# Surface organization of aptamers via diazonium grafting: A key parameter in label-free electrochemical sensing

Teodora Lupoi, Bogdan Feier, Florence Geneste, Cecilia Cristea, Yann R. Leroux

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Teodora Lupoi, Bogdan Feier, Florence Geneste, Cecilia Cristea, Yann R. Leroux. Surface organization of aptamers via diazonium grafting: A key parameter in label-free electrochemical sensing. Electro-chemistry Communications, 2025, 178, pp.108000. 10.1016/j.elecom.2025.108000. hal-05246529

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# Surface organization of aptamers via diazonium grafting: a key parameter in label-free electrochemical sensing

Teodora Lupoi¹,², Bogdan Feier², Florence Geneste¹, Cecilia Cristea², Yann R. Leroux¹*

¹ Univ Rennes, CNRS, ISCR, UMR 6226, F-35000 Rennes, France

² "Iuliu Hatieganu" University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Analytical Chemistry, 400349, Cluj-Napoca, Romania

*Correspondence: yann.leroux@univ-rennes.fr

## Abstract

The spatial arrangement of biorecognition molecules on the sensor surface plays a critical role in determining the performance of electrochemical biosensors. In this work, we report a covalent and tunable immobilization strategy using aryl diazonium chemistry to functionalize carbon electrodes with ethynyl groups protected by trimethylsilyl (TMS) or triisopropylsilyl (TIPS) moieties. After deprotection, an azide-modified aptamer (APT) specific to diclofenac (DCF) was immobilized via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Although the TMS and TIPS groups differ in size by only 1.7 Å, this small variation significantly influenced APT spacing and sensor performance. The TIPS-based sensor displayed a nearly fourfold increase in signal response compared to the TMS-based counterpart, achieving a limit of detection of 17.95 µM. These results underscore the importance of nanoscale molecular design in optimizing label-free aptasensor sensitivity.

**Keywords:** controlled immobilization, click-chemistry, diazonium grafting, electrochemical sensor, controlled immobilization

## 1. Introduction.

The performance of aptamer-based electrochemical sensors is strongly influenced by the method of APT immobilization and their spatial organization on the surface [1–4]. Covalent attachment strategies are generally preferred over non-covalent ones due to their improved stability, robustness, and control over the aptamer (APT) density. Among these, self-assembled monolayers (SAMs) on gold are widely used but often suffer from poor reproducibility, instability, and uncontrolled molecular distribution, which negatively impact sensor performance [5–7].

A key factor in optimizing aptasensor response is the controlled spacing between immobilized aptamers, which influences target accessibility and signal transduction. Recent studies have shown that increasing the distance between aptamers using nanostructured supports
or by tuning loading density can significantly enhance sensitivity [8,9]. However, these methods often require complex architectures or lack robust covalent attachment.

Click chemistry, particularly copper-catalyzed azide–alkyne cycloaddition (CuAAC), offers a reliable alternative for APT immobilization, forming stable triazole linkages under mild conditions [10–12]. To create surfaces bearing reactive alkyne groups, diazonium salt chemistry can be used to graft organic layers covalently onto carbon electrodes. Importantly, the structure of the diazonium salt, especially the steric hindrance of substituents, can be tailored to control the density and distribution of functional groups on the surface [13,14]. Lately, some pioneering work has been done on the immobilization of aptamers via click chemistry for the fabrication of electrochemical [15] and graphene field-effect transistor (GFET) biosensors [16,17]. While Onas et al. investigated the influence of the APT spacing in a system where the redox probe was immobilized on the APT, the control of spacing in label-free detection remains unexplored [15]. Label-free methods are faster, simpler, eliminate the need for costly and time-consuming labeling steps, and conserve the primary sequence of the aptamer and original affinity [18].

