# Detection of Mercury Ions at ng/L Scale by Surface Plasmon Resonance (SPR) on DNA Aptamer Biochips

Antoine Elie, Yann Niberon, Aurélien Bruyant, Laurent Arnaud, Claudia Cosio, Julien Proust

► To cite this version:

Antoine Elie, Yann Niberon, Aurélien Bruyant, Laurent Arnaud, Claudia Cosio, et al. Detection of Mercury Ions at ng/L Scale by Surface Plasmon Resonance (SPR) on DNA Aptamer Biochips. Journal of Physical Chemistry C, 2025, 129 (3), pp.1779-1788. 10.1021/acs.jpcc.4c04764. hal-05252409

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Distributed under a Creative Commons Attribution 4.0 International License
Detection of mercury ions at ng/L scale by
Surface Plasmon Resonance (SPR) on DNA
aptamer biochips

Antoine Elie,*,† Yann Niberon,† Aurélien Bruyant,† Claudia Cosio,‡ Laurent Arnaud,¶ and Julien Proust*,†

†Light, Nanomaterials, Nanotechnologies (L2n), Université de Technologie de Troyes
(UTT) & CNRS UMR 7076, 12 rue Marie Curie, Troyes, France

‡Université de Reims Champagne-Ardenne, UMR-I 02 INERIS-URCA-ULH SEBIO,
Reims, France

¶PhaseLab Instrument SAS

E-mail: antoine.elie@utt.fr; julien.proust@utt.fr

Abstract

Mercury pollution is an important environmental problem due to its toxicity for hu-
mans, even at very low concentrations. Presently, several methods enable the detection
of this element at environmental concentration. Although sufficiently sensitive in most
cases, measurements are typically made punctually. We present here the development
of a continuous flow detection system for two mercury forms: the mercuric ion (Hg<sup>2+</sup>)
and the methylmercury ion (CH<sub>3</sub>-Hg<sup>+</sup>). We used a compact SPR device based on gold
chips functionalized with tailored aptamers. Leveraging the specific affinity between
DNA aptamers and mercuric ions, this reliable technique allowed the detection of mer-
curic ion concentrations in the ng/L range. The high sensitivity is attributed to the
expected DNA folding into a hairpin shape in presence of the mercuric ions. Circular Dichroism (CD) spectroscopy confirmed the triggering of conformational DNA change for 3 mercuric ions per aptamer. This study highlights the strong potential of compact SPR associated with specific DNA aptamers for the realization of a portable real-time detection device.

Introduction

Mercury is a toxic metal for biota and as such is targeted by environmental legislation and is closely supervised. ¹,² According to the European framework directive 2008/105/CE,³ its annual average concentration should not exceed 0.05 µg/L and its admissible limit is fixed at 0.07 µg/L in water.

There are various mercury sources in the environment, including natural (soil emissions, volcanic eruptions, forest fires) and anthropogenic (coal-fired power generation, waste management, mineral extraction).⁴ Mercury is found mainly as elemental mercury (Hg), mercuric ion (Hg²⁺) or methylmercury ion (CH₃−Hg⁺) and show a relatively complex biogeochemical cycle.⁵⁻⁷ Once in the atmosphere, elemental mercury is oxidized to Hg²⁺ and is deposited on soils and in water. Under the action of microbes mainly in sediments, this ion is methylated into methylmercury. This methylmercury is then bioaccumulated and biomagnified in the food chain, reaching particularly high concentrations in fishes. It is then demethylated by microbes or photochemical processes to the elementary form which is re-emitted in the atmosphere.⁸

Numerous studies have been carried out on mercury-contaminated human families and populations, showing correlations between this contamination and dysfunctions or diseases of the human body.⁴,⁷,⁹⁻¹³ Furthermore, toxicological studies on mercury are also carried out on aquatic and terrestrial species (animals and plants). These studies demonstrate the complexity of exposure to pollutants and the need for more in-depth studies in this area.¹⁴,¹⁵

