Printed carbon electrode electrochemical review tamiya

Gold electrodes are some of the most prevalent electrochemical biosensor substrate materials because they are readily functionalized with thiolated biomolecules. Yet, conventional methods to fabricate gold electrodes are costly and require onerous equipment, precluding them from implementation in low-resource settings [LRS]. Recently, a number of alternative gold electrode fabrication methods have been developed to simplify and lower the cost of manufacturing. These methods include screen and inkjet printing as well as physical fabrication with common materials such as wire or gold leaf. All electrodes generated with these methods have successfully been functionalized with thiolated molecules, demonstrating their suitability for use in biosensors. Here, we detail recent advances in the fabrication, characterization and functionalization of these next-generation gold electrodes, with an emphasis on comparisons between cost and complexity with traditional cleanroom fabrication. We highlight gold leaf electrodes for their potential in LRS. This class of electrodes is anticipated to be broadly applicable beyond LRS due to their numerous inherent advantages.

Gold electrodes for biosensing purposes

Biosensors, which are devices that utilize a biorecognition element to detect an analyte of interest, are frequently used in point-of-care testing, as they enable rapid and accurate diagnoses with minimal equipment and personnel []. These attributes are crucial in low-resource settings [LRS] that lack the infrastructure required to perform laboratory-based tests [i.e. polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assays]. Electrochemical transduction is often used in combination with biosensors for point-of-care deployment because electrochemistry enables precise, sensitive, and quantitative readout with inexpensive, portable instrumentation. Gold is often employed as a substrate for such technologies because it is readily modified with thiolated molecules through the spontaneous formation of gold–thiol bonds. As thiols are readily incorporated into biomolecules ranging from proteins to nucleic acids, a wide range of biosensors can be made. For example, monolayers of thiolated DNA can be immobilized onto gold electrodes for detection of DNA, small molecules or enzymatic activity. Similarly, thiolated antibodies or antigens can be immobilized on gold in order to build immunosensors that detect specific biomarkers. Despite these advantages, the processing required for conventional gold electrode fabrication is costly and requires cumbersome, highly-specialized equipment, limiting the fabrication of such sensors to very few highly-specialized facilities.

Fig. 1 Typical subtractive photolithography methods involve laborious steps, specialized equipment, and expensive, dangerous chemicals. a. Gold is deposited on a substrate via vacuum deposition. b. The gold-coated surface is covered in photoresist. c. Photolithography is performed through a mask to selectively cure the photoresist. d. The photoresist is developed to selectively remove cured or uncured photoresist. e. The exposed gold is etched away. f. The photoresist is removed, and the substrate now contains a patterned gold coating. Figure reproduced and adapted from ref. [Sui et al., 2020] with permission. Copyright Journal of the Electrochemical Society 2020.

Conventional gold electrode fabrication

Conventional gold electrode fabrication involves deposition to add gold to a substrate and lithography to pattern it. Most of these techniques require cleanroom facilities, limiting fabrication to a few specialized labs. Chemical vapor deposition [CVD] and physical vapor deposition [PVD] are used to deposit gold, resulting in thin films of pure metal on a substrate. During CVD, a chemical reaction between a metal-containing precursor and a gas results in the deposition of the metal on a substrate, while in PVD, the source metal is vaporized prior to deposition. To pattern the gold, lithographic techniques are incorporated, including shadowmask and photolithography. In shadowmask lithography, a physical mask is placed in contact with the substrate, and PVD or CVD is performed. Only the exposed regions of the substrate are coated with gold. These methods are costly and not conducive to high-throughput prototyping. Photolithography, in contrast, generates micron or submicron features using photoresist, a set of chemicals whose properties change after light exposure. Photolithography enables high resolution patterning of fine features, but it is costly and laborious []. Lastly, dry and wet etching processes can be used. The most common form of dry etching is reactive ion etching [REI], during which reactive plasma etches away the exposed gold, but this method is difficult because the products of the reaction between gold and the etchant are largely non-volatile. Wet etching exposes the gold to an etching solution that contains a ligand that forms soluble Au[I] complexes and an oxidant that generates Au[II]. While wet etching is more cost effective than dry etching, it is only capable of precision for features larger than 5 μm.

A plethora of electrodes made with conventional fabrication techniques have been used to generate biosensors that detect DNA, proteins and molecules. Cheng-Chi Chou et al. sputtered a thin film of gold on a silicon substrate. This film was then treated with 6-hexane dithiol to support a monolayer of AuNPs, which were functionalized with thiolated single-stranded DNA [ssDNA]. These electrodes were used to detect genetically modified soybeans from real samples with a limit of detection [LOD] of 1.792 ng mL−1 and a linear range of 17.9–19.2 ng mL−1. The same technique was used by Wang et al. to create a monolayer of AuNPs on a sputtered thin film of gold. These NPs were immobilized with antibodies used to detect neutrophil gelatinase-associated lipocalin [NGAL] in urine samples. The sensor had an LOD of 0.47 ng mL−1 and a linear range of 1–100 ng mL−1, which were clinically relevant levels that could be used to detect kidney injury. These examples shed light on the versatility of gold substrates for biosensing purposes, as they can be functionalized with a wide range of biomolecules.

Despite their use in biosensor fabrication, electrodes fabricated using conventional cleanroom methods suffer from high capital costs and are inaccessible in most LRS; the specialized laboratory space required for cleanroom fabrication can cost up to USD $1 million. This number does not include instrumentation or the cost of the gold itself [which can require an initial investment of up to $2000]. Thus, significant costs are incurred during the time-consuming processes involved in cleanroom fabrication.

