a 10 | 30

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

LFA, which rehydrated the spotted aggregates. In the presence of adenosine, the AuNP-DNA2 was displaced and the aggregate disassembled. The liberated AuNP-DNA2 then migrated to the test line, where it was captured by immobilised streptavidin (Figure 8).

Figure 8: Schematic representation of lateral flow strip for adenosine / cocaine detection where target binding induced displacement and disassembly of aggregated gold nanoparticles (Adapted from [53])

TE D

In the absence of adenosine, the large aggregates were not able to transverse the membrane. The LOD was estimated to be 20 µM and other ribonucleosides were tested and no cross-reactivity was observed. A detection limit of 10 µM was obtained for cocaine using the same assay format. In addition, this assay was tested in spiked serum samples, demonstrating compatibility with biological samples.

AC C

EP

Shen et al. developed an assay for the detection of thrombin, using two bioconjugate pads on the strip [54]. On the first conjugate pad, a DNA1 probe-AuNP complex was absorbed (AuNP-DNA1), whilst on the second conjugate pad a complex between DNA2 probe, AuNPs and aptamer incorporating a PolyT sequence was absorbed (AuNPDNA2-aptamer). Anti-thrombin antibody was immobilised on the test line, whilst biotinylated DNA3 probe (polyA) was immobilised on the control line. AuNP-DNA1 was designed to bind specifically with the second AuNP-DNA2-Aptamer via hybridisation between the DNA1 and DNA2 sequences. When sample containing thrombin is added to the LFA, the solution migrates to the first conjugate pad and then to the second conjugate pad, where the aptamer interacts with thrombin, and simultaneously the DNA 1 sequence hybridises to the DNA 2 sequence. Thus, a complex of the AuNP-DNA1 / AuNP-DNA2- aptamer / thrombin is formed and moves to the test line where it is captured by the immobilised antibodies. The excess of the AuNP-DNA1 / AuNP-DNA2aptamer is captured in the control line by an immobilised DNA3 probe (Figure 9). Quantitative analysis was realised using a portable strip reader achieving an LOD of 0.25 nM, ten-fold better than using only the AuNP-DNA2-aptamer, thus justifying the complexity and additionally cost of the assay [40]. Specificity was confirmed by testing with other proteins, including prothrombin, casein, IgG and HSA. P á g i n a 11 | 30

RI PT

ACCEPTED MANUSCRIPT

M AN U

SC

Figure 9: Configuration of sandwich lateral flow strip using two conjugate pads (Adapted from [54])

3.3.3. Signal amplification using an enzymatic reaction

AC C

EP

TE D

Combining an aptamer-cleavage reaction with an enzyme amplification system for the detection of thrombin, Qin et al. achieved a visual LOD of 6.4 pM, which was improved to 4.9 pM using a strip reader [55], detection limits three orders of magnitude lower than a previously reported aptamer-AuNP based LFA [40], and two orders of magnitude lower than Shen’s approach [54]. A thiolated DNA sequence composed of a poly T and a few bases complementary to TBA1 (15-mer thrombin aptamer) was linked to AuNPs (DNA1-AuNP) and a DNA sequence with a few bases complementary to the aptamer and also bearing both biotin and horseradish peroxidase (HRP) moieties was also linked to AuNPs (Biotin-DNA2-AuNP-HRP). Streptavidin (SA) and a poly A oligo were dispensed on the test line and the control line, respectively. The particles were mixed and aptamer was added, forming an aggregate (DNA1-AuNP/Aptamer /BiotinDNA2-AuNP-HRP). In the presence of thrombin, the aggregate disassembles due to the interaction between the aptamer and thrombin. The particles with biotin (BiotinDNA2-AuNP-HRP) bind to streptavidin on the test line to form a red band. The excess of complex moves to the control line where poly A captures DNA1-AuNP, producing a second red band. Five minutes later, the authors added 3-amino-9-ethyl carbazole (AEC) and H2O2, to induce enzyme-catalysed reactions with HRP in the Biotin-DNA2AuNP-HRP bioconjugate producing more intense red bands (Figure 10).

P á g i n a 12 | 30

RI PT

ACCEPTED MANUSCRIPT

SC

Figure 10: Configuration of sandwich lateral flow strip combining an aptamer-cleavage reaction with an enzyme amplification system (Adapted from [55])

M AN U

3.3.4. Signal amplification by using isothermal amplification

AC C

EP

TE D

Fang et al. presented a simple and sensitive LFA for the detection of Salmonella enteritidis using a pair of aptamers and isothermal strand displacement amplification (SDA) [56]. One aptamer specific for the outer membrane of S.enteritidis (aptamer1) was linked to magnetic beads for enrichment. The other was used as a reporter (aptamer2), which following sandwich formation, was amplified by SDA and detected by LFA. Aptamer1 was biotinylated for target enrichment using streptavidin coated magnetic beads and aptamer2 was modified with a primer sequence and an Nb.BbvCI recognition site at the 3’ end for SDA amplification. In the presence of Salmonella, the aptamers bound to the bacterium, and this complex (Biotin-aptamer1/ bacterium/aptamer2) was captured by the magnetic beads. Subsequently, a strand displacement reaction, including primer, DNA polymerase and nicking enzyme, was initiated using aptamer2 as a template. During primer extension, the synthesised complementary strand was cut by a nicking enzyme at the Nb.BbvCI site of the amplification aptamer. The cut strand was then displaced and another round of polymerisation started and this cycle continued. Finally, the SDA amplicon was detected on a LFA via hybridisation between thiolated DNA1 chemisorbed on gold nanoparticles (DNA1-AuNP) and DNA2 immobilised on the test line, thus forming a sandwich (DN2/amplicon/DNA1-AuNP). The excess of DNA1-AuNP was then captured on the control line by DNA3, which was complementary to DNA1. In the absence of Salmonella, no amplification was performed, and DNA1-AuNP was only captured on the control line (Figure 11). The specificity of this assay was tested against other pathogens including Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa and Citrobacterfreundi, and no cross-reactivity was observed. In addition, the assay was applied to spiked milk samples, achieving a LOD of 101 CFU/ml. The sample pre-treatment of this assay is simpler than PCR based methods of pathogen detection with a comparable level of sensitivity [57–62], whilst avoiding the tedious cell lysis and DNA/RNA extraction, by using the two aptamers to capture and enrich specific bacterium, followed by direct addition into the reaction buffer for isothermal amplification, without any specialised instrumentation. The same group subsequently P á g i n a 13 | 30

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

demonstrated that this method can be applied to other bacterium including Escherichia coli O157:H7 [63], again achieving an LOD of 10 CFU/ml. The assay was tested with different real samples, including milk, water and apple juice.

