# Non-Competitive Determination of Small Analytes by Sandwich-type Lateral Flow Assay based on Aptamer Kissing Complex

Benoit Chovelon, Velu Ranganathan, Sathya Srinivasan, Erin Mcconnell, Patrice Faure, Emmanuelle Fiore, Corinne Ravelet, Eric Peyrin, Maria Derosa

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Benoit Chovelon, Velu Ranganathan, Sathya Srinivasan, Erin Mcconnell, Patrice Faure, et al. Non-Competitive Determination of Small Analytes by Sandwich-type Lateral Flow Assay based on Aptamer Kissing Complex. Analytical Chemistry, 2024, 96 (18), pp.6875-6880. 10.1021/acs.analchem.3c05472. hal-04993941

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Distributed under a Creative Commons Attribution 4.0 International License
Non-Competitive Determination of Small Analytes by
Sandwich-type Lateral Flow Assay based on Aptamer
Kissing Complex

Benoit Chovelon,¹,²,³* Velu Ranganathan,³ Sathya Srinivasan,³ Erin M McConnell,³ Patrice Faure,¹,² Emmanuelle Fiore,¹ Corinne Ravelet,¹ Eric Peyrin,¹* Maria DeRosa³*

¹ University Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble; CNRS, DPM UMR 5063, F-38041 Grenoble, France

² Biochemistry, Toxicology and Pharmacology Department, Grenoble site Nord CHU – Biology and Pathology Institute, F-38041 Grenoble, France

³ Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Canada

Corresponding authors:

*benoit.chovelon@univ-grenoble-alpes.fr

*eric.peyrin@univ-grenoble-alpes.fr

*MariaDerosa@Cunet.Carleton.Ca
**Abstract**

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a non-competitive manner, by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.
Introduction

Lateral Flow Assays (LFAs) are essential platforms for point-of-care testing. Given their
affordability, speed, robustness and low cost, they are attracting a great deal of interest, as
already demonstrated, for instance in pregnancy or group A streptococcal pharyngitis
diagnostics, but also, more recently, as indispensable devices for the worldwide detection of
SARS-COV2 virus infection. In its standard version, antibodies are most widely implemented
as molecular recognition elements. However, immunochromatographic strip tests are not free
from drawbacks, including notably the risk of irreversible denaturation of antibodies under
certain conditions and their well-known batch-to-batch variability.¹ In this context, other
functional materials have emerged to counterbalance these limitations. As highlighted in
many recent reviews,¹⁻³ aptamers are now being very extensively exploited in LFA as
alternative recognition agents for the determination of a wide range of targets.

In lines with the lateral flow immunoassays, two common formats are retrieved in the
aptamer-based LFA. On one hand, the sandwich mode, which involves the combination of
two aptamers (or one aptamer and one antibody) recognizing different binding sites of the
target, is typically devoted to high-molecular weight analytes. On the other hand, for the
detection of small compounds, the competitive format using either the target or the
complementary strand immobilized on the strip is often implemented.¹ These competitive
assays suffer nevertheless from a well-established drawback, namely that the signal at the test
line (T-line) decreases with the analyte amount increasing in the sample (signal-off assays).
To overcome this limitation, the target-induced strand displacement has been introduced by
many researchers through diverse variants.⁴⁻⁹ However, these approaches also rely inherently
on a principle of competition, which could result in sensitivity and precision decrease, and
may require additional steps prior to LFA.⁹ An elegant option is the split aptamer-based LFA
that works under sandwich-like conditions, allowing small analytes to be detected through a
non-competitive mechanism.¹⁰ Unfortunately, aptamer splitting is generally associated with a
significant loss of target-binding affinity.¹¹

