Elsevier

Bioelectrochemistry

Volume 146, August 2022, 108107
Bioelectrochemistry

A simple, low-cost instrument for electrochemiluminescence immunoassays based on a Raspberry Pi and screen-printed electrodes

https://doi.org/10.1016/j.bioelechem.2022.108107Get rights and content

Highlights

  • A Raspberry Pi can be used as the basis for a simple but analytically powerful ECL biosensor, at a fraction of the usual cost.

  • Picomolar levels of [Ru(bpy)3]2+ can be detected, which compares favourably with conventional ECL instrumentation.

  • Using carbon screen-printed electrodes, a limit of detection of 50 fg mL−1 for C-reactive protein was demonstrated.

Abstract

A powerful, yet low-cost and semi-portable electrochemiluminescence (ECL) biosensing device is described. It is constructed around a Raspberry Pi single-board computer, which serves as the controller and user interface. The Pi is interfaced with an expansion board that controls the potential applied to a disposable screen-printed electrode and facilitates data acquisition from a photomultiplier tube (PMT), which detects the ECL emission from the sensor surface. As proof-of-concept, we demonstrate that this arrangement can quantitate tris(2,2′-bipyridine)ruthenium(II) ([Ru(bpy)3]2+]) with an estimated limit of detection (LOD) of 20 pM, and C-reactive protein with an LOD of 50 fg mL−1. The analytical performance of the Raspberry Pi-based setup is comparable to a conventional ECL configuration (computer, potentiostat and photodetector). The Raspberry Pi-based setup can replace a conventional ECL setup, at a fraction of the cost, without sacrificing sensitivity or versatility. The combination of a single-board computer and a sensitive light detector represents a significant step towards translating ECL instruments into mobile, point-of-care diagnostic platforms.

Introduction

The Raspberry Pi is a credit card-sized, single-board computer (SBC) that is powerful and fully functional, yet cheap. For example, the Raspberry Pi 3B + boasts a 1.4 GHz 64-bit quad-core central processing unit (CPU), and costs just USD35 [1]. With more SBCs now available, they are becoming a disruptive technology of growing importance for a wide range of applications, especially for Internet of Things [2], [3], [4], [5].

SBCs are easily programmed using free, open-source software, and the hardware can be readily tailored to interface with external components via the 40-pin GPIO interface. SBC-based platforms are particularly suited to point-of-care and point-of-need applications, where size, flexibility, cost and portability are important. For example, Bustos et al. measured C-reactive protein electrochemically with a limit of detection (LOD) of 58 ng mL−1 using a Raspberry Pi-based instrument [6], while Lin et al. performed colorimetric analysis with a microfluidic biosensor and a Raspberry Pi to detect Salmonella with an LOD of 14 CFU mL−1 [92].

Electrochemiluminescence (ECL) is an electrochemically triggered chemiluminescence reaction. The luminophore’s excited state can arise through the annihilation pathway, involving the electrochemical generation of both the oxidised and reduced forms of the luminophore, or by using a co-reactant [7], [8], [9], [10], [11], [12], [13], [14]. The co-reactant pathway is critical for sensing in aqueous media as it enables ECL within the potential window of the solvent. A range of co-reactants can be used, including oxalate [15], [16], [17], persulfate [17], [18], and various amines [17], [19], [20], [21], of which tri-n-propylamine is the most important. There are multiple classes of electrochemiluminophores, including nanomaterials and metal complexes [7], [8], [9], [10], [11], [12], [13], [14]. The gold standard, tris(2,2′-bipyridine)ruthenium(II) ([Ru(bpy)3]2+), continues to be the most commonly used, although iridium complexes are emerging as a popular alternative [22], [23], [24], [25], [26], [27].

ECL combines the high temporal and spatial control of electrochemical methods with the high sensitivity and low background of chemiluminescence, and thus promises ultra-low (sub-pM) LODs that are far superior to those of fluorescence detection [7], [8], [9], [10], [11], [12], [13], [14], [28], [29], [30]. Such low LODs are critically important in medical diagnostics, especially for early disease diagnosis, and when fluids other than blood are sampled, where the analyte concentration is generally much lower [31], [32].

An ECL instrument, whether custom-made or commercial [33], [34], has two main components, in combination with a computer for control and display: a potentiostat, which controls the applied potential while monitoring the current, and a photodetector to measure the luminescence signal. However, the cost of a custom setup will typically exceed USD10,000, and commercial instruments usually cost considerably more.

Various studies have aimed to reduce the cost of ECL instruments, commonly by using cheaper luminescence detectors, including smartphones [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47]. Some report modifying the smartphone with external electronics to enable it to act as a potentiostat and initiate ECL, while others use the smartphone only as the photodetector [35], [37], [38], [39], [44], [45], [46], [47]. In a recent report, Liu and Chen et al. used a smartphone/printed circuit board-based low-cost potentiostat combination to monitor ECL from a sensor for 3-nitrotyrosine with an LOD of 8.4 nM [38]. Photodiodes (in combination with a potentiostat or functionally equivalent electronics) are another alternative [48], [49], [50]. However, the LODs achieved using photodiodes and smartphones are usually in the nM-μM range.

