# Iodinated Organic Molecule as Tag for Inductively Coupled Plasma-Mass Spectrometry Aptamer Assays

Blandine Billet, Benoit Chovelon, Erin Mcconnell, Dominique André, Laurence Puillet-Anselme, Emmanuelle Fiore, Patrice Faure, Corinne Ravelet, Maria Derosa, Eric Peyrin

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Blandine Billet, Benoit Chovelon, Erin Mcconnell, Dominique André, Laurence Puillet-Anselme, et al.. Iodinated Organic Molecule as Tag for Inductively Coupled Plasma-Mass Spectrometry Aptamer Assays. Talanta, 2024, 267, pp.125107. 10.1016/j.talanta.2023.125107. hal-04998704

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Distributed under a Creative Commons Attribution 4.0 International License
Iodinated Organic Molecule as Tag for
Inductively Coupled Plasma-Mass
Spectrometry Aptamer Assays

Blandine Billet,<sup>1,2</sup> Benoit Chovelon,<sup>1,2,3*</sup> Erin M. McConnell,<sup>3</sup> Dominique André,<sup>2</sup> Laurence
Puillet-Anselme,<sup>2</sup> Emmanuelle Fiore,<sup>1</sup> Patrice Faure,<sup>1,2</sup> Corinne Ravelet,<sup>1</sup> Maria C. DeRosa,<sup>3</sup>
and Eric Peyrin<sup>1*</sup>

Corresponding authors:

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

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

<footnote n="1"><sup>1</sup> University Grenoble Alpes, DPM UMR 5063, F-38041 Grenoble; CNRS, DPM UMR 5063, F-38041 Grenoble, France</footnote>

<footnote n="2"><sup>2</sup> Biochemistry, Toxicology and Pharmacology Department, Grenoble site Nord CHU – Biology and Pathology Institute, F-38041 Grenoble, France</footnote>

<footnote n="3"><sup>3</sup> Department of Chemistry, Carleton University, Ottawa, Canada</footnote>
Abstract

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) aptamer-based assays using
metallic nanostructures or chelates as exogenous tags have gained growing attention in the
last decade. We describe here a proof-of-concept study based on the exploitation of a simple
organic molecule as a tag, i.e. L-thyroxine carrying four iodine atoms detectable by ICP-MS.
A solid-phase assay involving the structure-switching format was deployed for the detection
of the small molecule L-tyrosinamide as model target. The overall design involved (i) a
reporter agent consisting of a DNA aptamer incorporating a single L-thyroxine label at its end
and (ii) a capture agent, which is a partially complementary strand, immobilized on a
microplate. Limit of detection in the nanomolar range was reported. The present labeling
approach was further developed for the detection of a model protein (α-thrombin), using a
sandwich mode, and proved effective in a biological matrix. We believe that the L-thyroxine
tagging method could become a simple and robust alternative to commonly used labeling
methods for ICP-MS aptamer-based assays.

Key-words: aptamers, ICP-MS, L-thyroxine tag, L-tyrosinamide, α-thrombin
# 1. Introduction

A huge variety of aptamer-based sensors and assays relying on surface-phase systems has been described over the last twenty years. Along the lines of typical immuno-assays, colorimetric or luminescent methodologies are most often implemented through microplate assays, magnetic bead-based devices or lateral flow strips [1-6]. Other techniques have been explored for improving the transduction efficiency. Notably, as the result of its very high sensitivity, element specificity and large dynamic range features, the Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) technique appears to be very promising for designing performant surface-phase aptamer assays.

To date, metallic nanoparticles (or nanoclusters) and lanthanide chelates have been privileged as elemental tags in aptamer assays, for quantifying proteins [7-9], small molecules [10, 11] or more complex systems [12, 13]. Metal nanomaterials exhibit high signal amplification capabilities while lanthanide chelates have all the necessary attributes for low-background and multiplexed analysis. However, these tagging approaches are not free of drawbacks. On one hand, the heterogeneous grade of metal nanostructures, the lack of batch-to-batch reproducibility as well as their changes in physico-chemical/surface properties over time and handling can lead to serious inconsistencies in the experiments and data [14, 15]. Furthermore, metal nanoparticles must be fully purified and characterized before use and their probe grafting process carefully controlled in order to obtain consistent results, making the analysis implementation cumbersome [16]. On the other hand, the labeling by lanthanide chelates can be quite complicated, including not only the chelating agent conjugation step but also the metal complexation step [13].