Herein, we describe aptamer strip tests that enable small molecules to be determined non-
competitively using an alternative sandwich-type mode, which has the added potential to
solve many of the issues described above. It builds on the kissing (or loop-loop) interactions¹²
and more specifically on the target-responsive aptamer kissing complex (AKC) concept.¹³ In
the AKC approach, the binding of a small target to its hairpin aptamer can lead to the
formation of a kissing complex with another hairpin through the incorporation of matching
loops into the nucleic acid structures. The hairpin aptamer (named aptaswitch) is likely to
switch from an unfolded to a folded state upon target binding. Only the folded, target-bound
aptaswitch combines with the second hairpin (called aptakiss) through kissing interactions,
which also contribute to stabilizing the binding complex. ¹³ Although the AKC strategy has
already found promising applications in various areas,¹⁴⁻¹⁹ the present work provides the first
demonstration of its exploitation in lateral flow testing.

The schematic representation of the AKC-based LFA is depicted in Figure 1. The aptaswitch is labeled with the FAM fluorophore to serve as reporter. Detection in LFA is often ensured by a colorimetric signal produced by functionalized metal nanoparticles, making it easy to visualize the test result with the naked eye. However, the conjugation of oligonucleotides to nanomaterials can be subject to variability, and the degree of grafting must be carefully controlled.²⁰ As with some previous strip tests,²¹,²² fluorescence signaling is favored here, not only because the fluorescent reporter is readily available from commercial sources, but also because of the reproducibility of its synthesis. The biotinylated aptakiss is immobilized on the T-line through interaction with pre-coated streptavidin for acting as the capture element. In the absence of analyte, the aptaswitch reporter cannot interact with the grafted aptakiss due to its incompetent conformation, so that no fluorescent signal is expected at the T-line. The unfolded aptaswitch then migrates to the control line (C-line), where strong electrostatic interactions with a deposited cationic polymer⁶ determine its accumulation and thus produce the control response. When the sample is challenged with the target, the bound aptaswitch adopts the folded shape compatible with the formation of a loop-loop complex. It is therefore captured by the aptakiss through a sandwich-type process, causing in turn a positive fluorescent signal at the T-line whose magnitude should be a direct function of the analyte concentration. Obviously, the excess aptaswitch reporter exceeds the T-line and is trapped on the C-line as for a non-target sample, ensuring the assay validity.

To establish the proof-of-principle of the present AKC-based LFA, we focused primarily on
adenosine as a target and the corresponding DNA aptaswitch and RNA aptakiss designed in
earlier works.¹⁷ The adenosine LFA was shown to achieve a detection limit in the micromolar
range. The assay was also used in the context of human urine sample analysis. We finally
showed the feasibility of the strip test for another (all-RNA) AKC system previously
engineered for the recognition of theophylline.¹⁵
# Experimental section

## Reagents

Potassium acetate, adenosine, cytidine, thymidine, guanosine, hypoxanthine, theophylline, caffeine, streptavidin, poly(diallyldimethylammonium chloride) (PDDA) (20%) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Sodium acetate, magnesium chloride, sodium chloride, tris hydrochloride, HEPES were purchased from Bioshop (Burlington, ON, Canada).

Absorption pad (CFSP223000) was obtained from Millipore Co, (Bedford, MA, USA).
Nitrocellulose (NC) membrane was purchased from Sartorius Stedin Biotech (Germany).
Pooled human urine (batch number: 22BC11) was purchased from ProBioQual (Lyon, France).

Water was obtained using a Q-POD® water purification system (Merck Millipore, Toronto, ON, Canada). The analyte stock solutions were prepared in deionized water.

