# Fine-tuning the Ratio of Tris(2-carboxyethyl) Phosphine and Mercaptohexanol Concentrations for Thiolated Oligonucleotide Immobilization on Platinum Electrodes within Microfluidic Platforms

Choayb Omar, Martina Freisa, Hiu Mun, Djamila Kechekche, Laurent Couraud, David Bouville, Téo Baptiste, Thi Hong Nhung Dinh, Isabelle Le Potier, Jean Gamby

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Choayb Omar, Martina Freisa, Hiu Mun, Djamila Kechekche, Laurent Couraud, et al. Fine-tuning the Ratio of Tris(2-carboxyethyl) Phosphine and Mercaptohexanol Concentrations for Thiolated Oligonucleotide Immobilization on Platinum Electrodes within Microfluidic Platforms. MNE 2024, Sep 2024, Montpellier, France. [hal-05347249](https://hal.science/hal-05347249v1)

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Submitted on 4 Nov 2025

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# Fine-tuning the Ratio of Tris(2-carboxyethyl) Phosphine and Mercaptohexanol Concentrations for Thiolated Oligonucleotide Immobilization on Platinum Electrodes within Microfluidic Platforms

Choayb Omar<sup>a</sup>, Martina Freisa<sup>a</sup>, Hiu Mun MAN<sup>a</sup>, Djamila Kechekche<sup>a</sup>, Laurent Couraud<sup>a</sup>, David Bouville<sup>a</sup>, Téo Baptiste<sup>a</sup>, Thi Hong Nhung Dinh<sup>a</sup>, Isabelle Le Potier<sup>a</sup>, and Jean Gamby<sup>a,*</sup>

<sup>a</sup> Centre de Nanosciences et de Nanotechnologies, Université Paris-Saclay, CNRS, Palaiseau 91120, France
e-mail: jean.gamby@universite-paris-saclay.fr

In this work, we present an approach aimed at examining how varying the ratio of tris(2-carboxyethyl)phosphine (TCEP) and mercaptohexanol (MCH) affects the efficiency of oligonucleotide functionalization on platinum microelectrodes in a microfluidic device. We have determined the optimal concentrations of these substances that promote efficient arrangement and uniform dispersion of probes and demonstrated the sensitivity of the DNA sensor in complex samples. These insights gained from microfluidic experimentation hold great potential for a wide array of applications in bioanalysis and clinical diagnostics.

The microfluidic device was fabricated following the work developed in our group ¹⁻⁴. The fabrication process includes the optical photolithography of electrode patterns on glass substrate, followed by the metal deposition of a 50 nm layer of platinum on top of 5 nm titanium. The fluidic channels were achieved by the lithography of PDMS on an SU8 mask. The PDMS and glass substrate were assembled by O₂ plasma-activated bonding. The fabricated, ready-to-use device can be seen in **Figure 1(a)**.

The DNA probe GP8 was first diluted in TCEP to prevent the reformation of disulfide bridges and the bare Pt electrodes were pretreated to ensure a clean surface. The electrodes were then immobilized in 10⁻⁶ M DNA probe with or without MCH for two hours as shown in **Figure 1(b)**. This is followed by an incubation of the complementary DNA sequence of GP8 (target DNA) for 30 minutes. An electrochemical characterization by cyclic voltammetry (CV) was carried out after each step described, in an electrolyte solution of 20 mM ferrocyanide/ ferricyanide in 0.5 M NaCl, under flow conditions at 0.5 µL s⁻¹.

**Figure 2(a)** shows the cyclic voltammograms recorded in 20 mM [Fe(CN)₃⁻] / [Fe(CN)₄⁻] at bare platinum electrode (black curve), after immobilization of target DNA without TCEP (red curve), and after immobilization of target DNA with TCEP (blue curve)⁵. This demonstrates the clear hindrance of platinum-thiol bonds caused by the disulfide bridges between the thiol functions at the ends of DNA strands. We have evaluated the influence of TCEP concentration on DNA probe functionalization. **Figure 2(b)** demonstrates the evolution of the surface coverage rate against the concentration of TCEP concentrations, with a fitted curve simulated using the Hill-Langmuir isotherm equation⁵. The optimal concentration was determined to be 5 mM and the obtained values of K<sub>a</sub> (TCEP concentration at half-occupancy) and n (the Hill cooperativity coefficient) are (0.28 ± 0.07) mM and (2.99 ± 0.69), respectively. Lastly, with the optimized ratio of MCH and TCEP, we have successfully detected the DNA hybridization of the complementary target sequence to GP8 probe ranging from 10⁻¹⁴ M to 10⁻⁷ M in the presence of 50% cell lysis solution (containing lysed bacteria *Escherichia coli* cells, TRIS-HCl buffer and lysing reagent BPER) by cyclic voltammetry as shown **Figure 3(a)**. **Figure 3(b)** shows the calibration curve against the logarithm of target concentration.

In conclusion, we have demonstrated the importance of optimizing TCEP and MCH concentrations in enhancing the efficiency of oligonucleotide functionalization in DNA biosensors, laying the groundwork for improved DNA hybridization processes, with excellent specificity and sensitivity. We also have shown the successful detection of DNA hybridization in the presence of real, complex samples (lysed *Escherichia coli* cells) over a wide range of concentrations. These results contribute to the advancement of direct nucleic acid biosensing, as well as the broader field in clinical diagnostics.

## REFERENCES:

[1] M.-C. Horny et al., Lab Chip, 16 (2016) 4373–4381.

[2] C. Poujouly et al., Electrochem. Comm., 137 (2022) 107262.

[3] C. Poujouly et al., Front. Chem., 10 (2022) 868909.

[4] M.-C. Horny et al., J. Chem. Educ., 101 (2024) 605–611.

[5] C. Omar, Ph.D. Thesis, Chapter 6 – DNA detection on Pt, C2N/Université Paris-Saclay, 2023.
<figure><img src="image_2.png" /><figcaption>Figure 1. (a) Ready-to-use fabricated microfluidic device consisting of 3 microfluidic channels and 6 pairs</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>of platinum microelectrodes. (b) Effect of MCH during the stages of construction of the DNA biosensor.</figcaption></figure>

<caption>**Figure 2.** (a) Voltammograms recorded in an electrolyte solution of 20 mM ferri-/ferro-cyanide in 0.5 M NaCl at bare platinum electrode (black curve), after immobilization of the probe DNA sequence without TCEP (red curve), after immobilization of the probe DNA sequence with TCEP (blue curve). (b) Effect of TCEP on the functionalization of DNA probes. Experimental evolution of the surface coverage rate simulated with the Hill-Langmuir isotherm equation.</caption>

<figure><img src="image_4.png" /><figcaption>**Figure 3** (a) Cyclic voltammograms recorded in 20 mM ferri-/ferro-cyanide in 0.5 M NaCl at bare platinum electrode (black curve), after immobilization of probe DNA with TCEP and MCH (red curve), 50% cell lysis solution (lysed Escherichia coli in Tris-HCL and BPER) (blue curve), and increasing concentrations of target DNA from 10 x 10<sup>-14</sup> M to 10 x 10<sup>-7</sup> M, inset shows a zoom to the curves at -0.2 V. (b) Calibration curve of the normalized currents from DNA hybridization of the target sequence complementary to the GP8 probe sequence in the presence of 50% cell lysis solution. The red point represents the “blank” recorded in 50% cell lysis solution without any target DNA. Each point corresponds to 6 experiments.</figcaption></figure>