Within this context, it seemed appropriate to us to explore the potential of utilizing alternative tags, of non-metallic nature, in ICP-MS-based aptamer assays. A rather obvious, very simple option would have been to assess the possibility of using, as an endogenous tag, the natural heteroelement phosphorus (P) of the aptamers, similar to what has been done for the quantification of nucleic acids [17]. However, we ruled out this possibility because the analytical signal would be dependent on the length of the aptamer and therefore not fully standardizable. Instead, we opted for the strategy of a generic molecular tag containing detectable heteroelements and covalently conjugated to a DNA binder, which would allow us to obtain an identical response regardless of the aptameric reagent used.

We looked at L-thyroxine (Figure S1), the synthetic form of the thyroid hormone, which potentially exhibits many attractive features. First, it contains four measurable iodinated
elements, which have already been successfully used as exogenous antibody or peptide tags for ICP-MS quantification of biomolecules [18-20]. Second, it is easily available at a high degree of purity (typically > 98-99%) and low cost (less than 0.1€/mg). Third, the presence of reactive groups (Figure S1) allows quite facile conjugation to biomacromolecules [21]. Moreover, the synthesis of L-thyroxine-tagged oligonucleotides is commercially being offered on request (see experimental section). Finally, its small size (MM = 777 g/mol) should minimize the impact of its terminal oligonucleotide attachment on both the active conformation of the aptamers and on target binding.

Given that the development of ICP-MS assays for small molecules is currently rather scarce [22], we decided to mainly focus on the detection of a small model target, namely L-tyrosinamide (L-Tym). Due to the small size of L-Tym, a competitive approach based on the structure-switching concept was proposed, similar to the route previously described for optical surface-phase assays [3] (Figure 1a). A biotinylated complementary DNA (c-DNA) was linked to the surface of a streptavidin-coated microplate (the capture agent). Subsequently, the sample solution, containing the reporter aptamer, was added to the wells. In absence of the target, the reporter hybridized with the immobilized cDNA. By adding nitric acid to the medium, the hybrid was disrupted and the released iodine elements were quantitated by ICP-MS. In the presence of target, a competition occurred between cDNA and target for binding to the aptamer site. The higher the target concentration, the lower the amount of cDNA-probe hybrid at the well surface so that an analyte-dependent decrease in the iodine signal was expected to be observed.

In an effort to demonstrate that the approach could be generalized, we further tested this labeling method for the determination of a model protein, namely α-thrombin (Thb). In this case, the sandwich format was appropriate as two described aptamers are capable of binding to different regions of the Thb protein [23]. Thus, an aptamer was immobilized on the microplate to serve as a capture agent. In the presence of the target, a ternary complex was formed with the reporter aptamer (Figure 1b). In contrast to the method dedicated to small molecules, the ICP-MS signal was assumed to be enhanced with increasing Thb analyte concentration.

Finally, the applicability of the L-thyroxine-labelled aptamer probe under practical conditions was established by the detection of Thb analyte in human urine.
## 2. Experimental section

### 2.1. Reagents

For L-Tym experiments: L-Tyrosinamide (L-Tym), Tris(hydroxymethyl)aminomethane and Tween 20 were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). D-Tyrosinamide (D-Tym) was provided from Iris Biotech (Marktredwitz, Germany). NaCl was purchased from Acros Organics (Geel, Belgium). MgCl₂ and NaH₂PO₄ were supplied by Fisher Scientific (Illkirch, France). Nitric acid 67%, ultrapure for trace metal analysis was obtained from VWR (Rosny-sous-Bois France). Iodine standard solutions were purchased from CPAchem (Bogomilovo, Bulgaria). Water was obtained using a Direct-Q® 3UV water purification system (Merck Millipore, Guyancourt, France). The analyte stock solutions were prepared in pure water. DNA BIND stripwell streptavidin-coated microplates were purchased from Thermofisher Scientific (New Jersey, USA).

