Engineering a Point-of-Care Paper-Microfluidic Electrochemical Device Applied to the Multiplexed Quantitative Detection of Biomarkers in Sputum

Abstract

Health initiatives worldwide demand affordable point-of-care devices to aid in the reduction of morbidity and mortality rates of high-incidence infectious and noncommunicable diseases. However, the production of robust and reliable easy-to-use diagnostic platforms showing the ability to quantitatively measure several biomarkers in physiological fluids and that could in turn be decentralized to reach any relevant environment remains a challenge. Here, we show the particular combination of paper-microfluidic technology, electrochemical transduction, and magnetic nanoparticle-based immunoassay approaches to produce a unique, compact, and easily deployable multiplex device to simultaneously measure interleukin-8, tumor necrosis factor-α, and myeloperoxidase biomarkers in sputum, developed with the aim of facilitating the timely detection of acute exacerbations of chronic obstructive pulmonary disease. The device incorporates an on-chip electrochemical cell array and a multichannel paper component, engineered to be easily aligned into a polymeric cartridge and exchanged if necessary. Calibration curves at clinically relevant biomarker concentration ranges are produced in buffer and artificial sputum. The analysis of sputum samples of healthy individuals and acutely exacerbated patients produces statistically significant biomarker concentration differences between the two studied groups. The device can be mass-produced at a low cost, being an easily adaptable platform for measuring other disease-related target biomarkers.

Keywords: chronic obstructive pulmonary disease; electrochemical biosensing; magnetic nanoparticle-based immunoassay; multiplexed detection; paper microfluidics; point-of-care rapid tests.

Figure 1

Components and assembly of the paper-microfluidic electrochemical device. (A) Photograph of the individual device components, including the two-electrode array chip containing five electrochemical cells and the paper component with five fluidic channels patterned by wax printing and the different parts of the PMMA cartridge. A spring-loaded connector and five neodymium magnets are inserted in the top and bottom layers, respectively. (B) Photograph of the assembled device, whose dimensions (W × L × H) are 48 × 67 × 19 mm³. (C) Scheme of the different components being aligned and assembled to construct the (D) overall device.

Figure 2

Electrochemical detection of biomarkers. (A) Reactions involved in the detection using ferrocenemethanol (Fc-MeOH) as redox mediator for both MPO biomarker and HRP label. The ferrociniummethanol cation [Fc-MeOH]⁺ generated by the enzymes is reduced back to Fc-MeOH at the electrode surface. (B) Cyclic voltammogram recorded with a single on-chip two-electrode electrochemical cell in an acetate buffer pH 4.5 solution containing both Fc-MeOH and [Fc-MeOH]⁺ at a scan rate of 50 mV/s. A set potential of −0.15 V was chosen for the subsequent chronoamperometric measurements. (C) Representative raw chronoamperometric responses recorded for 0 to 2000 pg/mL IL-8 solutions. The current responses recorded at 1.6 s were used as analytical signals.

Figure 3

Dose–response calibration curves in the semi-logarithmic plot. The current responses recorded at 1.6 s were used as analytical signals. Each data point represents the mean value of three replicates carried out consecutively for (A) MPO, (B) IL-8, and (C) TNF-α biomarkers. The standard deviation values of these replicates are represented as error bars.

Figure 4

Bar graphs showing the analytical current signals obtained in the analysis of the human sputum samples with the electrochemical device. “HI” corresponds to extracts from healthy individuals, and “COPD” corresponds to extracts from COPD patients. The analytical signals correspond to the mean current values of the chronoamperometric signals recorded at 1.6 s for (A) MPO, (B) TNF-α, and (C) IL-8. The standard deviation values of three replicates carried out consecutively are represented as error bars.