TE D

Figure 11: Schematic representation of signal amplification assay LFA using a pair of aptamers and isothermal strand displacement assay (SDA) (Adapted from [56])

AC C

EP

Jauset-Rubio et al. described an alternative method for the detection of β-conglutin via LFA, combining aptamer based detection with isothermal recombinase polymerase amplification (RPA) [46]. The assay exploited a competition assay between the target in solution and target immobilised on the surface of magnetic beads to bind to the aptamer. Following competition, the aptamer bound to the immobilised target on the beads was eluted via sonication and used as a template for amplification by RPA, an isothermal amplification technique performed at 37ºC, in just 20 minutes. The RPA reaction mix is composed of different proteins, including a recombinase protein, which binds to primers, forming filaments that can recombine with homologous DNA in a duplex target, forcing displacement of the non-complementary strand and provoking the formation of a D-loop. Single-stranded DNA binding proteins then attach to the displaced DNA strand, thus preventing the dissociation of primer and hybridisation of the duplex target. A polymerase then copies the DNA, adding bases onto the 3’ end of the primer, forcing the duplex helix open as it progresses. The cycle is repeated several times achieving exponential amplification [64]. The primers used for the amplification were designed with sequences specific to bind to extremes of eluted aptamer and flanked by single stranded tails, designed to be complementary to the capture and reporter probes on the lateral flow strip. These primers are referred to as “tailed” P á g i n a 14 | 30

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

primers, and resulted in amplicons of double stranded DNA with a single stranded DNA tail on each end. This was facilitated by a stopper, C3, located between the primerbinding site and the single stranded tail. The RPA product was mixed with thiolated reporter probe functionalised on AuNPs (DNA1-AuNP), and added to the lateral flow strip. Biotinylated probe complementary to the tail 5’ region of the amplified aptamer was immobilised on a streptavidin functionalised test line (DNA2), whilst probe complementary to the DNA1-AuNP was immobilised on the control line (DNA3). The DNA1-AuNP was complementary to the tail at 3’ end of the amplified aptamer and thus will bind to the amplified aptamer forming a sandwich on the test line (DNA1AuNP/aptamer/DNA2) and with the DNA3 on the control line (DNA1-AuNP/DNA3), generating a red band in both cases. The LOD of the assay was 9 fM with the complete assay being completed in just 30 minutes (Figure 12).

Figure 12: Schematic representation of signal amplified LFA combining aptamer detection with recombinase polymerase amplification (RPA) (Adapted from [46]).

P á g i n a 15 | 30

ACCEPTED MANUSCRIPT 3.3.5. Signal amplification using an aptamer-phage as reporter bioconjugate

EP

TE D

M AN U

SC

RI PT

An alternative method for ultrasensitive detection was reported by Adhikari et al. [65] for the detection of IgE based on the introduction of bacteriophage particles with aptamers as reporters for LFAs. For the reporter bioconjugate, M13 phage display AviTag peptide was biotinylated using biotin ligase, and covalently modified with HRP on the major coat protein pVIII, and the complex was then linked to neutravidin, and subsequently to biotinylated IgE aptamer. A sandwich assay was performed based on the immobilisation of anti-IgE antibody on the test line and anti-M13 antibody on the control line. When the IgE is added to the sample pad it bound with the aptamerphage bioconjugate and this complex was captured on the test line by the anti-IgE antibody. The excess of conjugate was then captured on the control line by the antiM13 antibody (Figure 13). The LOD of the assay was 0.7 pM (0.13 ng /ml) and the authors demonstrated better sensitivity than the ImmunoCAP Rapid test, a commercially available test for IgE detection with a sensitivity ranging from 0.72 ng/ml to 1.68 ng/ml. The specificity of the assay was also tested by spotting an unrelated murine IgG1 anti-lysozyme antibody at the test line, and no signal was obtained.

AC C

Figure 13: Schematic representation of signal amplification LFA using an aptamerphage as labelled aptamer (Adapted from [65])

3.3.6. Signal amplification by using an aptamer-gated silica nanoparticles

Finally, Özalp et al. [66] developed an interesting LFA based on aptamer-gated silica nanoparticles, facilitating sensitive detection of small molecules. As proof of concept ATP was the chosen target. To obtain an ATP molecular aptamer gate, the aptamer sequence was engineered into a hairpin by using a short-stem DNA complementary to one end of the sequence. This hairpin was immobilised on the surface of silica nanoparticles, which encapsulated the fluorophore Rhodamine B, within its pores and this complex was fixed on the test line of the membrane. At the control line, a mutated form of the aptamer sequence was used as a gating molecule. When ATP was added to the strip the hairpin was disrupted, resulting in a release of the encapsulated P á g i n a 16 | 30

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

fluorophores, producing a decrease in the fluorescence intensity at the test line, whilst on the control line no conformational change took place, and the fluorescence intensity was not affected (Figure 14). The LOD was determined to be 69 µM with the two main advantages of this approach being the avoidance of labelled target molecules, combined with signal amplification due to the multiple reporter molecules trapped inside the pores of silica nanoparticles.