For Thb experiments: Human α-thrombin was provided from Cryoprep (Montpellier, France). Tris(hydroxymethyl)aminomethane, NaCl, MgCl₂ were purchased from Bioshop (Burlington, ON, Canada). Bovine serum albumin (BSA), Tween 20 and KCl were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Iodine standard solution was purchased from VWR (Radnor, PA, USA) and nitric acid 68%-70% was obtained from Anachemia (Radnor, PA, USA). Water was obtained using a Q-POD® water purification system (Merck Millipore, Toronto, ON, Canada). Finally, pooled human urine (batch number: 22BC11) was purchased from ProBioQual (Lyon, France).

All DNA oligonucleotides (listed in Table S1) used in the present work were synthesized, HPLC-purified and identified by MALDI-TOF mass spectrometry by Eurogentec (Seraing, Belgium). The chemistry of the linkage between the L-thyroxine tag and the DNA extremity is confidential and proprietary. Briefly, the link consisted of a C6 chain for the L-Tym assay or a longer C6 + tetraethylene glycol (TEG) chain for the Thb assay. Of note, for capture oligonucleotides, a TEG spacer is introduced between biotin and DNA terminus. The oligonucleotide stock solutions were prepared in pure water at 10⁻³ M and stored at -20°C until use.

### 2.2. Structure-switching assay for L-Tyrosinamide sensing

Biotinylated cDNA (Biot-CS) working solution was prepared by adequate dilution of the stock solution in grafting buffer (10 mM Tris, 2 M NaCl, pH 7.5), heated at 80°C for 5 min
and left to stand at room temperature for 30 min. 100 µL/well cDNA solution were added to streptavidin-coated microplates at a concentration of 1 µM. After 90 min incubation at room temperature under small shaking, the plate was washed three times by filling each well with 200 µL of washing buffer (10 mM NaH₂PO₄, 100 mM NaCl, 10 mM MgCl₂, 0.05% Tween 20, pH 7,5) to remove the unbound cDNA.

L-thyroxine-functionalized aptamer (5'Thr-Apt-Tym) working solution was prepared by adequate dilution of the stock solution in binding buffer (10 mM NaH₂PO₄, 100 mM NaCl, 10 mM MgCl₂, pH 7.5), heated at 80°C for 5 min and left to stand at 4°C or room temperature for 30 min. Successive dilutions of L- or D-Tym stock solutions were made in the binding buffer. 100 µL of a mixture containing the aptamer (final concentration = 500 nM, unless otherwise stated) and the corresponding target (final concentration from 0 to 400 µM) were added into each well and incubated for 5 min at 4°C or 25°C under shaking. After washing, 125 µL of nitric acid diluted by half in pure water were added into each well and incubated for 40 min at room temperature under shaking. Samples were then taken for ICP-MS analysis.

## 2.3. Sandwich assay for Thrombin sensing

Biotinylated capture aptamer (5'Biot-Apt-Thb1) working solution was prepared by adequate dilution of the stock solution in grafting buffer (10 mM Tris, 2 M NaCl, pH 7.5), heated at 80°C for 5 min and left to stand at room temperature for 30 min. 100 µL/well cDNA solution were added to streptavidin-coated microplates at a concentration of 2 µM. After 90 min incubation at room temperature under small shaking, the plate was washed three times by filling each well with 200 µL of washing buffer (20 mM Tris, 120 mM NaCl, 10 mM KCl, 1 mM MgCl₂, 0.05% Tween 20, pH 7,4) to remove the unbound 5'Biot-Apt-Thb1.