All DNA oligonucleotides (Table 1) used in the present work were synthesized, HPLC-purified and identified by MALDI-TOF mass spectrometry by Eurogentec (Seraing, Belgium). Aptaswitches were FAM-labeled and aptakiss oligonucleotides were biotin-labeled.
On one hand, ADOsw3'B and THEsw4' were derived from the original hairpin aptamers binding adenosine ($K_d$ = 6 µM)$^{23}$ and theophylline ($K_d$ = 0.1 µM)$^{24}$, respectively. They were engineered by substituting a sequence prone to kissing interactions to the parent aptamer apical loop, as fully detailed in our previous works.$^{15,17}$ On the other hand, aptakiss-k3 corresponded to the TAR RNA hairpin$^{25}$ while aptakiss-k4 was the upper part of the pre-miRNA let7b.$^{26}$

## Construction of the strips

The LFA was constructed by overlapping two LFA pads, namely the absorption pad and NC membrane. A width of 5 mm LFA pads cut and pasted on an adhesive backing with a 2 mm overlap. NC membrane was used to immobilize PDDA (15% PDDA polymer, 2 µL.cm⁻¹) to develop the control line. Biotinylated aptakiss was grafted to the T line by interaction with immobilized streptavidin. In practical terms, approximately 1 µL of solution containing 65 µmol/L aptakiss and 3.2 mg/mL streptavidin is deposited on the T line per strip. The distance between the lines is approximately 4 mm. The device was loaded with reagent and dried for 30 minutes at room temperature prior to use. Once made, the strips were stored at -20°C (unless otherwise stated). Before use, they were removed from the freezer and placed at room temperature for one hour.
## Assays for adenosine and theophylline

The oligonucleotide stock solutions were prepared in deionized water at $10^{-4}$ or $10^{-5}$ M and stored at -20 °C. The working solutions of aptaswitches were prepared from the stock solutions by diluting them with the concentrated appropriate buffer (for adenosine: HEPES 20 mM, potassium acetate 140 mM, sodium acetate 20 mM, magnesium chloride 10 mM, pH 7.4; for theophylline: Tris hydrochloride 10 mM, magnesium chloride 10 mM, sodium chloride 100 mM, pH 7.5), heated at 80°C for 3 min and left to stand at room temperature for 30 min. Of note, this preliminary heating step was a factor to be taken into account to obtain a better test response (see Figure S1).

Test samples consisted of the aptaswitch in the presence or absence of the target. The assay began with the dipping step, where the strips were immersed in the samples (50 µL) for 2.5 minutes. For adenosine, strips were read directly 25 minutes after incubation. For theophylline, an additional step of washing the strips in buffer for 3 minutes was introduced before reading at 25 minutes.

Assays (three or four replicates) were carried out at 4°C for adenosine and theophylline, and at 22°C (room temperature) or 37°C for adenosine only. Strip photographs were taken with a simple camera installed in an AlphaImager system, working at 302 nm. We used ImageJ software in order to convert the C-line and T-line signal into quantitative intensity data. We then calculated the ratio (T-line intensity/C-line intensity) and, finally, the ratio was transformed into a percentage, giving a value of 100% for the highest analyte concentration.

For assessing the selectivity of the adenosine assay, the fluorescent aptaswitch was replaced by a FAM-labeled scrambled oligonucleotide (Table 1). To establish the involvement of the kissing interaction in the signaling system, a variant of the reporter containing a mutation in the apical loop (ADOsw3'B<sub>mut</sub>-5'FAM, see Table 1) was also challenged in place of the labeled native aptaswitch. In other experiments, different non-cognate molecules were tested using the FAM-tagged aptaswitch. Concentrations of guanosine and hypoxanthine were lower because of their poor solubility. For assessing the specificity of the theophylline assay, caffeine was used as non-cognate species.

The recovery of adenosine from spiked human urine was studied using the sample diluted directly (without any centrifugation step) in the buffer in order to obtain 10% urine. The adenosine was also diluted in the buffer. For the storage study, we investigated the functionality of adenosine strips stored for several days at either room temperature or -20°C.