Several methods allow the detection of mercury concentrations in the µg/L to pg/L
range. These include Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Cold Vapor Atomic Fluorescence Spectroscopy (CV-AFS) or Anodic Stripping Voltammetry (ASV). Despite a high sensitivity, ICP-MS and CV-AFS are relatively complex and expensive methods that are mainly used in laboratories and not deployed in situ for daily water analysis. The ASV was developed to overcome this limitation. Nonetheless, it is still difficult with this technique to characterize a complex sample such as water sampled in the environment.¹⁶ Table 1 presents a comparison of the performance of the various detection solutions outlining their respective advantages and disadvantages.

In this paper, we propose an accurate and accessible method to analyze solutions in continuous flow, a feature not included in the above mentioned techniques. Besides, this method is versatile and can potentially detect several contaminants, further increasing the benefits of this kind of measurement.

The approach is based on a portable Surface Plasmon Resonance (SPR) system using glass chips covered with a gold thin film. This metal layer is functionalized with aptamers sensitive to the presence of mercuric ions.
<table><caption>Table 1: Characteristics of current mercury detection methods<sup>17–25</sup></caption><thead><tr><th></th><th>ICP-MS<sup>a</sup></th><th>CV-AFS<sup>b</sup></th><th>CV-AES<sup>c</sup></th><th>CV-AAS<sup>d</sup></th><th>ASV<sup>e</sup></th><th>chelation<sup>f</sup></th><th>aptamer<sup>g</sup></th></tr></thead><tbody><tr><th>LOD (ng/L)</th><td>&lt; 1</td><td>&lt; 5</td><td>200</td><td>50<sup>h</sup></td><td>250</td><td>5 &times; 10<sup>-2</sup><br>(10 nM)</td><td>5 &times; 10<sup>-3</sup><br>(1 nM)</td></tr><tr><th>Advantages</th><td>- sensitivity<br>- selectivity<br>- range of detectable element</td><td>- sensitivity<br>- selectivity<br>- range of detectable element</td><td>- sensitivity<br>- range of detectable element</td><td>- sensitivity<br>- selectivity</td><td>- simple<br>- non expensive<br>- portable</td><td></td><td>- sensitivity<br>- selectivity<br>- stability<br>- regenerable<br>- ionic selectivity</td></tr><tr><th>Disadvantages</th><td>- expensive</td><td>- expensive<br>- complex sample preparation</td><td>- expensive<br>- complex (real) sample preparation<br>- spectral interferences</td><td>- expensive<br>- complex sample preparation</td><td>- difficulty with complex sample<br>- only amalgam forming</td><td>- not selective</td><td>- operating time</td></tr></tbody></table>

# Materials

## Choice of the aptamer probes

The choice of an aptamer containing thymine bases was inspired by the work of Miyake et al.<sup>26</sup> who demonstrated in 2006 the formation of a particular and selective bond between the mercuric ion and thymine by forming a complex T–Hg<sup>2+</sup>–T (THTc) (Figure 1a). Because methylmercury ion is monovalent, it can only form one bond with the thymine (Figure 1b).

<footnote><sup>a</sup>HPLC ICP-MS : High Performance Liquid Chromatography Inductively Coupled Plasma Mass Spectrometry<sup>17</sup></footnote>

<footnote><sup>b</sup>Cold Vapor - Atomic Fluorescence Spectrometry<sup>18</sup></footnote>

<footnote><sup>c</sup>Cold Vapor - Atomic Emission Spectrometry<sup>19,20</sup></footnote>

<footnote><sup>d</sup>Cold Vapor - Atomic Absorption Spectrometry<sup>21,22</sup></footnote>

<footnote><sup>e</sup>Anodic Stripping Voltammetry<sup>17</sup></footnote>

<footnote><sup>f</sup>on PEG<sup>23</sup></footnote>

<footnote><sup>g</sup>on gold nanoparticles (with aggregation)<sup>24,25</sup></footnote>