The working solution of L-thyroxine-functionalized aptamer (5'Thr-Apt-Thb2) was prepared by adequate dilution of the stock solution in binding buffer (20 mM Tris, 120 mM NaCl, 10 mM KCl, 1 mM MgCl₂, pH 7,4), heated at 80°C for 5 min and left to stand at room temperature for 30 min. Successive dilutions of human α-thrombin stock solutions were made in the binding buffer. 100 µL of a mixture containing the aptamer (final concentration = 1 µM) and the corresponding target (final concentration from 0 to 250 nM) were added into each well and incubated for 30 min at room temperature under shaking. After washing, 70 µL of nitric acid diluted by half in pure water were added into each well and incubated for 40 min at room temperature under shaking. Samples were then taken for following analysis. The assay specificity was evaluated by adding 500 nM BSA instead of thrombin.
Analysis of biological matrix assay was performed by using human urine. The 10% human urine sample was spiked with the Thb analyte at 62.5 nM and 500 nM.

## 2.4. Iodine measurement by ICP-MS

Iodine was determined using quadrupole ICP-MS apparatus (either Perkin Elmer NexION 2000, Waltham, MA, USA or Agilent 7900, Santa Clara, CA, USA). The samples, containing 50% nitric acid (v/v) (unless otherwise stated), were diluted in pure water 1:50 before analysis. Iodine standard solutions were prepared in 1% nitric acid (v/v). <sup>127</sup>I isotope was measured using <sup>103</sup>Rh as internal standard.

To note, iodine is usually determined in alkaline media to limit the memory effect and the volatility phenomenon that could generate a loss of element. However, as shown in Supplementary Data (Figure S2), the results of the external quality assessment (EQA) of our routine hospital practice in Grenoble (France) indicated that analysis in acidic medium is relevant when performed after sample preparation and when rinsing steps are introduced between sample runs. Rinsing steps were carried out with a 1% nitric acid solution. Total rinsing time was around 5 minutes, divided into three parts (three different rinsing bottles in succession).

# 3. Results and Discussion

## 3.1. Detection of L-tyrosinamide model target

A DNA aptamer targeting L-Tym was used as model system for small molecule sensing. The original aptamer has been extensively employed in sensing [24], especially as an efficient enantioselective DNA receptor for creating chiral platforms [25]. In addition, its structure-switching format has been reported in solution-phase conditions [26], allowing its facile translation to the surface-based format. To note, we exploited a truncated, optimized version of the parent aptamer [27]. It was functionalized at its 5' terminus by L-thyroxine (5'Thyr-Apt-Tym, see Table S1) for serving as reporter (Figure 1).

First, we set out to determine the conditions for adding nitric acid to the wells in order to destroy the pre-immobilized cDNA-aptamer hybrid and provide a reporter-dependent ICP-MS signal (Figure 1a). The hybrid was formed at 25°C using 3'Biot-CS10 and 5'Thyr-Apt-Tym at a final concentration of 1 µM and 500 nM, respectively. We evaluated the effects of nitric acid dilution as well as incubation time after nitric acid addition on the ICP-MS signal
generated by the reporter agent release. More ICP-MS signal was observed when nitric acid was diluted at 50% as compared to undiluted nitric acid (Figure 2). Since the signal obtained after 40 min or 15 h was similar, an incubation time of 40 min was preferred. We also evaluated lower amounts of nitric acid ranging from 6.25% to 50% (Figure 2), under the same conditions. We showed that the more concentrated the nitric acid, the higher the response. Therefore, incubation conditions in 50% nitric acid for 40 min were subsequently retained.

Before going any further in implementing the L-Tym assay, we decided to ensure that the method would not generate significant background noise. We first evaluated the possibility of non-specific adsorption of the 5'Thyr-Apt-Tym reporter to well surfaces. In this case, 1 µM of 5'Thyr-Apt-Tym was introduced into the wells in the absence of CS. Iodine was found to be undetectable by the ICP-MS instrument, indicating that non-specific adsorption of the reporter, if it exists, is negligible under the current washing conditions (Figure 3). To note, another experiment involving 1 µM 3'Biot-CS10 in the absence of 5'Thyr-Apt-Tym was also conducted. Again, no iodine signal was observed (Figure 3). It should also be pointed out that previous work has shown that the L-Tym target does not interact significantly with immobilized streptavidin [28].