TE D

Figure 14: Schematic representation of signal amplification LFA based on aptamergated silica nanoparticles (Adapted from [66])

4. Concluding remarks

AC C

EP

The use of aptamers in lateral flow assays has great potential, with aptamers possessing several advantageous properties as compared to their antibody counterparts. Aptamers are selected using an in vitro process, thus avoiding the use and sacrifice of animal hosts. This not only enables the selection of aptamers against non-immunogenic molecules, or molecules that could potentially kill the animal host (e.g. toxins), the selection of aptamers can also be carried out in non-physiological conditions akin to those conditions in which the aptamer will be applied e.g. presence of reducing agents, solvents. Whilst the selection process can be expensive, once the aptamer has been selected the cost of producing it via chemical synthesis is significantly lower than the cost of a monoclonal or even polyclonal antibody. These costs can be even further reduced if the aptamer is truncated, and an aptamer of just 8 bases with nanomolar affinity has been reported. Additionally, aptamers have been demonstrated to be highly stable, even in an immobilised form, over long periods of time stored at ambient temperatures, and this is of significant importance for LFAs as this property facilitates storage of the LFA in unrefrigerated conditions. Furthermore, aptamers can be easily modified or immobilised with no affect on its affinity, and due to their inherent nucleic acid nature, they can be exploited in formats not achievable P á g i n a 17 | 30

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

using antibodies, including molecular aptamer beacons, displacement assays and the combination of detection with nucleic acid amplification. However, despite all these clear advantages there are a limited number of reports of aptamer based lateral flow assays and not one single commercial product, to date. This can be attributed to some shortcomings of aptamers. In general, the preferred format for lateral flow immunoassays is that of a sandwich assay, with antibody against the target immobilised at the test line and an anti-idiotypic antibody against the labelled reporter antibody immobilised at the control line. An excess of this labelled reporter antibody is adsorbed on the conjugate pad and this excess allows formation of the immunocomplex at the test line (in the case the analyte is present) and at the control line. In the case of competitive assays, the control line is far more complicated as the labelled reporter molecule (antibody / antigen) must be present in limited amounts to facilitate competition and thus there is no excess to wick to the control line. Additionally, the use of two antibodies is known to improve specificity and sensitivity and this has also been demonstrated for aptamers, where dual aptamers against vaspin and thrombin where the use of dual aptamers resulted in an improvement of one and two orders of magnitude, respectively [67,68]. However, there are very few examples of dual aptamers, where a pair of aptamers bind to different binding sites on their cognate target. To address this, a combination of antibodies and aptamers in a sandwich format have been used, but the inclusion of the costly and less stable antibody invalidates the inherent advantages of using aptamers in lateral flow assays. An alternative solution is the use of split aptamers [69–72], exploiting an aptamer divided in two fragments, and using these fragments as as capture and reporter probes. Whilst this approach overcomes the use of antibodies, splitting an aptamer into fragments can result in a loss of affinity, as observed with an aptamer selected against adenosine [3,73]. Thus, for the successful implmentation of aptamers in lateral flow assays, it is highly desirable to select dual aptamers, which can be challenging and may require some innovative solutions, such as the use of alternative starting libraries or blocking aptatopes (aptamer binding sites). Additionally, the recent implementation of next generation sequencing to identify aptamer candidates can results in the identification of aptamers binding to different binding sites, which was not so accessible using cloning and Sanger sequencing. Aptamers could also be used in sandwich assays for small molecules, where aptamers could be selected against an aptamer-small molecule complex, or using the split aptamer approach, again highlighting the potential advantages of aptamers over antibodies.

However, competitive LFAs are still very useful and aptamers again show increased flexibility as compared to antibodies for their use in these assays. Two different types of competitive LFAs have mainly been reported. The first takes advantage of competition between immobilised target analyte and the analyte present in the introduced sample for binding to labelled aptamer adsorbed on the conjugate pad, whilst the second involves competition between the analyte present in the introduced sample and an oligonucleotide immobilised at the control line, which is partially P á g i n a 18 | 30

ACCEPTED MANUSCRIPT complementary to the aptamer sequence. It should be noted that the partially complementary sequence has to be very carefully designed to have the same or less affinity to the aptamer than its cognate target, or there will be no competition. In the case of immobilised target analyte, this can also infer cost and stability issues depending on the nature of the target.

TE D

M AN U

SC

RI PT

Various approaches for the realisation of the control line in both these types of competitive LFAs have been reported, including the incorporation of additional bases in the aptamer sequence, with a probe immobilised at the control line to hybridise to these incorporated sequences. However, this solution is problematic as the inclusion of further bases may interfere with the aptamer binding, and furthermore at very low concentrations of target all the labelled aptamer will bind to the immobilised target / oligonucleotide, with none available to bind at the control line, and at high concentrations of target analyte, all the aptamer will be bound to the analyte introduced in the sample, and again, no aptamer will be available for binding to the immobilised probe. Alternatively, an additional labelled sequence has been incorporated and stored together with the labelled aptamer at the conjugate pad, with its complementary sequence immobilised at the control line. Whilst this can be used to indicate the functionality of the LFA, the appearance of a band at the control line cannot guarantee that the labelled aptamer is functional or has correctly wicked along the LFA. An approach that directly takes advantage of the nucleic acid nature of aptamers has recently been reported, where a sequence fully complementary to the aptamer is immobilised at the control line. Following competition, aptamer bound to the target analyte wicks to the control line and is rapidly displaced due to the higher affinity of the aptamer for its complementary sequence as compared to the affinity the aptamer has for its cognate target. However, this approach also requires very careful optimisation of the labelled aptamer concentration to allow for binding at the control line when very low concentrations of the target analyte is present.