In this work, we investigated the effect of APT surface spacing on sensor performance using two structurally similar aryl diazonium salts bearing different protecting groups: TMS and TIPS. These led to monolayers with different steric hindrance and, upon deprotection, yield terminal alkyne groups suitable for CuAAC-based APT immobilization. Using a label-free electrochemical detection approach, we evaluated the impact of this nanoscale design on the sensitivity of a diclofenac (DCF) aptasensor. Our results showed that a small increase in spacer size (~1.7 Å) dramatically enhanced the electrochemical response, highlighting the critical role of APT distribution in target binding and signal generation. This study provides a simple, tunable, and robust method for improving aptasensor performance via molecular-level design of surface architecture.
<figure><img src="image_2.png" /><figcaption>Scheme 1. A. Schematic representation of the sensor fabrication. First, a carbon electrode is functionalized via electroreduction of ethynylaryl diazonium salt protected by two different groups (TMS or TIPS), followed by a chemical deprotection step and a copper-catalyzed click immobilization of the azide-modified aptamer. B. Sensor detection principle. The DPV oxidation current of an external redox probe (ferrocyanide) is recorded before and after diclofenac detection.</figcaption></figure>

## 2. Materials and methods.

### 2.1 Reagents.

All reagents were of analytical grade and used as received. NaCl, KCl, Na₂HPO₄, KH₂PO₄, tetrabutylammonium hexafluorophosphate (NBu₄PF₆), tetra-n-butylammonium fluoride (TBAF), CuSO₄, NaHCO₃, EDTA, L-ascorbic acid, DCF sodium salt, potassium ferrocyanide, THF, acetone and acetonitrile were purchased from Sigma-Aldrich. The diazonium salts 4-((triisopropylsilyl)ethynyl)benzenediazonium tetrafluoroborate (TIPS-Eth-ArN₂⁺) and 4-((trimethylsilyl)ethynyl)benzenediazonium tetrafluoroborate (TMS-Eth-ArN₂⁺) were synthesized according to the published procedure [19]. The APT sequence targeting DCF was selected from the literature, with a dissociation constant of 42.7 nM [20]: 5'-ATA-CCA-GCT-TAT-TCA-ATT-GCA-ACG-TGG-CGG-TCA-GTC-AGC-GGG-TGG-TGG-GTT-CGG-TCC-AGA-TAG-TAA-GTG-CAA-TCT-3'. It was modified at the 5' end with an azidobutyrate group for click chemistry immobilization. For additional immobilization investigations, the same APT sequence was used with a ferrocene label at the 3' end. Aptamers were synthesized and purified by HPLC-RP by Eurogentec (Belgium). They were received as lyophilized powder and dissolved in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4), aliquoted, and stored at
-20 °C. Before immobilization, aptamers were thermally folded by heating to 90 °C for 5 min, followed by rapid cooling to -20 °C.

## 2.2 Surface modification procedure.

Home-made glassy carbon electrodes (GCE, 3 mm diameter) were first polished with 0.3 µm alumina slurry, thoroughly rinsed with ultrapure water and electrochemically characterized in 1 mM ferrocyanide solution until a stable peak-to-peak separation (ΔE) close to 60 mV was obtained for each electrode. The GCEs were modified by electrochemical grafting of 10 mM diazonium salt in 0.1 M NBu₄PF₆ in acetonitrile via cyclic voltammetry (CV) (0.6 to -0.75 V vs. SCE, 5 cycles, 50 mV s⁻¹). Because the grafting of diazonium salts is a radical process, in which the generated aryl radicals can also react with already grafted aryl groups, protective groups such as TMS and TIPS were employed to sterically hinder multilayer formation. The surface was rinsed with acetonitrile and acetone, then dried under argon. For the subsequent deprotection of the alkyne group, 0.1 M TBAF in THF was used. The reaction was carried out for 20 minutes, after which the surface was thoroughly rinsed with THF and acetone. Click chemistry was performed by incubating the electrode with 1 µM azide-modified APT and in situ-generated Cu(I) (0.01 M CuSO₄ and 0.01 M sodium ascorbate) in PBS buffer for one hour. Excess copper was removed with 0.1 M EDTA, followed by rinsing in ultrapure water.

## 2.3 Analysis.

Electrochemical measurements were performed using an Originals potentiostat with a standard three-electrode setup. A saturated calomel electrode (SCE) and a Pt electrode serve as reference and counter electrode respectively. The DPV measurements had the following parameters: starting potential: -0.3 V vs SCE, end potential: 0.6 V vs SCE, modulation amplitude 50 mV, step potential 5 mV, pulse time 25 ms, scan rate 10 mVs⁻¹. X-ray photoelectron spectroscopy (XPS) analyses were performed using a Thermo Scientific NEXSA G2 spectrometer (Al Kα source, 1486.6 eV). Survey and high-resolution spectra were acquired and processed using CasaXPS. For XPS studies, pyrolyzed photoresist film (PPF) was used as the carbon substrate, prepared following a published protocol [21]. Silicon (Si) is often observed in XPS when using PPF as substrates, as the underlying surfaces used to make these PPF substrates are Si wafers.