<footnote><sup>h</sup>2 ng/L with preconcentration step<sup>21</sup></footnote>
<figure><img src="image_2.png" /><figcaption>Figure 1: Bonds of mercuric ion (a) and methylmercury ion (b) with thymine DNA base</figcaption></figure>

Herein, the selected sequence for the DNA strand is composed of 22 bases in a specific order: 5'-TTCTTTCTTCCCCTTGTTTGTT-3', as proposed by Ono et al.²⁷ in the context of Fluorescence Resonance Energy Transfer (FRET) measurement. This symmetric sequence induces a folding of the strand into a hairpin shape in the presence of mercuric ions, through the formation of THTc. In this case, one strand of DNA can bind 7 Hg²⁺ ions as shown in Figure 2a. In the sequence, we can see that guanine (G) and cytosine (C) bases are also used in addition to thymines (T). The introduction of other bases in the T sequences increases the stability of the mercury-DNA complex and eases the folding by C-G hybridization. On the other hand, in the presence of methylmercury ions, the strand will not be able to fold because of the monovalence of the ion (Figure 2b). The aptamer can then bind to 14 CH₃–Hg⁺ ions but without significant conformational change.
<figure><img src="image_3.png" /><figcaption>Figure 2: Behavior of the DNA strand with (a) mercuric ions and (b) methylmercury ions</figcaption></figure>

For SPR measurements on gold coated chip, this aptamer must be efficiently immobilized
on the gold surface. Therefore, the DNA strand is modified by a dithiol Phosphoramidite
(DTPA)²⁸ at the 5' end (5'-DTPA). When reduced by Tris(2-carboxyéthyl)phosphine hy-
drochloride (TCEP), DTPA exhibits 2 thiol (R-SH) functions, allowing it to attach to the
gold surface through coordinate covalent bonds (Au-S).

Measurement devices

To characterize the interaction between thymine and the two forms of mercury described
above, two devices were used. Most of the measurements and experiments were done using
the compact 2-channels SPR shown in Figure 3a, working at a wavelength of about 680 nm
and enabling phase-sensitive measurement ("IPSOLab" SPR from PhaseLab Instrument).
The plasmonic chips were made of gold coated prisms covered by two microfluidic channels
(Figure 3b), one for the measurement, one for the reference. The test channel, here marked
by a “1”, is functionalized with aptamers while the control one, marked by a “2”, remains
unfunctionalized and can be used to monitor non-specific interactions. The flow in the
microfluidic channels was controlled by a syringes driver in withdraw mode (Figure 3c). The
analyte solution is flown through the chip via a drilled coverglass (Figure 3e).

<figure><img src="image_4.png" /><figcaption>Figure 3: Illustrations of the entire measuring device. (a) General top view of the system, with the solution suction device (syringe driver) on the left and the compact 2-channel SPR on the right. (b) Prism with microfluidic cell (channel 1 is the test channel). (c) Location of prism in compact SPR 2 channels system and tubes used to flow solutions. (d) Front view of the entire system. (e) Schematic representation of a chip (gold coated prism covered with a microfluidic cell (adhesive + drilled coverglass)).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4: Focus on test channel (channel 1). (a) Before functionalization. (b) Measure of the resonance angle after the aptamer functionalization. (c) Measure of the resonance angle after binding between aptamer and Hg<sup>2+</sup> ions and folding of the aptamer.</figcaption></figure>

In this device, the binding events between the aptamer probes and mercuric ions are tracked by monitoring an effective refractive index shift occuring at the gold layer surface as the mercuric ions are trapped by the aptamers. The high sensitivity of index monitoring in SPR stems from the optical excitation of a Surface Plasmon Polariton (SPP) wave at the interface between the gold thin film and the dielectric medium.²⁹ The light-matter interaction being enhanced by SPP. Plasmonic metals in the form of nanoparticles²⁴,³⁰ or thin films are commonly used for trace sensing. The SPP surface wave appearing at the surface of the chip metal (a gold thin film of 45 nanometers in our case) is evanescent, i.e. featuring an exponential decay from the metal surface, where the electromagnetic field is maximum. The decay length $l_d$ depends on the excitation angle and refractive indices of the glass chip and the analyte. At resonance, when the light coupling is maximum $l_d$ is about 200 nm, while the SPPs typically propagate over tenth of microns in the form of a Transverse Magnetic (TM) waves.