AC C

EP

Stability of aptamers in biological matrices can also be a concern, especially in the case of RNA aptamers, which can be rapidly degraded by nucleases in minutes to sveral tens of minutes. However, the issue of aptamer stability has been widely studied, particularly for their application in therapeutics, and various modified forms of RNA, including 2’-Amino pyrimidine, 2’-fluoropyrimidine, 2’-O-methyl purine, 2’-O-methyl pyrimidine have been developed. However, these modifications are expensive and whilst this elevated cost can be justified for the therapeutic use of aptamers, it will hinder their implementation in lateral flow assays, and it can be anticipated that the vast majority of aptamer based LFAs will exploit DNA aptamers, which are stable to the nucleases present in biological matrices in the typical 5- 30 minute timeframe for assay execution. The choice of membrane for use in aptamer based LFAs has not been widely explored. The most widely reported method for immobilisation of oligonucleotides (DNA, aptamer) as capture molecules is based on biotin-streptavidin interactions, where the aptamer or oligonucleotide is modified with a biotin group which binds to a P á g i n a 19 | 30

ACCEPTED MANUSCRIPT

RI PT

nitrocellulose strip-immobilised streptavidin. To date, there has only been one report of the direct attachment of aptamer to the membrane itself, where a capture aptamer was immobilised via an amino terminal group using UV treatment [52]. Despite the fact that the membrane is known to have a huge effect on assay performance [39,74,75] as it controls both flow rate and biomolecule immobilisation, with an overly rapid flow-rate contributing to low sensitivity, whilst a too slow flow rate can result in non-specific binding or false positives. The conjugate pads and membranes optimised for and widely used in lateral flow immunoassays may not be the best membranes for aptamer based LFAs and this is an area that needs to be studied in depth in order to achieve maximum assay performance.

M AN U

SC

Whilst reports of aptamer based LFAs for the ultrasensitive detection of target analytes have been included in this review, it must be highlighted that these approaches no not directly meet the requirements for ASSURED devices for use at the point-of-need, as either they require multiple steps (e.g. combination of assay, isothermal amplification and LFA detection), are very expensive (e.g. phages, enzymes, quantum dots, silica nanoparticles) and may require infrastructure such as a fluorescent reader. However, these approaches may find application where extremely low limits of detection are required, and future efforts should focus on integration of the multiple steps on a single device and/or reducing the complexity and costs of these sophisticated assays.

TE D

Quantitative detection using LFA is of increasing importance and the use of Smartphones to capture an image of the LFA has been widely exploited. ImageJ software can be used to analyse the intensity of the band and whilst calibration curves have been constructed using multiple LFAs and the concentration of analytes in real samples achieved with another LFA, ongoing and future work is achieving true quantitative detection at the point-of-care/need using one single LFA.

AC C

EP

In conclusion, whilst various LFAs reported here have been demonstrated to be affordable, sensitive, portable tools, capable of detecting a wide range of diverse targets in a short time (5-30 minutes), with LODs similar to those achieved by laboratory based technniques [76,77], thus facilitating point of care / point of need applications, there are only a limited number of reports of aptamer based LFAs and not one single commercial product, to date. However, the clear advantages of aptamers over antibodies in terms of cost, stability, ease of modification and immobilisation should position aptamer LFAs as serious contenders to lateral flow immunoassays. The successful selection of dual aptamers for exploitation in sandwich type formats will greatly contribute to a greater uptake as this LFA format is the most robust, using an excess of labelled reporter aptamer, with an easy to achieve control line. Combined with a direct immobilisation of the capture aptamer on the membrane, the storage of any protein on the LFA can be completely avoided, reducing costs and facilitating refrigerator free storage. Furthermore, multiplexed aptameric detection will be significantly simplified using a sandwich rather than a competitive format. The combination of dual aptamers and direct immobilisation of aptamers onto different P á g i n a 20 | 30

ACCEPTED MANUSCRIPT types of membranes should assist in the successful implementation and commercial exploitation of aptamers in LFAs and an exponential increase in the number of reports and products of these types of LFAs can be anticipated.

RI PT

Acknowledgments The authors are grateful to the King Abdulaziz University, under the financing of the collaborative project “Selection and application of aptamers against anabolic steroids”. References

R.C. Conrad, L. Giver, Y. Tian, A.D. Ellington, In vitro selection of nucleic acid aptamers that bind proteins., Methods Enzymol. 267 (1996) 336–367.

[2]

C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase., Science. 249 (1990) 505– 510.

[3]

D.E. Huizenga, J.W. Szostak, A DNA aptamer that binds adenosine and ATP., Biochemistry. 34 (1995) 656–665.

[4]

M. McKeague, M.C. Derosa, Challenges and opportunities for small molecule aptamer development, J. Nucleic Acids. 2012 (2012). doi:10.1155/2012/748913.

[5]

M. Sassanfar, J.W. Szostak, An RNA motif that binds ATP., Nature. 364 (1993) 550–553. doi:10.1038/364550a0.

[6]

M. Famulok, Molecular Recognition of Amino Acids by RNA-Aptamers: An LCitrulline Binding RNA Motif and Its Evolution into an L-Arginine Binder, J. Am. Chem. Soc. 116 (1994) 1698–1706. doi:10.1021/ja00084a010.

[7]

D. Shangguan, Y. Li, Z. Tang, Z.C. Cao, H.W. Chen, P. Mallikaratchy, K. Sefah, C.J. Yang, W. Tan, Aptamers evolved from live cells as effective molecular probes for cancer study., Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 11838–43. doi:10.1073/pnas.0602615103.

[8]

P.K. Mallik, H. Shi, J. Pande, RNA aptamers targeted for human alphaA-crystallin do not bind alphaB-crystallin, and spare the alpha-crystallin domain., Biochem. Biophys. Res. Commun. 491 (2017) 423–428. doi:10.1016/j.bbrc.2017.07.085.

M AN U

TE D

EP

AC C

[9]

SC

[1]

F.J. Sanchez-Luque, M. Stich, S. Manrubia, C. Briones, A. Berzal-Herranz, Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies., Sci. Rep. 4 (2014) 6242. doi:10.1038/srep06242.

