Electrochemiluminescence in “Planar” Micropore for Application in Single Cell Cytokine Biosensing
Authors: Tawil Stéphane , Leroy Loic , Billon Martial , Sojic Neso , Bouchet-Spinelli Aurélie 
Published: August 2024

The immune response is a complex process involving interactions mediated by cellular secretions (cytokines). These key proteins dictate each cell’s behavior and are thus important markers to decipher the immune response. Produced in very low quantities, at the single cell level, their efficient detection is still a bottleneck for the complete understanding of the immune process.
Since the early 2000s, biosensors based on micro and nanopores have emerged for the detection and identification of biomolecules [1]. However, the main challenge of such biosensors remains the appropriate functionalization of pores for selectivity. Previsouly, we developed a contactless electrofunctionalization technique (CLEF) for the localized functionalization of the inner walls of a pore, drilled in a silicon membrane covered with SiO2 [2]. The main advantage of this technique is that it only functionalizes the pore and not the surrounding membrane. The functionalization mechanism is inspired by bipolar electrochemistry (BPE) where a voltage is applied between two electrodes positioned at each side of the pore, allowing electrochemical reactions exclusively at the restriction site, which is polarized under the applied voltage.
More recently, we have adapted CLEF to « planar » micropores i.e. restrictions in microfluidic channels, which are easier to integrate into microfluidic devices. We have successfully performed, inside of a silicon planar micropore, i) the bipolar electropolymerization of pyrrole [3] and ii) the bipolar electrochemiluminescence (ECL) [4,5], in a locally controlled manner. In this work, we aim to combine bipolar functionalization and bipolar ECL to conceive relevant biosensors for cytokine detection. To take into account the ECL requirements, polymers with higher over oxidation potential are selected. To achieve efficient localized BPE on Si, dedicate microfluidic design with two different canal thicknesses were developed and tested.
As a proof of concept, we illustrate reproducible analytes detection by ECL on silicon inside of a microfluidic device. The specific biorecognition of these analytes is achieved via aptamer and antibody recognition.
Two different strategies of functionalization were evaluated in terms of stability of the ECL behavior: 1) direct copolymerization of thiophene derivatives on bare Si and 2) copolymerization on electrodeposited Au. Moreover, since our final goal is to achieve detection of single cell secretions, the microfluidic setup was also simulated using a multiphysics simulation to ensure the device’s capability to trap and isolate single cells.
This platform should allow further development of devices for the detection of various cytokines at the single cell level.
Ref :
[1] M. Tsutsui, et al., Sci. Rep. 2017, 17371
[2] A. Bouchet et al. 2009 Small, 5: 2297-2303.
[3] Abdulghani Ismail et al. Adv.Mater. Technol.2021, 6, 2001154
[4] Silvia Voci et al. 2020 J. Electrochem. Soc. 167 137509
[5] Abdulghani Ismail et al., Analytical Chemistry 2019 91 (14), 8900-8907

Figure 1