In this context, SPR device consists in bringing the SPPs near the resonance condition by adjusting the excitation angle. The resulting signal is characterized by measuring the reflection coefficient as function of this angle (Figure 4a). At the resonance angle, we observe
a strong decrease in the reflected beam signal due to absorption of the incident light by the free electrons, resulting in characteristic curves for the reflection coefficient both in amplitude and phase (Figure 4b). The position of the resonance angle $\theta_R$ on the angular scans gives a direct estimation of the refractive index at the surface (Figure 4c), while eventual change in the shape of the curve relates to possible surface inhomogeneities. The location of $\theta_R$ coincide with the amplitude dip minimum or the highest slope in the phase signal which is detected trough a specific interferometric scheme. By tracking the evolution of $\theta_R$, the effective index change are then followed in real-time leading to conventional SPR sensorgrams (the refractive index evolution), revealing mercury association or dissociation occurring at the sample in presence of the aptamers. In this method, the choice of a DNA ligand with a good affinity for the ion analyte present in solution is therefore of great importance for obtaining quick association and determine trace concentrations.

Subsequently, circular dichroism measurements were used to confirm hypothesis concerning the folding and non-folding of the DNA strand in the presence of mercuric and methylmercury ions respectively.³¹ These measurements were performed with a CD spectrometer J-1500 from Jasco in 10 mm quartz cuvettes.

## Equipment cleaning

To ensure the absence of potential mercury traces on the equipment, the following cleaning procedure was carried out before the measurements. The glassware was oven dried at 200°C for at least 12 hours. The plastic material (pipette, caps) was cleaned with acid. To do this, the items to be cleaned were placed in an initial resealable glass vessel containing 10% (v/v) HNO₃ acid. This container was then placed in an ultrasonic bath for 10 min (frequency: 28 kHz, power: 900 W). It was then rinsed 3 times with ultrapure water (R ≈ 18 MΩ.m). The same process (acid/ultrasonic bath + rinsing) was then repeated 2 times in a vessel containing 10% (v/v) HCl acid, with the same thorough rinsing procedure after each HCl acid bath.
# Mercury solutions preparation

## - for SPR measurement

Nine solutions were made by successive dilutions, with a concentration ranging from $10^{-6}$ to $10^{-14}$ g/L for both $Hg^{2+}$ and $CH_3-Hg^+$ ions. The solutions were made in 0.5x PBS buffer in order to be in a quasi-neutral pH controlled medium.<sup>26,32,33</sup> The different concentrations were obtained starting from a 1 g/L stock solution, followed by careful successive dilutions as detailed in the Supporting Information S1.

## - for circular dichroism measurement

In order to compare the influence of mercuric ions and methylmercury on circular dichroism, three sample were made, each containing 2 mL of aptamer solution at 10 µM, diluted in 0.5x PBS. The three samples differ in the additional component they contain: one with 2 mL of 0.5x PBS (reference solution), another with 2 mL of $Hg^{2+}$ (70 µM), and the third with 2 mL of $CH_3-Hg^+$ (70 µM). These concentrations were chosen to be in optimal measurement conditions (i.e. 1 aptamer per 7 elements captured).