Commercial portable potentiostats and/or luminescence analysers have also been utilised, although they are still expensive [51], [52], [53]. Fanjul-Bolado et al. used a commercial portable potentiostat combined with either an avalanche photodiode or commercial micro-spectrometer to detect biotinylated alkaline phosphatase with LODs of 5 and 100 pM, respectively [53]. Purpose-built equivalents have been developed, such as by Yang and Tu et al., who combined a photomultiplier tube (PMT) ECL detector with home-made electronics and a laptop to measure methamphetamine with an LOD of 188 pg mL−1 [54]. Other studies that used a PMT also reported pg mL−1 LODs [55], [56]. These instruments required purpose-built electronics as the potentiostat, and a separate computer to control the experiment and acquire data, which adds cost and reduces versatility.

In this paper, we describe a simplified ECL instrument which is built around a Raspberry Pi SBC, and how it gains the advantages of having a small footprint and low cost without compromising sensitivity. In our device, an expansion board connects directly via the GPIO interface of the Raspberry Pi. It essentially becomes the potentiostat as it initiates ECL at low-cost, screen-printed electrodes by controlling the applied electrochemical potential. A fully functioning potentiostat is not required since the signal of interest in ECL is optical, rather than electrochemical. The Raspberry Pi-based instrument, itself a computer, also acquires the resulting luminescence data in the form of an amplified signal from a PMT. Various low-cost photodetection options were considered, including photodiodes and Raspberry Pi camera modules. However, these lacked sufficient sensitivity to detect weak luminescence signals [43], [49], [50], [53], [57], [58] and so we concluded that a PMT is required in order to obtain the ultra-low LODs that are necessary for most biomedical applications.

The configuration described is highly versatile because, unlike other purpose-built instruments, the Raspberry Pi is easily programmed using open-source software and can be repurposed for other applications simply by using a different sensor. Here, we use C-reactive protein (CRP) as a proof-of-concept analyte. CRP is indicative of inflammation which may arise as a result of a range of conditions including cardiovascular disease, sepsis and COVID-19 [59], [60], [61]. It is most commonly measured using optical or electrochemical detection methods, with LODs varying significantly [59], [61], [62], [63]. For example, Roche Diagnostics’ cobas b 101 analyser uses immunoturbidity to detect CRP with an LOD of 3 μg mL−1 [62], while Hu and Yang et al. electrochemically measured CRP with an LOD of 3.3 pg mL−1 [64]. A range of ECL-based sensors have also been developed [30], [65], [66], [67], including studies by O’Reilly and Dennany et al. [30] and Kim et al. [65] with reported LODs of 0.3 fg mL−1 and 4.6 pg mL−1, respectively, but are often complex from an instrumental point of view.

We demonstrate that our biosensor (or Raspberry Pi-o-sensor) can quantify C-reactive protein with an LOD of 50 fg mL−1. We show that the analytical performance of the Raspberry Pi-o-sensor, despite its low cost and small footprint, compares very favourably with a larger, more expensive custom-made ECL setup. To the best of our knowledge, this is the first time that an SBC-based instrument has been used as the basis for a miniaturised instrument to simultaneously initiate, monitor and record ECL.

Section snippets

Materials

Phosphate buffered saline (PBS) tablets (pH 7.4, Astral Scientific), dimethylformamide (DMF, anhydrous, Sigma Aldrich), Tween 20 (Sigma Aldrich), foetal bovine serum (FBS) (Assay Matrix Pty. Ltd.), tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate ([Ru(bpy)3]Cl2·6H2O) (98% grade, Strem chemicals), and Roche Diagnostics ProCell buffer (180 mM tri-n-propylamine (TPrA) and ≤ 0.1% undisclosed surfactant, in pH 6.8 phosphate buffer) were used without further purification. ProCell contains

Results and discussion

The Raspberry Pi-based biosensor system was first tested using varying concentrations of [Ru(bpy)3]2+, which is the standard electrochemiluminophore used in most commercial ECL systems. Solutions were prepared in the commercially sourced ProCell, which contains 180 mM of tri-n-propylamine (TPrA) as co-reactant. ProCell is commonly used in these types of biodiagnostic studies [25], [45], [55], [76]. An excitation signal, consisting of a 1 Hz square wave pulse cycle (as shown in Figure S1, top

Conclusions

Conventional ECL instruments consist of a computer interfaced with a potentiostat, coupled with a photodetector such as a photomultiplier tube (PMT) which is housed within a light-tight box. We demonstrate that these can be replaced with a Raspberry Pi-based instrument, or Raspberry Pi-o-sensor, which maintains high analytical performance despite the reduced cost. In the Raspberry Pi-o-sensor, a Raspberry Pi singe-board computer with a suitable expansion board replaces the potentiostat’s

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.

Acknowledgements

This work was supported by the ARC Training Centre for Portable Analytical Separation Technologies (ASTech) IC140100022; and ARC discovery grants DP200102947 and DP200100013. The research performed by LD which underpins this publication was undertaken while completing a PhD at the Biomedical and Environmental Sensor Technology (BEST) Centre, La Trobe University, Melbourne, Victoria.

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