Sensor response was calculated using equation (1), where I<sub>Blank</sub> corresponds to the peak current of K₄[Fe(CN)₆] recorded after APT immobilization and incubation in blank buffer (without DCF), and I<sub>DCF</sub> is the peak current recorded after incubation with DCF.

$$ (1) \quad \textit{Relative current increase (\%)} = \frac{|I_{\text{Blank}} - I_{\text{DCF}}|}{I_{\text{Blank}}} \times 100 $$

The LOD was determined based on 3 times the standard deviation of 5 blank measurements, following: St-Sb≥ 3σ, where St is the signal at the LOD, Sb is the average blank response and σ represents the variability in the blank signal.

# 3. Results and discussions.
### 3.1 Electrode functionalization and aptamer immobilization.

Our sensing strategy involved covalent modification of GCEs with terminal alkyne moieties via electroreduction of protected aryl diazonium salts, followed by click-chemistry immobilization of an azide-modified APT specific to DCF (Scheme 1). To favor monolayer formation and control inter-APT spacing, two diazonium salts were employed, bearing either a TMS or a TIPS protecting group.

CV during diazonium reduction revealed irreversible cathodic peaks with no return oxidation and no further activity in subsequent scans, consistent with aryl radical formation and covalent surface grafting (Figure 1A and B). This process leads to the formation of thin organic films, which, due to their high reactivity, can block surface electron transfer.

To monitor surface accessibility, ferrocyanide was used as a redox probe in solution. After diazonium grafting, its signal was completely suppressed, confirming surface passivation. Upon deprotection with TBAF, partial recovery of the ferrocyanide response was observed, particularly for the TIPS-modified surface (Figure 1C and D). This suggests the successful removal of the protecting group and exposure of the alkyne functionality without the complete removal of the organic film. The observed differences between the TMS and TIPS surfaces can be attributed to the relative sizes of the protecting groups. Estimated spherical diameters are 0.75 nm for TMS and 0.92 nm for TIPS, compared to 0.62 nm for ferrocyanide (Chem3D modeling). The larger TIPS group provides greater spacing between grafted sites, allowing more efficient redox probe access and improved molecular organization.

Click chemistry was then employed to immobilize a DCF-specific APT bearing a 5'-azidobutyrate group. After this step, a significant decrease in the ferrocyanide signal was again observed on both surfaces, indicating successful APT attachment and blockage of the surface. These electrochemical trends support the conclusion that both diazonium-based platforms enable robust and spatially defined APT immobilization. Blank experiments were performed on a non-grafted carbon electrode, showing no aptamer immobilization.
<figure><img src="image_3.png" /><figcaption>Figure 1. CVs of A) TMS-Eth-Ar-N₂⁺ and B) TIPS-Eth-Ar-N₂⁺ reduction from a 10 mM solution in 0.1 M NBu₄PF₆. CVs in 1 mM ferrocyanide solution in PBS buffer 0.15 M (pH 7.4) in water of the GCE surface modified with C) TMS-Eth-Ar-N₂⁺ and D) TIPS-Eth-Ar-N₂⁺. Bare GCE (black), after diazonium salt reduction (green), after TBAF deprotection (red), after immobilization of 1 µM APT solution for 1 h by click chemistry (blue).</figcaption></figure>

To further confirm APT presence, we decided to immobilize a redox-label APT specific to DCF bearing the same azidobutyrate group onto the 5'end position. With this new APT, the same protocol was applied to modify the carbon substrates. After immobilization of the APT, it is possible to detect its presence on the modified carbon surfaces by direct oxidation of the ferrocenyl redox label, as shown in Figure 2A. A linear dependence of the peak current with the scan rate highlights the effective immobilization of the modified aptamer onto the carbon surface. XPS was also conducted on the surfaces before and after APT grafting (Figure 2B). While only traces of nitrogen (N) can be observed on the carbon substrate before APT immobilization (except the presence of carbon (C) and oxygen (O) that come from the bare carbon substrate itself), an increase in N signal and appearance of peaks at binding energies corresponding to iron (Fe) and phosphorus (P) are observed once the carbon substrate is modified with the redox-label APT. P and N are elements associated with the APT DNA backbone, while trace Fe comes from the redox-labelling of the APT. These observations fully support the successful functionalization of carbon substrate with aptamers via the presented two-step strategy.
<figure><img src="image_4.png" /><figcaption>Figure 2. A) CVs recorded at various scan rates in PBS (pH 7.4) of the GCE modified with the aptamer functionalized with azide and ferrocene. Inset: linear dependence of the anodic peak current ($R^2$ = 0.996) and the cathodic peak current ($R^2$ = 0.992) on the scan rate. B) XPS survey before (black) and after (blue) the immobilization of the ferrocene-modified APT.</figcaption></figure>