Finally, to determine the number of mercuric ions required to induce DNA strand folding, we prepared several aptamer solutions with increasing concentration of mercuric ions, ranging from a 1:1 to 1:10 ratio. To do this, each solution consisted of 2 mL of aptamer solution diluted in 0.5x PBS down to 10 µM, mixed with 2 mL of a mercuric ion solution diluted in 0.5x PBS in concentration ranging from 10 to 100 µM as detailed in the Supporting Information S1.
# Results and discussions

## Surface modification monitoring

The gold surface functionalization with the DNA strands induces a substantial refractive index change at chips surface. A typical sensorgram obtained while flowing the aptamer solution in concentration $c_A = 1.67$ µM (details in Supporting Information S1) is shown in Figure 5.

<figure><img src="image_6.png" /><figcaption>Figure 5: Real-time monitoring of the gold surface modification with the selected aptamers through SPR sensorgrams given in RIU (Refractive Index Unit).</figcaption></figure>

The surface modification induced a variation of about 550 µRIU, measured with a high signal to noise ratio. The observed time constant $\tau \approx 13.5$ min was obtained by fitting the DNA-gold association with an exponential fit. This monitoring ensures a good coverage of the sensor chip is attained in about one hour as a saturation plateau is almost reached. As expected, no dissociation is observed after rinsing due to the presence of dithiol at one of the DNA strand extremity. The dissociation rate $k_{OFF}$ is therefore close to 0 and the association rate $k_{ON} \approx 1/\tau c_A$ was determined to be about $7.4 \times 10^{-4}$ µM$^{-1}$s$^{-1}$.
Trace sensing of mercury ions

While sensorgrams can be used to monitor the DNA amount at the chip surface (Supporting Information S4), the presence of mercury ions in trace concentration where found to be better accounted for through the reflection angular scans of the SPR resonance. During the flow of solutions at different concentrations (associations and dissociations of analytes), the relatively low concentrations and the presence of potential clusters made it difficult to use the sensorgrams for these steps.

Therefore, in this case, we focused our study on the analysis and interpretation of the angular scans obtained before and after mercury injection. The second scan was performed about 15 minutes to let the transient signal stabilize. The resonant angle values are available in Supporting Information S2.

These SPR angles were determined using two methods: a “classical” one, which consists of measuring the shift of the minimum in amplitude, and a method using the complex plane, which involves amplitude and phase in the expression of the complex number and consists of characterising the shift of the minimum of the norm of these complex numbers (Supporting Information S3).
<figure><img src="image_7.png" /><figcaption>Figure 6: The key steps in the SPR experiments</figcaption></figure>

The following results come from two experiments using mercuric ion and methylmercury solutions at variable concentrations. Figure 6 shows the different steps of these experiments. After air calibration of the chip, initial rinsings are performed (ethanol, water, 0.5x TBE buffer). The gold surface in channel 1 is functionalized with DNA aptamers at a slow speed (10 µL/min) until the surface is saturated, followed by rinsing with PBS 0.5x. Then, a reference angle scan is made to determine the initial resonant angle. The measurement cycles then begin, with an increment in the analyte concentration between each turn. Each analyte solution (mercury ions) is introduced simultaneously into both channels at a speed of 50 µL/min. The angle response is then measured on both channels after 15 minutes, in order to reach a steady response. A rinsing is then performed with PBS 0.5x for 15 min at
the same speed condition, before increasing the analyte concentration.

- SPR response for methylmercury CH<sub>3</sub>-Hg<sup>+</sup>

<figure><img src="image_8.png" /><figcaption>Figure 7: Detection of CH<sub>3</sub>-Hg<sup>+</sup> at different concentrations. (a) Angle scan in amplitude. (b) Angle scan in phase. (c) SPR angle shift as a function of the ion concentration obtained from the scan, using classical and complex method. Bottom inset: schematic representation of ions binding with aptamer without folding. In (c), the error bars correspond to the angle measurement uncertainty given by 2 times the standard deviations (σ = 5 × 10<sup>-3</sup> degrees) obtained on repeated angle measurements (made in a separated experience).</figcaption></figure>
Figures 7a-b represent the angle-dependent scans of the reflected light in both amplitude and phase. The observed resonant angle shift between the steps is small but rather monotonic. Small fluctuations in the dip's minimum are observed on the amplitude scans (Figure 7a), while the phase signal exhibits a more steady gradual change (Figure 7b). We can note that fluctuations in the amplitude level would make slightly erroneous the determination of a sensorgram at fixed angle, as such sensorgram assumes pure angle shift occurs, without deformation in the angle response. The interpolated angle shift across the concentration range is shown in Figure 7c showing a noticeable response over several order of magnitudes in terms of concentration.