We also assessed potential interferences from the iodine element, which may be potentially present in some complex matrices. Experiments were carried out using unmodified or CS-functionalized streptavidin wells, in the absence of the iodinated aptamer probe. Samples spiked with 1 µM standard iodine were subjected to analysis. Whatever the conditions, the signal obtained in ICP-MS was very limited, not exceeding 10% of that found with the immobilized hybrid (Figure 3). This suggests that the presence of endogenous iodine found, for instance, in biological media should not cause harmful interferences.

Previous work has demonstrated the importance of cDNA length for sufficient hybridization without compromising target-aptamer complex formation [3, 26]. As such, the 3'Biot-CS10 capture agent as well as a longer cDNA (3'Biot-CS14) were used to identify the one that produces the best assay response. In the presence of 100 µM target and 500 nM 5'Thyr-Apt-Tym, we found that the signal gap was slightly larger with 3'Biot-CS10 (data not shown). This cDNA was therefore selected for further optimization experiments.

The amount of 5'Thyr-apt-Tym added to the wells may also have a major role in assay response. Considering that the aptamer-target incubation time of 5 min is sufficient to reach equilibrium [27], dose-response curves were recorded with the reporter aptamer at a final concentration ranging from 250 to 2000 nM (Figure S3). The highest response was achieved for an aptamer concentration of 500 nM, with a maximum signal decrease of about 55-60%
(Figure 4a). Furthermore, we obtained a K₁/₂ value (target concentration needed to obtain 50% of the absolute signal gap [27]) of about 2.9 µM. Given that the in-solution dissociation constant of the complex between L-Tym and Apt-Tym is around 0.8 µM in similar binding buffer conditions (10 mM Mg²⁺, ambient temperature) [27], this data indicates that the designed structure-switching assay weakly affected the overall affinity of Apt-Tym for its target.

The temperature for the target-aptamer incubation was subsequently investigated. It has been previously reported that the interaction between L-Tym and its binding aptamer is very greatly enhanced at low temperature [25]. In this context, we also conducted experiments at 4°C, under the same reaction conditions. A noticeable shift of the dose-response curve to the left was observed at low temperature (Figure 4a). A K₁/₂ value of about 0.4 µM was obtained while maintaining the maximal signal gap. The specificity features of the sensing platform were assessed by testing the non-cognate D-Tyrosinamide (D-Tym) enantiomer [25, 27]. As depicted in Figure 4a, the addition of D-Tym did not cause any response, demonstrating that our assay is selective for the L-Tym detection. Under these optimized conditions, the limit of detection (LOD) for L-Tym (determined from the 3σ method, where σ is the standard deviation of the response in absence of target) was estimated to be 60 nM.

By way of comparison, we summarized in Table S2 certain key features of the previously described ICP-MS assays for the detection of small molecules, using either antibodies or aptamers as molecular recognition elements. Although a comparable LOD (in the nanomolar range) is reported in some papers, lower detection limits in the pM range are most often encountered, especially when metal nanomaterial tags are implemented. There is no doubt that the signal amplification inherent in the use of metallic nanosystems contributes to such improved detection capability. Nevertheless, the present assay involving L-thyroxine labeling offers a number of practical advantages over these earlier approaches.

First, let's compare the competitive immunoassay modes with that of the structure-switching strategy proposed herein (Figure 1a). Small-molecule immunoassays involve either the tagged antibody as reporter with the antigen bound to solid support or the labeled antigen-protein (or labeled antigen) as tracer using the antibody immobilized on solid-phase [22] (Table S2). Whichever the scheme, the attachment of the small-sized analyte to a solid surface or a tag has to be adapted on a case-by-case basis, depending on the nature of its own reactive groups. In addition, as reported by Grindlay and co-workers [29], the presence of large proteins when using antigen-protein conjugate probes can seriously interfere with antibody recognition. In contrast, the CS used as competitor in the structure-switching format is totally independent of
the target analyte. It is easily supplied by standard oligonucleotide synthesis methods with the
desired modifications at precise locations for directed functionalization, usually at the DNA
ends to avoid adverse effects on binding properties [27, 28]. Another limitation of
immunoassays that specifically implement antibody tracers is associated with their degree of
tagging, which is rather difficult to handle and laborious to characterize [22], whereas aptamer
labeling by chemical synthesis is controlled and reproducible [22].