## Results and discussion
Initially, we sought to establish the proof-of-concept of the system by taking adenosine as small-molecule target model using its previously engineered DNA aptaswitch/RNA aptakiss pair<sup>17</sup> (ADOsw3'B/aptakiss-k3, see experimental section and Table 1). It has been demonstrated that the interaction between free ADOsw3'B and aptakiss-k3 is negligible, whereas the presence of adenosine leads to the formation of a highly stable AKC (K<sub>d</sub> of around 5 nM at saturating target concentration).<sup>17</sup>

Earlier works have suspected that the formation of aptamer kissing complexes is favored at low temperatures.<sup>14,18</sup> We therefore started our work at 4°C for the dipping step. In addition, SPR studies have previously shown that target-bound aptaswitches associate with surface-grafted aptakisses within seconds, provided their concentration is sufficiently high (5 µM).<sup>13,15</sup> As such, we performed LFA experiments by introducing 10 µM ADOsw3'B into the working solution (also containing different concentrations of adenosine) to ensure proper ternary complex formation over the assay timescale, and thus efficient signaling.<sup>27</sup>

As depicted in Figure 2a, the T-line signal was enhanced when the adenosine concentration increased, in accordance with the principle of the sandwich-type mode (Figure 1). A minimal signal was observed in the absence of a target, as expected from the recognition properties mentioned above. Through the analysis of strips by the imageJ software, we converted the fluorescent output into a quantitative response for constructing calibration curves (Figure 2b). The detection limit (based on 3σ/S, where σ is the standard deviation of the response in absence of target and S the slope of calibration curve linear part, Figure S2) was equal to 20 µM.

We then performed selectivity experiments by implementing a scrambled oligonucleotide (ADOsw3'B<sub>scr</sub>, see Table 1) as reporter instead of ADOsw3'B. In such case, no fluorescence signal was detectable at the T-line, revealing that the adenosine recognition by aptamer was responsible for the output (Figure S3). We also assessed the effects of introducing a mutation point into the apical recognition loop of ADOsw3'B (ADOsw3'B<sub>mut</sub>, see Table 1). We found that the ADOsw3'B<sub>mut</sub> probe was unable to produce any perceptible signal at the T-line (Figure S3). This demonstrates that the kissing interaction between the reporter and capture agents was unquestionably involved in the signaling scheme.

We evaluated the specificity of the test by analyzing the response caused by various non-cognate ligands. The signals observed were close to that of blank (Figure 2c,d), indicating that the assay was specific to adenosine.

As adenosine is known as capable of interacting with streptavidin,<sup>28</sup> we additionally performed a negative control using strips devoid of aptakiss, i. e. with only streptavidin
immobilized on the T-line. In the presence of 2000 µM adenosine, these strips produced a truly negligible signal, which reveals that this potential interaction was insignificant in the present conditions (Figure 2c,d).

Rapid tests are widely used for health-related biological applications. Therefore, we aimed at using our LFA in the context of urine sample analysis (Figure S4). We spiked samples containing 10% raw urine (not pre-treated) with various concentrations of adenosine. The fluorescence signal at the C- and T-lines was found to be somewhat diminished compared with that retrieved in buffered medium (compare Figure S4 with Figure 2a). However, the intensity ratio was maintained, enabling good levels of recovery (ranging from 93 to 116%). This means that the AKC-based test could be adapted to the analysis of real-life samples.

We subsequently studied the functionality of the newly manufactured strips after storage under two different conditions, *i. e.* at room temperature or at -20°C. We found both a significant increase in background noise as well as deterioration of the signal-to-background after 4 days of storage at room temperature (Figure S5). In strong contrast, the strips responded identically after two weeks of -20°C storage (Figure S5). These data show that freezer storage may be a good way to preserve functional strips.

We then assessed the possibility of using our strips while working at room temperature, which would be much more advantageous for routine practice. Although the intensity ratio between the T-line and the C-line was of lesser magnitude with increasing temperature (Figure S6), we noticed an improvement in assay precision (Figures 2f and S2), which resulted in a lowering of the detection limit to 13 µM. Encouraged by this result, we raised the test temperature even more, working at 37°C. We observed a further drop in the signal ratio (Figure S6), which was now associated with a marked alteration in assay performance, as evidenced by the lack of variation in fluorescence at the T-line for low concentrations of target (Figure S2).

