Dual-Target Additively Manufactured Electrochemical Sensor for the Multiplexed Detection of Protein A29 and DNA of Human Monkeypox Virus

Abstract

Herein, we present the first 3D-printed electrochemical portable biodevice for the detection of monkeypox virus (MKPV). The electrochemical device consists of two biosensors: an immunosensor and a genosensor specifically designed for the detection of the protein A29 and a target DNA of MKPV, respectively. The electrodes were manufactured using lab-made ultraflexible conductive filaments composed of carbon black, recycled PLA from coffee pods, and castor oil as a plasticizer. The sensors created through 3D printing technology exhibited good reproducibility and repeatability of analytical responses. Furthermore, both the immunosensor and genosensor demonstrated excellent MKPV detection capabilities, with a linear range from 0.01 to 1.0 μmol L^-1 for the antigen and 0.1 to 20.0 μmol L^-1 for the DNA target. The biosensors achieved limits of detection of 2.7 and 29 nmol L^-1 for the immunosensor and genosensor, respectively. Interference tests conducted with the biosensors demonstrated their selectivity for MKPV. Moreover, analyses of fortified human serum samples showed recoveries close to 100%, confirming the absence of significant matrix effects for MKPV analysis. Therefore, the 3D-printed multiplex device represents a viable and highly promising alternative for on-site, portable, and rapid point-of-care MKPV monitoring.

Figure 1

(a) Illustrative diagram of the biosensors preparation steps. (WE1 - immunosensor) step 1 - EDC:NHS immobilization; step 2 - anchoring of the specific antibody; step 3 - blocking with BSA and step 4 - label-free detection of the specific MKPV antigen. (WE2 - genosensor) step 1 - immobilization of EDC:NHS; step 2 - anchoring the capture DNA together with blocker/spacer; step 3 - hybridization with MKPV target DNA for label-free analysis.

Figure 2

SEM images of the electrode surface (a) before polishing and (b) after polishing at different magnifications. (a-b) 31× and (b-b’) 1000 ×, respectively. (c) FTIR spectra for CB-rPLA, and (d and d’) water contact angle measurement for unpolished and polished electrodes, respectively. Inset: real images of the 3D printed multiplex system.

Figure 3

Cyclic voltammograms obtained with WE1 and WE2 in the presence of 1.0 mmol L^-1 FcMeOH in 0.1 mol L^-1 KCl. (a) Immunosensor steps: (black line) CB-rPLA; (red line) EDC:NHS; (blue line) Ab; (pink line) BSA and (green line) detection 0.01 μmol L^–1 MKPV antigen. (b) Genosensor steps: (black line) CB-rPLA; (red line) EDC:NHS; (green line) DNA target + blocking and (blue line) detection 1.0 μmol L^–1 MKPV target DNA. (a’) and (b’) bar graph of the anodic peak currents obtained in each stage of modification of the immunosensor and genosensor. Scan rate: 50 mVs^–1.

Figure 4

Cyclic voltammograms obtained with WE1 and WE2 sensors (a) immunosensor: varying the concentration of antigen from 0.01 to 1.0 μmol L^-1; (b) genosensor: varying the concentration of target DNA from 0.1 to 20.0 μmol L^-1. Calibration curves for the (a’) immunosensor and (b’) genosensor, obtained for variations in analyte concentration as a function of -ΔI.

Figure 5

Cyclic voltammograms obtained with WE1 (a) and WE2 (b) for interference tests in the presence of 1.0 mmol L^-1 FcMeOH in 0.1 mol L^-1 KCl. (a’) (black line) CB-rPLA; (red line) immunosensor; immunosensor in the presence of (blue line) 1.0 μmol L^-1 protein S1 SARS-CoV-2; (pink line) 1.0 μmol L^-1 generic protein (BSA) and (green line) 1.0 μmol L^-1 MKPV antigen. (b) (black line) CB-rPLA; (red line) genosensor; genosensor in the presence of (blue line) 20.0 μmol L^-1 Influenza A target cDNA target; (pink line) 20.0 μmol L^-1 SARS-CoV-2 target cDNA; and (green line) 20.0 μmol L^-1 MKPV DNA target. (a’) and (b’) bar graph obtained from the anodic peak current of each analysis. Scan rate: 50 mV s^-1.