Furthermore, metallic nanomaterials, which are most often used in ICP-MS immunoassays and aptamer-based assays (Table S2), may be prone to a number of limiting factors such as their size heterogeneity, non-specific binding, aggregation phenomena and limited stability, as recently pointed out in reference [22]. In the face of these considerations, we can emphasize that the 5'Thr-Apt-Tym reporter is not liable to any heterogeneity due to its molecular nature, is not affected by non-specific adsorption (see Figure 3) and is stable over time. Moreover, the same batch of the reporter was shown to retain its functionality for a very long period under conventional oligonucleotide storage conditions, as highlighted in Figure 5.

**3.2. Detection of α-thrombin model target and potential for complex matrix analysis**

In order to assess the generalizability of our reporting method, we sought to simply extend the approach to the detection of a protein target, by implementing another assay scheme. Following the lines of the previously reported strategy [23], a sandwich assay was deployed for Thb by using two aptamers (Apt-Thb1 and Apt-Thb2, see Table S1), which bind to different regions of the analyte. To note, we requested a linker of different chemical nature and longer length (see experimental section) for evaluating the versatility of the labeling approach.

The operating conditions were close to those above optimized for the L-tyrosinamide assay.
However, the binding buffer (and so the washing buffer) was different to approximate the
selection conditions of the two aptamers. To improve the sensitivity, we increased the
concentration of 5'Biot-Apt-Thb1 and 5'Thr-Apt-Thb2 two times compared to the L-
tyrosinamide assay. Under these conditions, a linear calibration curve from 0 to 500 nM was
obtained in the buffer (Figure 4b). The detection limit for Thb (based on the 3σ/S method,
where S is the slope of the linear part of the dose-response curve) was estimated to be about
40 nM. The assay specificity was assessed by using BSA at the 500 nM concentration. No
signal was obtained with this non-cognate protein, suggesting that the assay response is
specific (Figure 4b).
We then studied the ability of the assay to function under real-life conditions by assessing the
application in human urine for Thb sensing. Urine physiologically contains many species
likely to generate interferences in our bioassay. The urine used here had a protein
concentration of 0.14 g/L, which was close to the limit of physiological protein concentration
in urine (<0.15 g/24h). A 10% human urine sample was spiked with the Thb analyte at two
different concentrations (62.5 and 500 nM). As shown in Figure 4b, the normalized response
was comparable to that recorded in buffered solution. Since the urinary concentration of
endogenous Thb is in the picomolar range [30], the signal can be considered to come from the
added target. This result shows that the assay could retain its activity in diluted complex
matrices.

**4. Conclusion**

In summary, we have established proof of principle that the L-thyroxine molecule can be an
effective tag for ICP-MS aptamer-based assays. The method enables detection of a small
molecule *via* a simple structure-switching format. The assay can be extended to the
determination of a protein analyte in a sandwich configuration, suggesting a potentially wider
field of practice for this labeling approach. The method also proves effective under practical
conditions. Future directions will focus in particular on the feasibility of this labeling strategy
for other types of target-aptamer pairs, as well as its application to other complex matrices of
biomedical or environmental interest.

**Funding**

We acknowledge the support of Labex Arcane and CBH-EUR-GS (ANR-17-EURE-0003) and Polynat Carnot Institute (ANR-11-CARN-007-01). MCD acknowledges the Natural Science and Engineering Council of Canada (RGPIN-2021-03817) for funding. We would also like to thank Prof. J. Smith, Prof. R. Amos and K. Wasslen for the ICP-MS analysis at Carleton.

**Declaration of competing interest**

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

**Data availability**

Data will be made available on request.
## CRediT authorship contribution statement

Blandine Billet: Methodology, Validation, Data curation, Investigation, Writing – original draft. Benoit Chovelon: Conceptualization, Supervision, Methodology, Validation, Data curation, Investigation, Writing – original draft, Writing – review. Erin McConnell: Supervision, Writing – review. Dominique André: Methodology, Investigation. Laurence Puillet-Anselme: Methodology, Investigation. Emmanuelle Fiore: Methodology, Investigation. Patrice Faure: Funding acquisition, Supervision, Writing – review. Corinne Ravelet: Funding acquisition, Supervision, Writing – review. Maria C. ReRosa: Supervision, Funding acquisition, Writing – review. Eric Peyrin: Conceptualization, Project administration, Funding acquisition, Writing – review & editing.