We finally investigated the applicability of our LFA using another AKC system, for which all oligonucleotide components are part of the RNA series (THEsw4'/aptakiss-k4, see experimental section and Table 1), to detect the theophylline analyte. As non-specific interactions between free THEsw4' and aptakiss-k4 could occur,¹⁴ we added a strip washing step after dipping strips in sample. Another difference with the adenosine system is that the concentration of labeled THEsw4' was reduced to 3.75 µM. We constructed a calibration curve as above-mentioned for adenosine (Figure S7). Despite the additional strip washing step, a certain level of background noise was apparent. However, a satisfactory detection limit of 10 µM was attained. Given that caffeine did not generate a signal different from that of blank, the assay was demonstrated to be specific to theophylline (Figure S7). These findings
suggest the possibility of extending the LFA to different hairpin aptamers, including all-RNA
systems.

While the strip test developed for adenosine can be considered primarily as a model case, it appears that the LFA-based detection of theophylline could be of interest in a clinical chemistry context. Indeed, this drug, used to treat asthma, has a narrow therapeutic window (in the range 55 µM-110 µM)²⁹ so that monitoring serum levels is crucial. In support of this point, it is worth noting that an LFA based on the CRISPR-Cas system has been recently developed for the determination of theophylline.³⁰ The detection limit of theophylline achieved in our work is well below the concentrations corresponding to its therapeutic index, making it possible to envisage applications in the area.

If we place our assay in a more general context, it turns out that the reported sensitivity is lower than that obtained for a variety of small-analyte LFAs, where detection limits in the nM range or even lower can be achieved (see recent review³¹). This result can be ascribed, at least in part, to the basic signaling approach we adopted. In the framework of this proof of concept, we simply used the gel imaging system available in the laboratory (see experimental section). The excitation at 302 nm was however far from optimal, given that the FAM reporter has an excitation spectrum maximum at around 495 nm. We are confident that the test performance could be improved by implementing devices with proper excitation facilities or, better still, specifically dedicated to reading the fluorescence of test strips.²²,³² Further gains are also expected by replacing the FAM label with quantum dots or dye-doped nanoparticles that offer superior fluorescence capabilities.³¹,³²

Conclusion

In summary, we have developed a new approach to LFA that could broaden the scope of
aptameric strips for small molecules. This has the potential to circumvent many of the issues
associated with signal-off methods and competitive processes, thanks to an alternative
sandwich-type mode, without the disadvantage of the significant loss of affinity that may
occur in a split aptamer configuration. Our results also demonstrate that capture RNA-
functionalized strips function suitably even under practical conditions, which to our
knowledge has never been tested before in aptamer-based LFA.

One of the limitations of the AKC approach is that it is currently only applicable to stem-loop
motifs. Given the large number of hairpin aptamers already described for small molecules of
biomedical<sup>33</sup> or environmental<sup>9,34,35</sup> interest, however, the potential for applications appears
vastly extended.

Acknowledgments

We acknowledge the support of Polynat Carnot Institute (ANR-11-CARN-007-01), Labex
Arcane and CBH-EUR-GS (ANR-17-EURE-0003) and CDP CerCoG program. We also
acknowledge the Natural Sciences and Engineering Research Council (NSERC) for a
Discovery Grant (RGPIN 2021-03817).

Notes

The authors declare no competing financial interest.

**Supporting Information**

Figures S1-S7 as mentioned in text.
References

(1) Wang, T.; Chen, L.; Chikkanna, A.; Chen, S.; Brusius, I.; Sbuh, N.; Veedu, R. N. Development of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective point-of-care diagnosis. *Theranostics* **2021**, *11*, 5174-5196.

(2) Huang, L.; Tian, S.; Zhao, W.; Liu, K.; Ma, X.; Guo, J. Aptamer-based lateral flow assay on-site biosensors. *Biosens. Bioelectron.* **2021**, *186*, 113279.