[10]

K.M. Song, S. Lee, C. Ban, Aptamers and their biological applications, Sensors. 12 (2012) 612–631. doi:10.3390/s120100612.

[11]

S. Tombelli, M. Minunni, M. Mascini, Analytical applications of aptamers., Biosens. Bioelectron. 20 (2005) 2424–2434. doi:10.1016/j.bios.2004.11.006.

[12]

A. Geiger, P. Burgstaller, H. von der Eltz, A. Roeder, M. Famulok, RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high P á g i n a 21 | 30

ACCEPTED MANUSCRIPT enantioselectivity., Nucleic Acids Res. 24 (1996) 1029–1036. R.D. Jenison, S.C. Gill, A. Pardi, B. Polisky, High-resolution molecular discrimination by RNA., Science. 263 (1994) 1425–1429.

[14]

T. Mairal, P. Nadal, M. Svobodova, C.K. O’Sullivan, FRET-based dimeric aptamer probe for selective and sensitive Lup an 1 allergen detection, Biosens. Bioelectron. 54 (2014) 207–210. doi:10.1016/j.bios.2013.10.070.

[15]

J.A. Cruz-Aguado, G. Penner, Fluorescence polarization based displacement assay for the determination of small molecules with aptamers., Anal. Chem. 80 (2008) 8853–8855. doi:10.1021/ac8017058.

[16]

C. Wu, L. Yan, C. Wang, H. Lin, C. Wang, X. Chen, C.J. Yang, A general excimer signaling approach for aptamer sensors., Biosens. Bioelectron. 25 (2010) 2232– 2237. doi:10.1016/j.bios.2010.02.030.

[17]

R. Yamamoto, T. Baba, P.K. Kumar, Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV-1., Genes Cells. 5 (2000) 389–396.

[18]

Y.S. Kim, J.H. Niazi, M.B. Gu, Specific detection of oxytetracycline using DNA aptamer-immobilized interdigitated array electrode chip., Anal. Chim. Acta. 634 (2009) 250–254. doi:10.1016/j.aca.2008.12.025.

[19]

B.R. Baker, R.Y. Lai, M.S. Wood, E.H. Doctor, A.J. Heeger, K.W. Plaxco, An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids., J. Am. Chem. Soc. 128 (2006) 3138–3139. doi:10.1021/ja056957p.

[20]

E.E. Ferapontova, E.M. Olsen, K. V Gothelf, An RNA aptamer-based electrochemical biosensor for detection of theophylline in serum., J. Am. Chem. Soc. 130 (2008) 4256–4258. doi:10.1021/ja711326b.

[21]

Y. Du, B. Li, H. Wei, Y. Wang, E. Wang, Multifunctional label-free electrochemical biosensor based on an integrated aptamer., Anal. Chem. 80 (2008) 5110–5117. doi:10.1021/ac800303c.

[22]

C.-C. Huang, Y.-F. Huang, Z. Cao, W. Tan, H.-T. Chang, Aptamer-modified gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors., Anal. Chem. 77 (2005) 5735–5741. doi:10.1021/ac050957q.

AC C

EP

TE D

M AN U

SC

RI PT

[13]

[23]

J. Liu, Y. Lu, Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes., Nat. Protoc. 1 (2006) 246–252. doi:10.1038/nprot.2006.38.

[24]

E. Fukusaki, T. Hasunuma, S. Kajiyama, A. Okazawa, T.J. Itoh, A. Kobayashi, SELEX for tubulin affords specific T-rich DNA aptamers. Systematic evolution of ligands by exponeential enrichment., Bioorg. Med. Chem. Lett. 11 (2001) 2927–2930.

[25]

S. Tombelli, M. Minunni, E. Luzi, M. Mascini, Aptamer-based biosensors for the detection of HIV-1 Tat protein., Bioelectrochemistry. 67 (2005) 135–141. doi:10.1016/j.bioelechem.2004.04.011.

[26]

A. Bini, S. Centi, S. Tombelli, M. Minunni, M. Mascini, Development of an optical P á g i n a 22 | 30

ACCEPTED MANUSCRIPT RNA-based aptasensor for C-reactive protein., Anal. Bioanal. Chem. 390 (2008) 1077–1086. doi:10.1007/s00216-007-1736-7. N.O. Fischer, T.M. Tarasow, J.B.-H. Tok, Protein detection via direct enzymatic amplification of short DNA aptamers., Anal. Biochem. 373 (2008) 121–128. doi:10.1016/j.ab.2007.09.035.

[28]

M. Svobodova, T. Mairal, P. Nadal, M.C. Bermudo, C.K. O’Sullivan, Ultrasensitive aptamer based detection of β-conglutin food allergen, Food Chem. 165 (2014) 419–423. doi:10.1016/j.foodchem.2014.05.128.

[29]

M. Jauset-Rubio, J. Sabaté del Río, T. Mairal, M. Svobodová, M.S. El-Shahawi, A.S. Bashammakh, A.O. Alyoubi, C.K. O’Sullivan, Ultrasensitive and rapid detection of β-conglutin combining aptamers and isothermal recombinase polymerase amplification, Anal. Bioanal. Chem. (2016). doi:10.1007/s00216016-9973-2.

[30]

K.M. Koczula, A. Gallotta, Lateral flow assays., Essays Biochem. 60 (2016) 111– 120. doi:10.1042/EBC20150012.

[31]

P. Gina, P.J. Randall, T.E. Muchinga, A. Pooran, R. Meldau, J.G. Peter, K. Dheda, Early morning urine collection to improve urinary lateral flow LAM assay sensitivity in hospitalised patients with HIV-TB co-infection., BMC Infect. Dis. 17 (2017) 339. doi:10.1186/s12879-017-2313-0.

[32]

N. Johnson, J.L. Ebersole, R.J. Kryscio, R.J. Danaher, D. 3rd Dawson, M. AlSabbagh, C.S. Miller, Rapid assessment of salivary MMP-8 and periodontal disease using lateral flow immunoassay., Oral Dis. 22 (2016) 681–687. doi:10.1111/odi.12521.