The rather continuous response is attributed to the absence of folding of the DNA probes despite the progressive attachment of methylmercury. As mentioned before, the expectations are different for the mercuric ion which can bond to two thymine bases, entailing the onset of a folding process.

- SPR response for mercuric ion Hg<sup>2+</sup>

Similar measurements were made with mercuric ions solutions to characterize the interaction with thymine and to compare these results with those obtained with methylmercury.
<figure><img src="image_9.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 8: Detection of Hg<sup>2+</sup> at different concentrations. (a) Angle scan in amplitude. (b) Angle scan in phase. (c) SPR angle shift for the investigated range, obtained from the angle scans using classical (amplitude only) and complex method. Data points of the complex method are fitted with a Langmuir isotherm. The initial resonant position is indicated with a blue dashed line to serve as reference. Bottom inset: schematic representation of the Hg<sup>2+</sup> induced DNA folding at low concentration. The error bars were obtained as for Figure 7.</figcaption></figure>

The Figure 8a-b represent the associated SPR angle scans. In addition to a concentration-
related angle shift, more fluctuations are visible in the amplitude response. In this case, the
study of the phase (Figure 8b) somehow facilitates the reading as it is easier to observe the
appearance of a step (a shift in resonance) for concentrations greater than or equal to 1 ng/L. Beside the angle shift occurring with the concentration increases, we can note that the phase slopes also change in the inflection region. The figure 8c shows the angle shift obtained from the angle scans.

Unlike for the methylmercury ion, the presence of a sudden angular shift is clearly observed on this graph. The increase in shift initiates for concentrations ranging between $10^{-9}$ and $10^{-10}$ g/L. This observed trend was qualitatively fitted to a Langmuir isotherm (Equation 1).

$$y = \Delta_{max} \times \frac{\frac{1}{K_D} \times x}{1 + \frac{1}{K_D} \times x}, \quad (1)$$

with the fitting parameters:

* $\Delta_{max} \approx 0.0347^\circ$: the maximum amplitude shift of the isotherm,

* $K_D \approx 3.5721 \times 10^9$ g/L: the dissociation constant $\frac{k_{OFF}}{k_{ON}}$, given by the abscissa of the inflection point (at ordinate $\frac{\Delta_{max}}{2}$).

To account for the experimental data, it is necessary to include an offset term ($\approx 0.0472^\circ$) in this equation. One could interpret this offset as an interaction between the gold surface and mercury. However, previous experiments were made demonstrating the absence of any change in the SPR signal at such low concentrations of mercuric ions, when the bare gold is in contact with the analyte. It was assumed that this offset could be induced by small, hardly avoidable morphological changes in the DNA due to the presence of minute traces of mercuric ions, despite using the same solvent.

The experiences with both mercuric ion and methylmercuric ions were repeated several times and provided always similar shift behaviour in the SPR response. Given the optical response difference with the methylmercury case in presence of mercuric ions, it is tempting to interpret this observation as indicative of a DNA strand folding (cf. schematic representation Figure 8). This interpretation is further investigated using circular dichroism spectroscopy
in the next section.

Circular dichroism

We showed in the previous section that in the presence of mercuric ions, the DNA strand exhibited a hairpin folding. To further support this observation, we conducted UV circular dichroism measurements on aptamer solutions with varying concentrations of mercury ions.