(3) Jaisankar, A.; Krishnan, S.; Rangasamy, L. Recent developments of aptamer-based lateral flow assays for point-of-care (POC) diagnostics. *Anal. Biochem.* **2022**, *655*, 114874.

(4) Liu, J.; Mazumdar, D.; Lu, Y. A Simple and Sensitive “Dipstick” Test in Serum Based on Lateral Flow Separation of Aptamer-Linked Nanostructures. *Angew. Chem. Int. Ed.* **2006**, *45*, 7955-7959.

(5) Alsager, O. A.; Kumar, S.; Hodgkiss, J. M. Lateral Flow Aptasensor for Small Molecule Targets Exploiting Adsorption and Desorption Interactions on Gold Nanoparticles. *Anal. Chem.* **2017**, *89*, 7416-7424.

(6) Velu, R.; DeRosa, M. C. Lateral flow assays for Ochratoxin A using metal nanoparticles: comparison of “adsorption-desorption” approach to linkage inversion assembled nano-aptasensors (LIANA). *Analyst* **2018**, *143*, 4566.

(7) Dalirirad, S.; Steckl, A. J. Lateral flow assay using aptamer-based sensing for on-site detection of dopamine in urine. *Anal. Biochem.* **2020**, *596*, 113637.

(8) Dalirirad, S.; Han, D.; Steckl, A. J. Aptamer-Based Lateral Flow Biosensor for Rapid Detection of Salivary Cortisol. *ACS Omega* **2020**, *5*, 32890-32898.

(9) Du, Y.; Liu, D.; Wang, M.; Guo, F.; Lin, J. S. Preparation of DNA aptamer and development of lateral flow aptasensor combining recombinase polymerase amplification for detection of erythromycin. *Biosens. Bioelectron.* **2021**, *181*, 113157.

(10) Zhu, C.; Zhao, Y.; Yan, M.; Huang, Y.; Yan, J.; Bai, W.; Chen, A. A sandwich dipstick assay for ATP detection based on split aptamer fragments. *Anal. Bioanal. Chem.* **2016**, *408*, 4151-4158.

(11) Debiais, M.; Lelievre, A.; Smietana, M.; Muller, S. Splitting aptamers and nucleic acid enzymes for the development of advanced biosensors. *Nucleic Acids Res.* **2020**, *48*, 3400-3422.

(12) Szczepanski, J.T.; Joyce, G.F. Binding of a Structured D-RNA Molecule by an L-RNA Aptamer. *J. Am. Chem. Soc.* **2013**, *135*, 36, 13290-13293.

(13) Durand, G.; Lisi, S.; Ravelet, C.; Dausse, E.; Peyrin, E.; Toulmé, J. J. Riboswitches Based on Kissing Complexes for the Detection of Small Ligands. *Angew. Chem. Int. Ed.* **2014**, *53*, 6942-6945.

(14) Chovelon, B.; Durand, G.; Dausse, E.; Toulmé, J. J.; Faure, P.; Peyrin, E.; Ravelet, C. ELAKCA: Enzyme-Linked Aptamer Kissing Complex Assay as a Small Molecule Sensing Platform. *Anal. Chem.* **2016**, *88*, 2570-2575.

(15) Durand, G.; Dausse, E.; Goux, E.; Fiore, E.; Peyrin, E.; Ravelet, C.; Toulmé, J. J. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers. *Nucleic Acids Res.* **2016**, *44*, 4450-4459.

(16) Zhang, K.; Wang, K.; Zhu, X.; Xie, M. A label-free kissing complexes-induced fluorescence aptasensor using DNA-templated silver nanoclusters as a signal transducer. *Biosens. Bioelectron.* **2016**, *78*, 154-159.