[33]

N. De Giovanni, N. Fucci, The current status of sweat testing for drugs of abuse: a review., Curr. Med. Chem. 20 (2013) 545–561.

[34]

X. Yang, L. Liu, Q. Hao, D. Zou, X. Zhang, L. Zhang, H. Li, Y. Qiao, H. Zhao, L. Zhou, Development and Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Assay for Quantitative Detection of NT-proBNP in Blood., PLoS One. 12 (2017) e0171376. doi:10.1371/journal.pone.0171376.

[35]

J.C. Phan, J. Pettitt, J.S. George, L.S. 3rd Fakoli, F.M. Taweh, S.L. Bateman, R.S. Bennett, S.L. Norris, D.A. Spinnler, G. Pimentel, P.K. Sahr, F.K. Bolay, R.J. Schoepp, Lateral Flow Immunoassays for Ebola Virus Disease Detection in Liberia., J. Infect. Dis. 214 (2016) S222–S228. doi:10.1093/infdis/jiw251.

[36]

C.M. Plotz, J.M. Singer, The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis, Am. J. Med. 21 (1956) 888–892. doi:10.1016/0002-9343(56)90103-6.

[37]

B. O’Farrell, Lateral Flow Immunoassay Systems: Evolution from the Current State of the Art to the Next Generation of Highly Sensitive, Quantitative Rapid Assays, in: Immunoass. Handb., 2013: pp. 89–107. doi:10.1016/B978-0-08097037-0.00007-5.

[38]

Millipore, Rapid lateral flow test strips: consideration for product development: P á g i n a 23 | 30

AC C

EP

TE D

M AN U

SC

RI PT

[27]

ACCEPTED MANUSCRIPT http:www.millipore.com/techpublications/tech1/tb500en500, (2009). A. Chen, S. Yang, Replacing antibodies with aptamers in lateral flow immunoassay, Biosens. Bioelectron. 71 (2015) 230–242. doi:10.1016/j.bios.2015.04.041.

[40]

H. Xu, X. Mao, Q. Zeng, S. Wang, A.N. Kawde, G. Liu, Aptamer-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis, Anal. Chem. 81 (2009) 669–675. doi:10.1021/ac8020592.

[41]

G. Liu, X. Mao, J.A. Phillips, H. Xu, W. Tan, L. Zeng, Aptamer-nanoparticle strip biosensor for sensitive detection of cancer cells, Anal. Chem. 81 (2009) 10013– 10018. doi:10.1021/ac901889s.

[42]

N.H. Ahmad Raston, V.-T. Nguyen, M.B. Gu, A new lateral flow strip assay (LFSA) using a pair of aptamers for the detection of Vaspin., Biosens. Bioelectron. 93 (2017) 21–25. doi:10.1016/j.bios.2016.11.061.

[43]

J.G. Bruno, M.P. Carrillo, A.M. Richarte, T. Phillips, C. Andrews, J.S. Lee, Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of arboviruses, BMC Res Notes. 5 (2012) 633. doi:10.1186/1756-0500-5-633.

[44]

H. Minagawa, K. Onodera, H. Fujita, T. Sakamoto, J. Akitomi, N. Kaneko, I. Shiratori, M. Kuwahara, K. Horii, I. Waga, Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Subnanomolar Affinity for a Salivary Biomarker., Sci. Rep. 7 (2017) 42716. doi:10.1038/srep42716.

[45]

C. Zhu, Y. Zhao, M. Yan, Y. Huang, J. Yan, W. Bai, A. Chen, A sandwich dipstick assay for ATP detection based on split aptamer fragments., Anal. Bioanal. Chem. 408 (2016) 4151–4158. doi:10.1007/s00216-016-9506-z.

[46]

M. Jauset-Rubio, M. Svobodova, T. Mairal, C. McNeil, N. Keegan, M.S. ElShahawi, A.S. Bashammakh, A.O. Alyoubi, C.K. O’Sullivan, Aptamer Lateral Flow Assays for Ultrasensitive Detection of beta-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers., Anal. Chem. 88 (2016) 10701– 10709. doi:10.1021/acs.analchem.6b03256.

AC C

EP

TE D

M AN U

SC

RI PT

[39]

[47]

L. Wang, W. Chen, W. Ma, L. Liu, W. Ma, Y. Zhao, Y. Zhu, L. Xu, H. Kuang, C. Xu, Fluorescent strip sensor for rapid determination of toxins, Chem. Commun. 47 (2011) 1574–1576. doi:10.1039/c0cc04032k.

[48]

L. Wang, W. Ma, W. Chen, L. Liu, W. Ma, Y. Zhu, L. Xu, H. Kuang, C. Xu, An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection, Biosens. Bioelectron. 26 (2011) 3059–3062. doi:10.1016/j.bios.2010.11.040.

[49]

W. Zhou, W. Kong, X. Dou, M. Zhao, Z. Ouyang, M. Yang, An aptamer based lateral flow strip for on-site rapid detection of ochratoxin A in Astragalus membranaceus., J. Chromatogr. B, Anal. Technol. Biomed. Life Sci. 1022 (2016) 102–108. doi:10.1016/j.jchromb.2016.04.016. P á g i n a 24 | 30

ACCEPTED MANUSCRIPT W.B. Shim, M.J. Kim, H. Mun, M.G. Kim, An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1, Biosens. Bioelectron. 62 (2014) 288– 294. doi:10.1016/j.bios.2014.06.059.

[51]

S.F. Cheung, S.K.L. Cheng, D.T. Kamei, Paper-Based Systems for Point-of-Care Biosensing., J. Lab. Autom. 20 (2015) 316–333. doi:10.1177/2211068215577197.

[52]

J.G. Bruno, Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold., Pathog. (Basel, Switzerland). 3 (2014) 341–355. doi:10.3390/pathogens3020341.