Screen printed gold electrodes

Gold screen-printed electrodes [SPEs] are a more affordable alternative to cleanroom-fabricated platforms. Screen printing involves the application of the appropriate metal ink, through a mask, on a substrate []. The shape of the mask determines the shape of the electrode. Screen printing is favorable because it can be performed on a diverse array of substrate materials, including plastic, polymers, ceramic and paper, while allowing for versatility in electrode design. Screen printed electrodes are highly reproducible and readily mass-produced.

Fig. 2 Screen printed electrodes are made by screen printing conductive ink onto a substrate. [a] A mask is first used to screen print the reference and contact electrodes. [b] A separate mask is used to screen print the gold working and counter electrodes. [c] The final device is assembled with a hydrophobic barrier covering the contact electrodes. Figure reproduced and modified from ref. [Ozkan et al., 2015] with permission. Copyright Springer Berlin Heidelberg 2015.

SPE surfaces can be functionalized to make immuno, genetic, and enzymatic sensors, demonstrating their versatility and suitability for biosensor construction. Furthermore, the surface morphology and roughness of the electrode can be altered by varying the gold ink used. This ability to generate multiple surface topologies broadens the applicability of the SPEs. For example, the most commonly used commercial SPEs are made by Dropsens and use either high temperature curing inks [AT] or low temperature curing inks [BT]. The AT electrodes are suitable for assays where steric hindrance is not concern. For example, Dizaji et al., used the DropSens AT electrodes to detect whole-cell antibiotic-susceptible bacteria. They immobilized thiolated vancomycin molecules on the SPEs and monitored the binding of the vancomycin-susceptible bacteria S. aureus. The LOD of the sensor was 34 CFU mL−1 and the linear range was 10–108 CFU mL−1. The low temperature curing inks have a rougher surface morphology, which allows for more sensitive biosensors due to decreased steric hindrance effects. Bialobrzeska et al., used the Dropsens BT electrodes to detect the cancer antigen AGR2 in lysed cell samples with a LOD of 0.093 fg mL−1 and a linear range of 0.001 fg mL−1 to 0.900 fg mL−1. These examples shed light on the potential of SPEs to be used in biosensor fabrication.

Despite these advantages, mass production of SPEs require an initial investment of at least $30

000, which remains inaccessible to labs with limited funding. Gold ink itself is also expensive, which can cost over $1000 per oz []. Furthermore, electrodes must be cured at elevated temperatures [130–800 °C] prior to use, necessitating additional equipment that is not available outside of select labs. An additional disadvantage of screen printing is that this technology does not reproducibly produce micrometer-scale electrodes, which may be necessary to achieve improved signal/noise ratios and faster mass-transport rates. Furthermore, the inks can contain impurities that interfere with sensor performance.

Table 1 Capital costs for various types of gold electrode fabrication

Inkjet-printed gold electrodes

Inkjet printing of gold electrodes is a contact-less, mask-less procedure []. Compared to screen-printing, inkjet printing offers more control over gold ink deposition and micron-resolution features. A wide variety of materials have been used as substrates for inkjet-printed gold electrodes, including paper, polyethylene teraphalate [PET], kapton film, cyclein olefin copolymer, and cellulose, making them favorable for flexible electronics. Depending on the fabrication method, some inkjet-printed gold electrodes exhibit as high as tenfold improved sensitivity as compared to sputtered gold, as measured by differential pulse voltammetry [DPV].

Fig. 3 Inkjet printed electrode fabrication. [a] Gold nanoparticles [AuNPs] are inkjet printed onto a substrate. [b] The AuNPs are sintered to form a continuous surface. [c] Excess AuNPs are washed away. Figure adapted from ref. [Ko et al., 2007].

Inkjet-printed gold electrodes have been used to detect proteins, protein–nucleic acid interactions, DNA hybridization, antioxidants, glucose and bacteria, demonstrating their broad utility in biosensor fabrication. Kant et al., 2021 made inkjet-printed gold electrodes on paper that detected glucose from serum without any enzymes. They achieved a LOD of 10 μM and a linear range of 0.05–35 mM. This sensor is critical because it demonstrates a proof-of-concept for low-cost glucose sensors that, once fabricated, can be easily deployed in LRS, especially because enzyme stability is not a concern. In another study, Sui et al., 2019 developed a novel class of nanoparticle inks that are sintered with plasma rather than high temperatures. This allowed for the development of a biosensor for amyloid beta, a critical marker for Alzheimer's disease, on a PET substrate. While limits of detection were not reported for this sensor, the sensor demonstrated that gold electrodes can be inkjet printed on a flexible PET substrate, which is not possible when using traditional AuNP inks that require sintering at high temperatures above the glass transition temperature of PET, thereby extending the number of substrates that can be used for inkjet printing gold.

Despite the recent advances in inkjet printing, similarly to SPEs, the costly fabrication of inkjet-printed electrodes limits their production to well-funded labs. While commercial inkjet printers can be repurposed to print metallic inks, the FUJIFILM Dimatix Materials Printer [$40

000] offers greater control over the printing process and is the most common printer for inkjet printing gold. Gold ink is similarly costly, costing around $1100 per oz, which fluctuates with commodities markets. Another disadvantage of inkjet-printed electrodes is that they require sintering prior to use to transform the ink into uniform, conductive electrodes. Gold nanoparticle [AuNP] inks are the are most commonly used. Such inks are stabilized with capping agents that prevent excessive clustering of the NPs and improve their wettability, jettability and adhesion. Yet, the capping agents act as insulators and must be removed to generate a conductive surface. This removal is most commonly performed via thermal sintering [∼130–440 °C] but can also be done with photonic curing, chemical, plasma, laser or IR sintering for substrates with lower glass transition temperatures. These processes all necessitate complex equipment that is not available in LRS. Recently, a group developed an alternative ink comprised of inorganic salts that could be activated via plasma at low temperatures [