3D printed electrode-microwell system: a novel electrochemical platform for miRNA detection

Abstract

3D printing has enabled the ability to make creative electrochemical well designs suitable for a wide field of electrochemical sensing. The demand for robust electrochemical systems is particularly high in diagnostics, where the rapid detection of emerging biomarkers associated with severe diseases is critical for rapid medical decision-making. This study is aimed at developing a fully 3D-printed electrochemical sensing device featuring a three-electrode system fabricated from conductive printing materials and incorporating a microwell as the sensing platform. The assay principle of a robust electrochemical screen-printed sensor was adapted for this platform, incorporating a well-structured design to enhance fluid control. This structure ensured the uniform distribution of reagents across the sensing surface, improving the reproducibility and consistency of measurements and enabling the reliable detection of a microRNA target associated with lung cancer. The detection process was based on the hybridization of the target miRNA with an immobilized DNA probe labeled with methylene blue as a redox mediator. The sensor was thoroughly characterized and optimized, achieving a dynamic detection range of 0.001 to 400 nM and a lower limit of detection compared to screen-printed sensors, down to the picomolar level. Furthermore, the sensor demonstrated high selectivity for the target miRNA compared to other miRNA sequences, proving its specificity. These results highlighted the potential of 3D printing technology for the development of sensitive and selective tools for biomarker detection, making it a valuable complementary method in the field of diagnostics.

Keywords: 3D printing; Conductive PLA; Diagnostics; Electroanalysis; MiRNA; Square wave voltammetry.

Fig. 1

A Production of FFF microwell with click-in electrodes. The figure shows the printed three-electrode setup, with a silver chlorinated reference and the well as printed. The electrodes are clicked into the microwell, with the ohmic connection produced through the embedding of a platinum wire for connection to the potentiostat. B Each stage of the process is illustrated, from initial design and fabrication to the assay’s detection principle

Fig. 2

Characterization of microwell for gold deposition using ferricyanide. A Cyclic voltammograms of 5 mM ferricyanide in 1 M KCl at 0.05 V s⁻¹ before (black line) and after (red line) gold electrodeposition. B Histograms of current and ∆Ep values before and after gold deposition. Data shown as mean ± SD, where n = 6

Fig. 3

Anti-miRNA probe optimization study. Various probe concentrations ranging from 25 to 200 nM were tested in the presence of 20 nM of target miRNA-4676. Histograms were plotted for each probe concentration against the corresponding signal change %. Data are shown as mean ± SD, n = 3

Fig. 4

A Calibration curve obtained in PBS with increasing concentrations of miR-4676, ranging from 0.001 to 400 nM. The inset shows the SWV curves corresponding to each concentration. The blank sample is represented by the black line. SWV parameters: teq: 5 s; Estart: 0.1 V; Estep: 0.001 V; amplitude: 0.01 V; frequency: 50 Hz. B A selectivity study of 50 nM anti-miR-4676 specific DNA probe was performed in the presence of 20 nM of the target mir-4676 and 20 nM of three interferent miRNAs, namely: mir-625-5p, mir-224-5p, and mir-101-5p. Data are shown as mean ± SD, n = 3

Fig. 5

Histograms showing the signal change percentage for 1% spiked commercial serum with miRNA target concentration ranging from 0.02 to 100 nM. The inset shows the SWV curves corresponding to each concentration. Data are shown as mean ± SD, n = 3