(17) Goux, E.; Dausse, E.; Guieu, V.; Azema, L.; Durand, G.; Henry, M.; Choisnard, L.; Toulmé, J. J.; Ravelet, C.; Peyrin, E. A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex. *Nanoscale* **2017**, *9*, 4048-4052.

(18) Chovelon, B.; Fiore, E.; Faure, P.; Peyrin, E.; Ravelet, C. Mirror-image aptamer kissing complex for arginine-vasopressin sensing. *Anal. Chim. Acta* **2018**, *1001*, 143-150.
(19) Sett, A.; Zara, L.; Dausse, E.; Toulmé, J. J. Engineering Light-Up Aptamers for the Detection of RNA Hairpins through Kissing Interaction. *Anal. Chem.* **2020**, *92*, 9113–9117.

(20) Bear, D. R. The Chameleon Effect: Characterization Challenges Due to the Variability of Nanoparticles and Their Surfaces. *Front. Chem.* **2018**, *6*, 145.

(21) Lin, H.; Fang, F.; Zang, J.; Su, J.; Tian, Q.; Kumar Kankala, R.; Lin, X. A Fluorescent Sensor-Assisted Paper-Based Competitive Lateral Flow Immunoassay for the Rapid and Sensitive Detection of Ampicillin in Hospital Wastewater. *Micromachines* (Basel) **2020**, *431*, 11.

(22) Zhu, C.; Zhang, G.; Huang, Y.; Yang, S.; Ren, S.; Gao, Z.; Chen, A. Dual-competitive lateral flow aptasensor for detection of aflatoxin B₁ in food and feedstuffs. *J. Hazard Mater.* **2018**, *344*, 249-57.

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

(24) Jenison, R.D.; Gill, S.C.; Pardi, A.; Polisky, B. High-resolution molecular discrimination by RNA. *Science* **1994**, *263*, 1425-1429.

(25) Darfeuille, F.; Sekkai, D.; Dausse, E.; Kolb, G.; Yurchenko, L.; Boiziau, C.; Toulmé, J. J. Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature. *Comb. Chem. High Throughput Screening* **2002**, *5*, 313-325.

(26) Griffiths-Jones, S.; Grocock, R.J.; van Dongen, S.; Bateman, A.; Enright, A.J. miRBase: microRNA sequences, targets and gene nomenclature. *Nucleic Acids Res.* **2006**, *34*, D140-D144.

(27) liu, Y.; Zhan, L.; Qin, Z.; Sackrison, J.; Bischof, J. C. Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis. *ACS Nano* **2021**, *15*, 3593-3611.

(28) Ravelet, C.; Michaud, M.; Ravel, A.; Grosset, C.; Villet, A.; Peyrin, E. Streptavidin chiral stationary phase for the separation of adenosine enantiomers. *J. Chromatogr. A.* **2004**, *1036*, 155-60.

(29) Weinberger, M.; Hendeles, L. Theophylline in asthma. *New Engl. J. Med.* **1996**, *334*, 1380-1388.

(30) Jiang, W.; Aman, R.; Ali, Z.; Mahfouz, M. Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline. *Front. Bioeng. Biotechnol.* **2023**, *11*, 1118684.

(31) Nuntawong, P.; Putalun, W.; Tanaka, H.; Morimoto, S.; Sakamoto, S. Lateral flow immunoassay for small-molecules detection in phytoproducts: a review. *J. Nat. Med.* **2022**, *76*, 521-545.

(32) Nguyen, V.T.; Song, S.; Park, S.; Joo, C. Recent advances in high-sensitivity detection methods for paper-based lateral-flow assay. *Biosens. Bioelectron.* **2020**, *152*, 112015.

(33) Nakatsuka, N.; Yang, K. A.; Abendroth, J. M.; Cheung, K. M.; Xu, X.; Yang, H.; Zhao, C.; Zhu, B.; Rim, Y. S.; Yang, Y.; Weiss, P. S.; Stojanovic, M. N.; Andrews, A. M. Aptamer-field-effect transistors overcome Debye length limitations for small-molecule sensing. *Science* **2018**, *362*, 319-324.

