A versatile 3D printed multi-electrode cell for determination of three COVID-19 biomarkers

https://doi.org/10.1016/j.aca.2023.341169Get rights and content

Highlights

  • A fully 3D-printed six-working electrode cell is proposed for detection of biomarkers.

  • The working electrodes were printed with polylactic acid and carbon black.

  • Detection of three COVID-19 biomarkers is investigated, aiming to achieve the whole viral window.

  • The 3D-printed set-up allowed the detection of N protein, SRBD protein and anti-SRBD.

  • The multiplex sensors were selective to serum and saliva samples.

Abstract

3D-printing has shown an outstanding performance for the production of versatile electrochemical devices. However, there is a lack of studies in the field of 3D-printed miniaturized settings for multiplex biosensing. In this work, we propose a fully 3D-printed micro-volume cell containing six working electrodes (WEs) that operates with 250 μL of sample. A polylactic acid/carbon black conductive filament (PLA/CB) was used to print the WEs and subsequently modified with graphene oxide (GO), to support protein binding. Cyclic voltammetry was employed to investigate the electrochemical behaviour of the novel multi-electrode cell. In the presence of K₃[Fe(CN)₆], PLA/CB/GO showed adequate peak resolution for subsequent label-free immunosensing. The innovative 3D-printed cell was applied for multiplex voltammetric detection of three COVID-19 biomarkers as a proof-of-concept. The multiple sensors showed a wide linear range with detection limits of 5, 1 and 1 pg mL−1 for N-protein, SRBD-protein, and anti-SRBD, respectively. The sensor performance enabled the selective sequential detection of N protein, SRBD protein, and anti-SRBD at biological levels in saliva and serum. In summary, the miniaturized six-electrode cell presents an alternative for the low-cost and fast production of customizable devices for multi-target sensing with promising application in the development of point-of-care sensors.

Introduction

The fused deposition modelling 3D printing technique is based on the extrusion process of thermoplastics that allows the production of three-dimensional materials using layer-by-layer deposition of conductive and/or non-conductive filaments. In electrochemistry, this breakthrough technology has shown promising applications in energy storage, microfluidic systems, and sensor devices [[1], [2], [3]]. From a manufacturing point of view, 3D printing is an appropriate approach for the fabrication of low-cost sensors with a capacity of production of more than 1000 electrodes/day. Moreover, additive manufacturing can be employed to create versatile and customizable electrochemical apparatus for flow-injection cells, integrated lab-on-a-chip, and wearable sensors [[4], [5], [6]]. It also allows the production of miniaturized electrochemical cells for portable analysis that can operate with only a few microliters of sample and supporting electrolyte [7,8]. Those characteristics are a key aspect in the development of point-of-care diagnostics ensuring operational capacity and high analytical frequency in biomedical assays.

3D-printed electrochemical sensors are commonly produced with conductive filaments of polylactic acid and carbonaceous nanomaterials [9,10]. Carbon black/polylactic acid (PLA/CB) is among the high-performance materials for conductive filaments because of its electrocatalytic properties, high surface-to-volume ratio, electrical conductivity, and low-cost. For example, the PLA/CB filaments can create electrochemical sensors with a cost of $ 0.015 dollars per electrode. Despite the low-cost and promising application, the polymeric content of 3D-printed filaments can cover the carbon conductive sites, reducing the kinetics of charge transfer onto the electrode surface. To avoid this problem, chemical and mechanical activation have been proposed in the literature as an alternative to expose the carbon sites in conductive filaments [11,12] Electrode modification with graphene oxide, and gold nanoparticles can be an additional strategy to improve the electrochemical performance and chemical functionality of PLA/CB for the production of (bio)sensors [11].

