Pad-printed Prussian blue doped carbon ink for real-time peroxide sensing in cell culture

Highlights

• Prussian blue mediated printed carbon electrodes allows in-situ determination of peroxide.

• Pad-printed Prussian blue electrodes demonstrate capacity to monitor both the exogenous production of peroxide and elimination via Fenton-like processes.

• Capable of linearly detecting peroxide found below, across and above cellular concentrations of peroxide.

• Constructed electrodes proven capable of reliably determining peroxide in complex cell culture media

Abstract

Hydrogen peroxide has important roles within cellular functions, as a prevalent form of Reactive Oxygen Species, detection within mammalian cells is of metabolic importance; typically requiring cell lysis or fluorescence-based methods to quantify.

Herein, we explore the novel use of Prussian blue mediated, pad printed carbon electrodes to allow the indirect detection of cellular peroxides in bulk culture media, which facilitates non-invasive, real-time detection.

Electrodes demonstrated capacity to detect H₂O₂ with a linear range of 1-200 μM in CMEM (R² = 0.9988), enabling detection of peroxides found in culture media and lysate. Developed electrodes had a Limit of Detection (LOD) of 0.41 μM H₂O₂ in Britton-Robinson Buffer (BRB), 0.38 μM in Eagle's Minimum Essential Medium (EMEM) and 9.19 μM in Dulbecco's Modified Eagles Medium (DMEM). Electrodes were tested in a conventional 5% serum supplemented EMEM (CMEM) and demonstrated an LOD of 0.5 μM and LOQ of 0.9 μM.

The results demonstrate proof of concept for monitoring H₂O₂ in complex culture media with potential long-term use and reusability using simple, pad printed Prussian Blue / Carbon electrodes. The lack of further modification, and cost-effectiveness of these disposable electrodes could offer great advancement to monitoring of peroxides in complex media.

Introduction

Hydrogen peroxide (H₂O₂) has well-established roles in cell signalling [1] and cell death [2]; its presence can be an indicator of intracellular and extracellular responses to potentially damaging stimuli, such as direct or indirect reactive oxygen species (ROS) inducing agents [3]. In biological systems, H₂O₂ is utilised and generated through several biochemical reactions, such as those catalysed by oxidase enzymes and superoxide dismutases, and as such are a vital component of cell metabolism [1]. Whilst H₂O₂ is necessary for cellular function, fluctuations in concentration can induce cell dysfunction, upregulate the antioxidant response of the cell and/or cause cell death/ mutation [4].

As such, the ability to detect H₂O₂ continuously in real-time within cell culture media holds significance for academic, industrial, and pharmacological application/ purposes [5]. Electrochemical methods using non-modified metal electrodes for the measurement of H₂O₂ are limited by the high over-potential required for oxidation (~0.7 V versus Ag/AgCl), and several biologically relevant electroactive substances, such as glucose, ascorbic acid, uric acid, tryptophan and tyrosine are typically oxidised at similar potentials.

Whilst the measurement of H₂O₂ itself is well established as a fundamental unpinning of many biosensors either as analytical target or product of an immobilised enzyme, they still require mediating compounds to effectively shuttle electrons. A challenge to using enzyme-based biosensors arises due to potential issues with stability, complexity and cost. Therefore, the use of non-enzymatic methods for electrochemical detection of hydrogen peroxide is particularly advantageous and has been previously facilitated through a range of electrode modifiers, such as: metal hexacyanoferrates (e.g. iron, copper, nickel, cobalt, chromium, vanadium, manganese and ruthenium [6,7]), carbon nanotubes and graphene [[8], [9], [10], [11], [12]], and various nanoparticles (e.g. AgO, ZnO,Fe₃O₄ and CuO [[13], [14], [15], [16]]).

Prussian Blue (PB) and its analogues have commonly been exploited to allow peroxide sensing through deposition onto various carbon electrode surfaces via electropolymerisation in a highly concentrated acidic solution (such as H₂SO₄ or HCl) or drop casting [17]. Reduced PB is capable of reducing H₂O₂ to various forms, literature cites either hydroxyl ions, water or a combination. This process oxidises PB, which is then in turn reduced by the electrode, mechanism shown in Fig. 1.

Though PB can be polymerised or solvent cast onto the electrode surface easily, desorption occurs rapidly and therefore requires binding agents/ co-polymers to assist with electrode adhesion [18]. Hence, the semi-conductive polymer poly(O-phenylenediamine)(PoPD) has been widely used as a supporting polymer [19], as has Nafion [20], and pyrrole [21].

However, due to the increase in popularity of printed electrodes, ink/paste formulations with incorporated mediators have previously been developed with results comparable to modified electrodes [22]. Prussian blue has widespread use in H₂O₂ sensing with a redox window between −0.2 to 0.4 V, with several studies utilising ~0.0 V for amperometric measurements [20,23,24].

H₂O₂ detection via PB has been utilised in combination with additional modifiers in an attempt to enhance sensitivity and selectivity towards H₂O₂, including pyrrole [25], Single-walled carbon nanotubes (SWCNTs) [26], Multi-walled carbon nanotubes (MWCNTs) [27], nanoparticles [28], and various polymers [18]. Commonly, modifiers are utilised to form polymers or as a supporting matrix for PB, deposited onto the electrode surface through electropolymerisation or solvent evaporation.

Table 1 outlines a selection of carbon electrodes modified with PB for H₂O₂ detection. Linear ranges vary between electrodes, with most demonstrating a limit of detection (LOD) of ~0.1–1.0 μM and a limit of quantification (LOQ) of ~0.5–2 μM. This sensitivity illustrates the potential for this sensing approach to be developed towards the detection of low concentrations of H₂O₂ released from cells into the surrounding medium – either through cell lysates or bulk culture media analysis (i.e. without sampling or preparation).

Bulk analysis of culture media would allow for the non-invasive detection of specific markers associated to changes in cellular behaviour/ metabolism. Hence, a system with the sensitivity to detect these small changes in-situ would be advantages in long term monitoring of cell metabolism. The bulk solution, in this context, relates to the whole aqueous environment that both supplies the cells with essential nutrition, environmental protection (from dehydration and heating), and allows for the exocytosis of waste materials, which can be compared to similar biological behaviours found In-vivo. The bulk medium cells are grown in is a complex mixture of salts, amino acids, proteins, sugars and a few additional components that assist with pH balance (NaHCO₃) and as an indicator (phenol red). Two of the most common media used for this application are Eagle's minimum essential medium (EMEM) and Dulbecco's minimal essential medium (DMEM), the latter of which is an adaption to original EMEM recipe by increasing glucose concentration and amino acids, which presents issues for detection via electrochemical means. As such, the implementation of an electrode capable of accurately and reliably detecting and quantifying the concentration of H₂O₂ offers great advantage over conventional methods, such as chromatography or assays.

The research presented herein documents the characterisation of pad-printed commercially-available Prussian Blue doped Carbon ink to simply fabricate disposable electrodes, and their potential use for H₂O₂ quantification and monitoring was assessed. This emerging technology may be useful to help understand and study the role of H₂O₂ in cell culture and offer novel means to monitor stress induction/ fluctuation in bulk media in real-time.