[53]

J. Liu, D. Mazumdar, Y. Lu, A simple and sensitive “dipstick” test in serum based on lateral flow separation of aptamer-linked nanostructures., Angew. Chem. Int. Ed. Engl. 45 (2006) 7955–7959. doi:10.1002/anie.200603106.

[54]

G. Shen, S. Zhang, X. Hu, Signal enhancement in a lateral flow immunoassay based on dual gold nanoparticle conjugates, Clin. Biochem. 46 (2013) 1734– 1738. doi:10.1016/j.clinbiochem.2013.08.010.

[55]

C. Qin, W. Wen, X. Zhang, H. Gu, S. Wang, Visual detection of thrombin using a strip biosensor through aptamer-cleavage reaction with enzyme catalytic amplification., Analyst. 140 (2015) 7710–7717. doi:10.1039/c5an01712b.

[56]

Z. Fang, W. Wu, X. Lu, L. Zeng, Lateral flow biosensor for DNA extraction-free detection of salmonella based on aptamer mediated strand displacement amplification, Biosens. Bioelectron. 56 (2014) 192–197. doi:10.1016/j.bios.2014.01.015.

[57]

S.C. Donhauser, R. Niessner, M. Seidel, Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis., Anal. Chem. 83 (2011) 3153–3160. doi:10.1021/ac2002214.

[58]

R. Kumar, P.K. Surendran, N. Thampuran, Rapid quantification of Salmonella in seafood by real-time PCR assay., J. Microbiol. Biotechnol. 20 (2010) 569–573.

[59]

S.H. Lee, B.Y. Jung, N. Rayamahji, H.S. Lee, W.J. Jeon, K.S. Choi, C.H. Kweon, H.S. Yoo, A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats., J. Vet. Sci. 10 (2009) 43–51.

AC C

EP

TE D

M AN U

SC

RI PT

[50]

[60]

N. McCarthy, F.J. Reen, J.F. Buckley, J.G. Frye, E.F. Boyd, D. Gilroy, Sensitive and rapid molecular detection assays for Salmonella enterica serovars Typhimurium and Heidelberg., J. Food Prot. 72 (2009) 2350–2357.

[61]

S.H. Park, I. Hanning, R. Jarquin, P. Moore, D.J. Donoghue, A.M. Donoghue, S.C. Ricke, Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples., FEMS Microbiol. Lett. 316 (2011) 7–15. doi:10.1111/j.15746968.2010.02188.x.

[62]

C.F. Pui, W.C. Wong, L.C. Chai, H.Y. Lee, A. Noorlis, T.C.T. Zainazor, J.Y.H. Tang, P á g i n a 25 | 30

ACCEPTED MANUSCRIPT F.M. Ghazali, Y.K. Cheah, Y. Nakaguchi, M. Nishibuchi, S. Radu, Multiplex PCR for the concurrent detection and differentiation of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium., Trop. Med. Health. 39 (2011) 9–15. doi:10.2149/tmh.2010-20. W. Wu, S. Zhao, Y. Mao, Z. Fang, X. Lu, L. Zeng, A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification., Anal. Chim. Acta. 861 (2015) 62–68. doi:10.1016/j.aca.2014.12.041.

[64]

O. Piepenburg, C.H. Williams, D.L. Stemple, N.A. Armes, DNA detection using recombination proteins, PLoS Biol. 4 (2006) 1115–1121. doi:10.1371/journal.pbio.0040204.

[65]

M. Adhikari, U. Strych, J. Kim, H. Goux, S. Dhamane, M.V. Poongavanam, A.E. V Hagström, K. Kourentzi, J.C. Conrad, R.C. Willson, Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays, Anal. Chem. 87 (2015) 11660–11665. doi:10.1021/acs.analchem.5b00702.

[66]

V.C. Ozalp, D. Cam, F.J. Hernandez, L.I. Hernandez, T. Schafer, H.A. Oktem, Small molecule detection by lateral flow strips via aptamer-gated silica nanoprobes., Analyst. 141 (2016) 2595–2599. doi:10.1039/c6an00273k.

[67]

N.H. Ahmad Raston, M.B. Gu, Highly amplified detection of visceral adipose tissue-derived serpin (vaspin) using a cognate aptamer duo., Biosens. Bioelectron. 70 (2015) 261–267. doi:10.1016/j.bios.2015.03.042.

[68]

S. Rinker, Y. Ke, Y. Liu, R. Chhabra, H. Yan, Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding., Nat. Nanotechnol. 3 (2008) 418–422. doi:10.1038/nnano.2008.164.

[69]

B. Yuan, Y. Sun, Q. Guo, J. Huang, X. Yang, Y. Chen, X. Wen, X. Meng, J. Liu, K. Wang, High Signal-to-Background Ratio Detection of Cancer Cells with Activatable Strategy Based on Target-Induced Self-Assembly of Split Aptamers., Anal. Chem. 89 (2017) 9347–9353. doi:10.1021/acs.analchem.7b02153.

[70]

H. Yu, J. Canoura, B. Guntupalli, X. Lou, Y. Xiao, A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva., Chem. Sci. 8 (2017) 131–141. doi:10.1039/c6sc01833e.

AC C

EP

TE D

M AN U

SC

RI PT

[63]

[71]

S. Auslander, D. Fuchs, S. Hurlemann, D. Auslander, M. Fussenegger, Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay., Nucleic Acids Res. (2016). doi:10.1093/nar/gkw117.

[72]

Q. Wang, X. Yang, X. Yang, K. Wang, H. Zhang, P. Liu, An enzyme-free colorimetric assay using hybridization chain reaction amplification and split aptamers., Analyst. 140 (2015) 7657–7662. doi:10.1039/c5an01592h.

[73]

K. Sergelen, B. Liedberg, W. Knoll, J. Dostalek, A surface plasmon field-enhanced fluorescence reversible split aptamer biosensor., Analyst. 142 (2017) 2995– 3001. doi:10.1039/c7an00970d.