Since the beginning of the COVID-19 pandemic, multiple technologies have emerged in the field of SARS-CoV-2 detection, including colorimetric immunoassays, surface-enhanced Raman scattering, and 3D-printed electrochemical sensors. In the last few years, the use of 3D-printed technology has also emerged in the field of biosensors for electrochemical detection of Hantavirus, SARS-CoV-2 and, Influenza [[13], [14], [15]]. From this perspective, versatile electrochemical approaches can be employed to design multiplex sensors, contributing to a fast response with a lower sample volume. Multiplex detection can also reduce the source of experimental errors [16]. In addition, sequential or simultaneous screening of multiple biomarkers can provide more detailed information about the patient's condition, supporting medical decision-making and precision diagnostics [17]. Different geometries of miniaturized 3D-printed cells containing multiple working electrodes can be an important tool for precision medicine, ensuring the low-cost and fast production of multiplex biosensors for viral diseases [18]. From this perspective, multiplex diagnostic of SARS-CoV-2 biomarkers can improve the confidence level of COVID-19 diagnostics reducing the possibility of false negative results.

Herein, we propose a miniaturized 3D-printed electroanalytical approach containing six working electrodes coupled to a multi-channel analogical controller that operates with only 250 μL of sample. As a proof-of-concept, the novel 3D-printed cell was applied for multiplex voltammetric detection of three main COVID-19 biomarkers: N protein, SRBD protein, and anti-SRBD in saliva and serum samples. The analytical validation is discussed in detail in terms of linear range, accuracy, interference response and multi-target detection. The low-cost 3D-printed cell is a simple and scalable approach for accurate multiplex electrochemical sensing.

Section snippets

Reagents and materials

All chemical reagents were analytical grade with high purity and the solutions were prepared using distilled water. Potassium ferrocyanide, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC), N-Hydroxysuccinimide sodium salt (NHS) and sodium hydroxide were purchased from Sigma Aldrich. Bovine Serum Albumin (BSA), SARS-CoV-2 receptor binding domain (SRBD) 0.12 mg L−1, anti-SRBD protein 15.4 μg L−1, Hantavirus araucaria nucleoprotein (Np) 1.18 mg mL−1, anti-nucleocapsid 6.6 mg mL−1

Electrochemical performance of multi-electrode cell

The novel 3D-printed electrochemical cell set-up was designed to provide a circular symmetric geometry, as can be seen in Scheme 1. This configuration ensures that the six working electrodes are equally distributed with the same distance between the shared reference and auxiliary electrodes, providing a homogeneous charge transfer in the presence of the lowest volume of supporting electrolyte. The ring format used as an auxiliary electrode was designed to present a larger surface area, aiming

Conclusion

In this work, we proposed a novel 3D-printed multi-electrode portable cell for multiplex electrochemical sensing of COVID-19 biomarkers. First, the novel cell configuration was investigated to ensure its adequate operation. PLA/CB was successfully modified with graphene oxide (PLA/CB/GO), providing an adequate surface for electrochemical immunosensing. Cyclic voltammetry was employed to stepwise investigate the construction of the biosensors for N-protein, SRBD protein, and anti-SRBD. Under

CRediT authorship contribution statement

Franciele de Matos Morawski: Conceptualization, Methodology, Investigation, Formal analysis, Writing – original draft. Gustavo Martins: Conceptualization, Methodology, Investigation, Formal analysis, editing, and reviewing the original draft. Maria Karolina Ramos: Formal analysis. Aldo J.G. Zarbin: Resources, Writing – review & editing. Lucas Blanes: Methodology, Funding acquisition, Resources, Writing – review & editing. Marcio F. Bergamini: Funding acquisition, Resources, Supervision, Project

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (financial code 001 and CAPES September 2020 Epidemias 88887.504861/2020–00), Conselho Nacional de Pesquisa (CNPq) (grants 408309/2018-0; 311290/2020-5, 309803/2020-9 and 402195/2020-5) and Fundação Araucária (PBA2022011000056 and PDT2020221000003). The authors are also grateful for the technical and financial support of the Carlos Chagas Institute–FIOCRUZ/PR.

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