### 3.2 Diclofenac detection and effect of surface architecture.

All functionalization steps were also monitored by DPV, and the results were consistent with the CV findings. Additional peaks were observed, likely corresponding to surface oxygen-containing species, which are known to influence the electrochemical behavior of carbon electrodes in a manner that depends on both the electrode and the experimental conditions. Label-free detection of DCF was evaluated via DPV using ferrocyanide as the redox probe in solution. Both TMS- and TIPS-based aptasensors were incubated with DCF and subsequently rinsed. At 10 µM DCF, no clear signal change was observed. However, at 50 µM, an increase in ferrocyanide oxidation current was detected, more pronounced for the TIPS-based sensor (Figure 3). The signal change observed in the presence of DCF is due to the aptamer conformational changes, reducing surface hindrance and allowing increased access of the external redox probe ($K_4[Fe(CN)_6]$) to the electrode surface. Quantitatively, the relative current increase for the TIPS sensor reached 92.6%, compared to 23.5% for the TMS sensor, a nearly fourfold difference. This enhancement demonstrates the critical role of APT spacing: despite a seemingly minor 1.7 Å difference between protecting groups, the resulting molecular organization significantly affects target accessibility and sensitivity. The TIPS platform enables a more open surface configuration, facilitating effective DCF binding.
<figure><img src="image_5.png" /><figcaption>Figure 3. DPV analysis in 1 mM ferrocyanide solution in PBS buffer in water of the GCE surface after: electroreduction of protected diazonium salt with A) TMS and B) TIPS (green), deprotection with TBAF (black), APT immobilization by click chemistry (blue), blank incubation (grey) and incubation with 10 µM DCF (orange) and 50 µM DCF (red).</figcaption></figure>

A calibration curve was established using the TIPS-modified platform (Figure 4A to C). The response increased with DCF concentration up to 70 µM, after which a plateau was observed, indicating saturation of APT binding sites. This is typical of a Langmuir-like profile. From the linear region, the limit of detection (LOD) was calculated as 17.95 µM. These results confirm the advantage of rational molecular spacing in aptasensor design, leading to superior performance in a label-free configuration.

<figure><img src="image_6.png" /><figcaption>Figure 4. A) DPV response in 1 mM ferrocyanide solution in PBS buffer of the aptasensor after incubations in different DCF concentrations. B) The percentage of increase in current intensity after DCF incubations in the range of 20 – 90 µM. C) The linear calibration curve. Error bars represent standard deviations and are based on 3 replicates.</figcaption></figure>

**4. Conclusions.**
This study demonstrates that fine-tuning of the spatial organization of aptamers through the rational design of surface-bound molecular spacers significantly impacts the performance of label-free electrochemical aptasensors. By employing diazonium salts bearing TMS and TIPS protecting groups, we achieved covalent functionalization of carbon electrodes with controlled APT spacing. Despite a minimal size difference of only 1.7 Å between the two protecting groups, the TIPS-based platform exhibited markedly enhanced sensitivity toward DCF detection, highlighting the critical role of steric effects and target accessibility. These findings underline the importance of nanoscale control in biosensor surface engineering and offer a straightforward strategy for improving sensor performance without altering the biorecognition element itself. Ongoing research in our laboratory focuses on other parameters influencing biosensor performances, with the aim of producing effective, sensitive, and robust biosensors.

**Funding:** This work received financial support under the EUR LUMOMAT project and the Investments for the Future program ANR-18-EURE-0012 Nanosens.

**Acknowledgments:** T.L. thanks the program "Bourses du Gouvernement Français France Excellence" for her scholarship. XPS measurements have been performed on ASPHERYX platform (ScanMAT, UAR 2025, University of Rennes-CNRS; CPER MAT &Trans 2021-2027). The authors are grateful to Dr. C. Lagrost for her assistance in recording the XPS spectra.

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