## Supplementary Data

Tables S1-S2 and Figures S1-S3 as mentioned in text.
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**Figure 1.** Schematic representation of present aptamer assays. (a) For small molecule (L-Tym) assay, we implemented a structure-switching approach (indirect sensing of target). This is an adaptation of the general scheme previously reported for the optical detection of some small target analytes by heterogeneous-phase assays on microplates [3]. In the absence of target, the thyroxine-tagged aptamer hybridized with the biotinylated cDNA immobilized onto the streptavidin surface of microplates. The aptamer-cDNA hybridization was broken upon nitric acid addition, and iodine measurement by ICP-MS was performed. In the presence of the small molecule of interest, the formation of the target-aptamer complex is favored at the expense of the hybrid, leading to a target-dependent decrease in the signal. (b) For protein (Thb) assay, a sandwich format was introduced (direct sensing of target). Upon addition of target, a ternary complex formed with one streptavidin-immobilized capture aptamer and a second reporter aptamer. After washing and then addition of nitric acid, the signal of released iodine was measured by ICP-MS, which increased when the protein concentration was increased.

<figure><img src="image_2.png" /></figure>
**Figure 2.** Effects of dilution of nitric acid added to the wells, as well as incubation time on the disruption of the hybrid formed by 3'Biot-CS10 and 5'Thr-Apt-Tym (in the absence of L-Tym), as assessed by the ICP-MS measurement of iodine element. (a) Iodine concentration as a function of two different incubation times for either 50% nitric acid or undiluted nitric acid. (b) For the 40 min incubation time, iodine concentration as a function of lower nitric acid amounts from 6.25 to 50% (Note that the experiments in 50% nitric acid were repeated once again and gave results not significantly different from those found in (a)). Experimental conditions: 1 µM 3'Biot-CS10, 500 nM 5'Thr-Apt-Tym, 25°C temperature. Error bars represent the standard deviation from at least two independent experiments.

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
**Figure 3.** Assessment of potential interferences by studying the ICP-MS iodine signal originating from different control experiments (from left to right): 1 µM 5'Thr-Apt-Tym only, 1 µM 3'Biot-CS10 only, 1 µM 3'Biot-CS10 in the presence of 1 µM iodine, and 1 µM iodine only. As reference, the signal obtained with the immobilized hybrid (1 µM 3'Biot-CS10, 500 nM 5'Thr-Apt-Tym) is also depicted. Experimental conditions: incubation with 50% nitric acid for 40 min, 25°C temperature. Error bars represent the standard deviation from at least two independent experiments.

<figure><img src="image_5.png" /></figure>
**Figure 4.** (a) Dose-response curve for the L-Tym structure-switching assay at 25 and 4°C. The behavior of the non-cognate D-Tym species is depicted for the 4°C temperature conditions. (b) Linear range of the dose-response curve for the Thb sandwich assay in buffer. Responses for the non-cognate BSA protein (500 nM, buffer) and Thb (62.5 and 500 nM, 10% urine) are presented. The response was normalized, where [Iodine]~analyte~ and [Iodine]~free~ were the iodine concentration in the presence and absence of analyte, respectively. Error bars represent the standard deviation from at least two independent experiments.

<figure><img src="image_6.png" /></figure>
Figure 5. Response from structure-switching assays conducted 15 months apart for two different L-Tym concentrations (1.56 and 25 µM; left: first experiment and right: 15 months later) using the same batch of the 5'Thr-Apt-Tym reporter (which was stored at -20°C). Experimental conditions: 1 µM 3'Biot-CS10, 500 nM 5'Thr-Apt-Tym, incubation with 50% nitric acid for 40 min, 4°C temperature. The response is normalized, where [Iodine]~analyte~ and [Iodine]~free~ is the iodine concentration in the presence and absence of analyte, respectively. Error bars represent the standard deviation from at least two independent experiments.

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