[74]

B. O’Farrell, Chapter 2.4 - Lateral Flow Immunoassay Systems: Evolution from P á g i n a 26 | 30

ACCEPTED MANUSCRIPT the Current State of the Art to the Next Generation of Highly Sensitive, Quantitative Rapid Assays A2 - Wild, David BT - The Immunoassay Handbook (Fourth Edition), in: Elsevier, Oxford, 2013: pp. 89–107. doi:https://doi.org/10.1016/B978-0-08-097037-0.00007-5. J. O’Farrell, B, Bauer, Developing highly sensitive, more reproducible lateral flow assays. Part 1: New approaches to old problems, IVD Technol. 10 (2006) 11–15.

[76]

G. Liang, S. Cai, P. Zhang, Y. Peng, H. Chen, S. Zhang, J. Kong, Magnetic relaxation switch and colorimetric detection of thrombin using aptamer-functionalized gold-coated iron oxide nanoparticles., Anal. Chim. Acta. 689 (2011) 243–249. doi:10.1016/j.aca.2011.01.046.

[77]

J. Yin, A. Zhang, C. Dong, J. Ren, An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes., Talanta. 144 (2015) 13–19. doi:10.1016/j.talanta.2015.05.034.

AC C

EP

TE D

M AN U

SC

RI PT

[75]

P á g i n a 27 | 30

ACCEPTED MANUSCRIPT

Table 1 Aptamers in lateral flow assays Format

Test line

Adenosine / Cocaine

20 µM Adenosine 10 µM cocaine

Direct assay (2 AuNP-conjugates)

SA

0.32 nM

Competitive assay

SA

------

Sandwich assay (Pair of aptamers)

Aflatoxin B1 (AFB1)

Control line

Bioconjugate

Ref.

------

AuNPs-DNA 1 + BiotinDNA 2-AuNPs-DNA 2 + Aptamer

[53]

Anti-Cy5 antibody

--------

[50] [43]

SA-biotin-aptamer

-------

AuNPs-SA-biotin-DNA complementary

SA-biotin-split aptamer 2

SA-biotin DNA probe

AuNPs-split aptamer 1

[45]

Aptamer gated silica nanoparticles loaded rhodamine B dye SA-Biotin-DNA probe

Mutated aptamer gated nanoparticles loaded rhodamine B dye

-------

[66]

SA-Biotin-DNA probe

AuNPs-DNA probe

[63]

Anti-digoxigenin antibody

QD-aptamer 1digoxigenin

[52]

RI PT

LOD

SC

Target

0.5 µM

Sandwich assay (Split aptamer)

ATP

69 µM

Competition assay

E. coli O157:H7

10 CFU /ml

Strand displacement amplification assay

E.coli O157:H7

3000 live cells

Sandwich assay

Amino-aptamer 2

E.coli 8739

6000 live cells

Sandwich assay

Amino-aptamer 2

Anti-digoxigenin antibody

QD-aptamer 1digoxigenin

[52]

IgE

0.7 pM

Sandwich assay

Anti-IgE antibody

Anti-M13 antibody

Aptamer-phage

[65]

Ochratoxin A (OTA)

4.7 nM

Competitive assay

SA-biotin-cDNA

SA-biotin-polyT

QD-aptamer

[47]

AC C

EP

TE D

M AN U

Arbovirus (Chikungunya virus and TBEV) ATP

P á g i n a 28 | 30

ACCEPTED MANUSCRIPT

LOD

Format

Test line

Control line

Bioconjugate

Ref.

Ochratoxin A (OTA)

2.48 nM; 0.45 nM (strip reader)

Competitive assay

SA-biotin-polyT

AuNPs-aptamer

[48]

Ochratoxin A (OTA) in Astragalus membranaceus

2.48 nM

Competitive assay

SA-biotincDNA SA-biotincDNA

SA-biotin-polyA

AuNPs-aptamer

[49]

Ramos cells

4000 Ramos cells visual and 800 Ramos cells in strip

Sandwich assay (Pair of aptamers)

SA-biotin-TE02 aptamer

SA-biotin-Control DNA

AuNPs-aptamer

[41]

Sandwich assay (Antibody / aptamer pair) Strand displacement amplification assay Sandwich assay (Pair of aptamers)

Anti-sAA antibody

sAA protein

AuNPs-SA-biotinaptamer

[44]

SA-Biotin-DNA probe

AuNPs-DNA probe

[56]

SA-biotin-DNA complementary primary aptamer SA-biotin-polyA

AuNPs-primary aptamer

[40]

2.5 nM

Thrombin

0.25 nM

Thrombin

6.4 pM visual; 4.9 pM strip reader 0.137 nM

β-conglutin

55 pM

β-conglutin

9 fM

SA-Biotin-DNA probe SA-biotinaptamer

Sandwich assay (2 AuNPs-conjugates) Aptamer-cleavage + enzymatic reaction

Anti-thrombin antibody SA

SA-Biotin-cDNA 1

Sandwich assay (Pair of aptamers) Competitive assay

SA-biotinaptamer β-conglutin

SA-biotin-DNA complementary aptamer SA-biotin full DNA complementary aptamer

Competitive assay + recombinase polymerase amplification

SA-Biotin-DNA probe

SA-biotin-DNA probe

AC C

Thrombin

Vaspin

SC

M AN U

101 CFU /ml

TE D

------

EP

Salivary α-amilase (sAA) Salmonella enteritidis

RI PT

Target

AuNPs-DNA 1 /DNA 2-AuNPs-Aptamer AuNPs-DNA 1 / Biotin-DNA 2AuNPs-HRP AuNPs-aptamer

[54]

AuNPs-aptamer

[46]

AuNPs-DNA probe

[46]

[55] [42]

P á g i n a 29 | 30

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

P á g i n a 30 | 30