(34) Niu, C.; Zhang, C.; Liu, J. Capture-SELEX of DNA Aptamers for Estradiol Specifically and Estrogenic Compounds Collectively. *Environ. Sci. Technol.* **2022**, *56*, 17702-17711.

(35) Sun, L.; Zhao, Q. Direct fluorescence anisotropy approach for aflatoxin B1 detection and affinity binding study by using single tetramethylrhodamine labeled aptamer. *Talanta* **2018**, *189*, 442-450.
<table><caption>**Table 1.** Sequences of oligonucleotides used in this study. RNA bases are in bold. Bases of the apical loops are underlined. The mutation point (C→G) in the apical loop of ADOsw3'B is highlighted by a grey rectangular box (ADOsw3'B<sub>mut</sub>).</caption><thead><tr><th>Name</th><th>DNA/RNA</th><th>5' → 3'</th></tr></thead><tbody><tr><td>Aptakiss-k3-5'biot</td><td>RNA</td><td>biotin-TEG-CGAGCCUGGGAGCUCG</td></tr><tr><td>ADOsw3'B-5'FAM</td><td>DNA</td><td>6-FAM-GTTGGGGGACACACTCCCATTCTGGGAGGAAAC</td></tr><tr><td>ADOsw3'B<sub>scr</sub>-5'FAM</td><td>DNA</td><td>6-FAM-TGCCACACGGTAGTCAGATGCGACAGGACGTGT</td></tr><tr><td>ADOsw3'B<sub>mut</sub>-5'FAM</td><td>DNA</td><td>6-FAM-GTTGGGGGACACACTCGCATTCTGGGAGGAAAC</td></tr><tr><td>Aptakiss-k4-3'biot</td><td>RNA</td><td>GGGCAGUGAUGUUGCCC-TEG-biotin</td></tr><tr><td>THEsw4'-5'FAM</td><td>RNA</td><td>6-FAM-GGCGAUACCAGACAUCACCCCUUGGCAGCGUC</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Schematic representation of the sandwich-type LFA relying on the AKC approach.</figcaption></figure>
**Figure 2.** (a) Photographs of aptakiss-k3-functionalized strips tested with increasing concentrations of adenosine (A) (from 0 to 2000 µM) using ADOsw3'B as reporter (at 4°C). (b) The corresponding calibration curve constructed from the ImageJ integration of the fluorescence signal at the T-line and C-line. (c) Photographs of aptakiss-k3-functionalized strips tested with various non-cognate ligands (cytidine (C), 1000 µM; thymidine (T), 1000 µM; guanosine (G), 250 µM; hypoxanthine (H), 250 µM) and streptavidin-coated strip (no aptakiss-k3) challenged with 2000 µM adenosine (strip marked with asterisk), each time using ADOsw3'B as reporter (at 4°C). For proper comparison, experiments at 4°C using aptakiss-k3-functionalized strips and the ADOsw3'B reporter in the presence of 2000 µM adenosine were repeated once more (strip without asterisk). (d) The corresponding test responses. (e) Photographs of aptakiss-k3-functionalized strips tested with increasing concentrations of adenosine (from 0 to 2000 µM) using ADOsw3'B as reporter (at room temperature, 22°C). (f) The corresponding calibration curve, constructed from the ImageJ integration of the fluorescence signal at the T-line and C-line.
<figure><img src="image_3.png" /><figcaption>(a)</figcaption></figure>

C-line

T-line

<figure><img src="image_4.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_5.png" /></figure>

C-line

T-line

<figure><img src="image_6.png" /><figcaption>(e)</figcaption></figure>

<figure><img src="image_7.png" /></figure>

C-line

T-line

<figure><img src="image_8.png" /><figcaption>(f)</figcaption></figure>
SYNOPSIS TOC

For TOC only

<figure><img src="image_9.png" /></figure>