Rapid, Low‑Cost Microfluidic Electrochemical Sensor Fabricated Directly on Screen‑Printed Electrodes for Ultra‑Sensitive PSA Detection

Background

Microfluidic systems manipulate volumes from nanoliters to picoliters within micrometer‑scale channels, enabling high‑throughput, reagent‑efficient analyses. Their integration with electrochemical sensors confers remarkable sensitivity, rapid response, and minimal instrumentation requirements, making them ideal for on‑site biomedical diagnostics.

Electrochemical readouts are inherently compatible with microfluidics, eliminating bulky optics and simplifying fabrication. The synergy of microfluidics and SPEs—already widely used in clinical point‑of‑care devices—promises a fully integrated, disposable sensor platform.

In this work, we fabricate CASPE‑MFDs by bonding PDMS microchannels onto a glass‑coated SPE via oxygen plasma activation. The device is tested for PSA quantification in phosphate buffer and human serum using chronoamperometry (CA) and square‑wave voltammetry (SWV).

a Fabrication of PDMS microchannels via SU‑8 photolithography.b Assembly of the CASPE‑MFD: PDMS channels bonded to a glass‑coated SPE containing two gold working/counter electrodes and a silver pseudo‑reference electrode.c Photograph of the completed CASPE‑MFD.

Materials and Methods

Reagents and Materials

PSA, HRP‑conjugated anti‑PSA, biotinylated anti‑PSA, streptavidin magnetic beads, BSA, hydroquinone, Tween‑20, H₂O₂, and ferrocenecarboxylic acid were sourced from commercial suppliers (Petsec Energy Ltd., Fisher Scientific, Sigma‑Aldrich). PDMS pre‑polymer and curing agent were obtained from Dow Corning. All buffers were prepared with ultrapure water (Millipore Milli‑Q).

Instrumentation

Fluorescence imaging: Olympus U‑CMAD3. Plasma cleaning: Harrick PDC‑32G. Electrochemical measurements: CHI 760B potentiostat (three‑electrode setup: gold working/counter, silver pseudo‑reference).

Microfluidic Chip Fabrication

PDMS channels were patterned on a silicon wafer coated with SU‑8 2075, exposed to UV light, and cured. The SPE substrate was coated with a thin glass layer via a sol‑gel mixture (TEOS:MTES:ethanol:water = 1:1:1:1) and cured at 65 °C overnight. Both PDMS and SPE surfaces were activated by 30 s O₂ plasma before alignment and bonding, forming a hermetic seal.

Chronoamperometric Experiments

Measurements were performed in 1× pH 7.4 PBS containing 4.5 mM hydroquinone and 0.1 mM H₂O₂. The CASPE‑MFD was first activated by cycling between 0.5–1.5 V in 0.5 M H₂SO₄. Then, 50 µL of magnetic‑bead‑conjugated anti‑PSA antibody was introduced at 50 µL min⁻¹, followed by blocking, PSA incubation, HRP‑anti‑PSA addition, and finally the electroactive solution. Chronoamperometry was carried out at –2.0 mV versus the silver pseudo‑reference, recording the steady‑state current after 30 s.

Results and Discussion

Device Characterization

Fluorescent microbeads injected into the CASPE‑MFD filled the channels uniformly without bubbles, confirming robust fluidic performance at flow rates up to 100 µL min⁻¹ (Figure 2).

a Fluorescent microbead infiltration on the SPE. b Bright‑field image of the filled CASPE‑MFD. c Enlarged view of the fluorescence distribution.

Cyclic voltammetry with 0.5 mM ferrocenecarboxylic acid confirmed reversible electron transfer (E_pa–E_pc = 62 mV) and diffusion‑controlled kinetics (i_pa ∝ √v). The currents in the CASPE‑MFD matched those in bulk solution, indicating that the microfluidic architecture does not compromise sensitivity (Figure 3).

a CVs at scan rates 10–350 mV s⁻¹. b Calibration plots of i_pa and i_pc versus √scan rate.

PSA Detection Performance

Chronoamperometric curves (Figure 5a) show a monotonic increase in current with PSA concentration (0–10 ng mL⁻¹). The linear range (0.001–10 ng mL⁻¹) yields a regression of I (µA) = 14.87 + 3.927 × log C_PSA (R² = 0.9985). The LOD of 0.84 pg mL⁻¹ surpasses commercial assays (0.1 ng mL⁻¹) by >100‑fold and outperforms comparable microfluidic sensors.

Square‑wave voltammetry (SWV) produced parallel results, confirming the robustness of the electrochemical readout (Figure 5c).

a Chronoamperometry for 0–10 ng mL⁻¹. b Linear plots in buffer (blue) and human serum (red). c SWV curves. d Linear fit for SWV (R² = 0.9884).

Selectivity and Real‑Sample Analysis

In human serum, the CASPE‑MFD maintained the same linearity and LOD, demonstrating compatibility with complex biological matrices. No significant interference was observed from potential matrix components, underscoring the assay’s selectivity.

Conclusions

The CASPE‑MFD combines low‑cost fabrication, microfluidic control, and SPE electrochemistry to deliver ultra‑sensitive PSA detection (<0.85 pg mL⁻¹) in a portable format. Its ease of assembly, reproducibility, and potential for multiplexing make it a promising platform for point‑of‑care diagnostics of cancer biomarkers, electrolytes, and nucleic acids.

Abbreviations

MFDs

Microfluidic devices

CASPE-MFDs

Screen‑printed electrode‑based microfluidic devices

PDMS

Polydimethylsiloxane

PSA

Prostate‑specific antigen

CA

Chronoamperometry

SWV

Square wave voltammetry

LOD

Limit of detection

HRP

Horseradish peroxidase

TEOS

Tetra‑ethoxy silane

MTES

Metyl‑triethoxysilane

BNP

B‑type natriuretic peptide