In Figure 9, a modification in circular dichroism, compared to the reference, was observed
only for the solution containing mercuric ions (blue curve). Consequently, we concluded that
methylmercury does not cause a change in the configuration and thus a folding of the DNA
strand. This observation aligns with the results obtained from SPR characterization.

<figure><img src="image_12.png" /><figcaption>Figure 9: Circular dichroism measurements in UV range for aptamer solution only (dash curve), aptamer with Hg²⁺ (blue curve) and aptamer with CH₃–Hg⁺ (orange curve). All curves are raw data and the path length is equal to 10 mm.</figcaption></figure>

We also analyzed further the strand folding generated by the mercuric ions to determine
the number of mercuric ion required to generate the folding. First, the raw data obtained
were then interpolated and smoothed to accurately determine the position of the circular dichroism maximum (Figure 10a). The shift in wavelength of the maximum CD from the reference (set to 0) is shown on the Figure 10b.

<figure><img src="image_13.png" /><figcaption>Figure 10: (a) Circular dichroism for different [Hg²⁺] at 20°C (with a path length of 10 mm). (b) Corresponding circular dichroism maximum shift, also plotted on the top as relative number of Hg²⁺ by DNA strand.</figcaption></figure>

Above a certain mercury concentration, a clear step is observed in the spectral position of the circular dichroism maximum. This step, indicative of DNA strand folding, occurs when the DNA to mercuric ions ratio is equal to or higher than 1:3. To verify the stability of the folded strand, the measurements were performed with a sample temperature of 20°C and 40°C (not shown). The change in temperature had almost no impact on the measurement which confirms the stability of the formed THTc and thus of the DNA strand folding.

We note that the concentration required to observe folding with circular dichroism is naturally much higher than in the SPR measurement. The occurrence of folding, triggered by the aptamer-to-analyte ratio, necessitates much higher DNA concentrations for a discernible signal using circular dichroism due to the limited technique sensitivity. However, in SPR as well, the concentration threshold at which the signal significantly increases must also be
directly related to the DNA strands concentration on the chip surface.

Conclusions and perspectives

In this study, we have demonstrated the interaction of a specific aptamer with two distinct
forms of mercury found in the environment. The characterization of this interaction was
conducted using an SPR device and validated through circular dichroism. Both measurement
techniques supported the probable observation of the aptamer folding in the presence of
mercuric ions but not in the presence of methylmercury. The non-folding behavior of the
aptamer in the presence of methylmercury can be attributed to the valence of one for this
ion, preventing the formation of a second bond once attached on one side. This is in contrast
to mercuric ions, which have a valence of two.

The SPR measurement combined with mercury-specific DNA aptamers enabled the de-
tection of mercury traces at concentrations in the ng/L range, close to advanced detection
technique such as CV-AFS (Table 1). Moreover, the advantages of the used method and SPR
system, such as real-time measurement capability, small footprint, “low” cost, or preparation
simplicity³⁴ make this technique a promising detection method for mercury trace sensing.
As more mercury ions should be required to fold a longer DNA strand, one can expect that
modifying the strand length could offer a way to adjust the measurement sensitivity. Finally,
the same sensing platform could be tested with other DNA sequences to explore alternative
interactions with mercury ions or elements, allowing for the diversification of detectable
analytes.

Acknowledgement

Financial support of Nano'Mat (www.nanomat.eu) by the "Ministère de l'enseignement supérieur et de la recherche", the "Fonds Européen de Développement Régional" (FEDER), the "Region Grand-Est" under EPIRNA and RANGE consortium, and the "Conseil général
de l'Aube" are acknowledged. This project was supported by the Agence nationale de la recherche (ANR), under Project NOSE (Grant ANR-21-CE39-0014). This work was partly supported by the nano'mat platform and the French RENATECH+ network. This work is supported by the Graduate School NANO-PHOT (École Universitaire de Recherche, PIA3, contract ANR-18-EURE-0013).

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