Review—Inkjet Printing of Metal Structures for Electrochemical Sensor Applications: A Review

Inkjet printing has emerged as a leading technology for additive manufacturing of electronic devices. It is particularly attractive in applications that require low waste, low-cost fabrication techniques. Most printing processes for electronic device applications involve the fabrication of metal structures owing to the availability of metal-based inks and the high quality structures that can be produced using these inks. As a result of rapid advances in the development of sensor-dependent technology areas like IoT and ﬂ exible/wearable electronics, inkjet printing has recently expanded into the sensor area due, in part to its inherent compatibility with a wide variety of polymer substrates and roll-to-roll processing. This review focuses on the development of inkjet-printed elemental metal structures for electrochemical applications. Included in this paper is a review of commonly used and newly emerging ink technologies, post printing sintering processes, functionalization of printed metal surfaces to enhance sensing capabilities and a variety of inkjet-printed electrochemical sensors including gas sensors, ion sensors, pH sensors, glucose sensors, and biomolecule sensors. Metal structures essential wide of H1 processed by N-succinimidyl S-acetylthioacetate (SATA) form a thiol-link. Compared with traditional SAM methods that take 24 – 48 h to incubate, protein immobilization by this method takes only 1 h. The functionalized Au electrode was tested using different nucleic acid oligonucleotides by differential pulse voltammetry (DPV) in the presence of ferricyanide to quantitatively analyze protein-DNA binding interaction. Similar bioconjugation was also applied to DNA aptamer to functionalize a printed Au electrode for trace mercury detection in water and organic solvents. The Au was using a nanoparticle ink and thermally sintered at 200 °C. The electrode functionalization was performed by drop-casting a DNA aptamer modi ﬁ ed with a thiol linker activated by disul ﬁ de bond reduction using Tris(2carboxyethyl)phosphine hydrochloride (TCEP). The functiona- lized electrode was tested by impedance spectroscopy in a trace mercury solution and the results were noticeably different than those obtained from sensors exposed to arsenic, lead and cadmium solutions, indicating the electrode had a high speci ﬁ city. method. 117 The electrode for the DNA hybridization sensor was fabricated using an Au nanoparticle ink. The electrode was functionalized using two different methods: the ﬁ rst method involved the sequential deposition of a biotinylated SAM, streptavidin and biotinylated DNA; the second method used a thiol- functionalized DNA and subsequent back ﬁ ll with a 11-mercapto-1-undecanol (MUOH) SAM. The binding capacity and selectivity of both methods were assessed using SPR analysis. It was found that the electrode functionalized with the thiol-DNA probe had a higher binding capacity. The impedimetric analysis showed that the hybridization enables detection in pM range with both electrodes. using Salmonella solutions of different tested and the

Metal structures comprise many of the essential components in a wide range of electronic devices including integrated circuits, 1,2 batteries, 3,4 sensors, 5,6 actuators, 7 light-emitting diode arrays, 8 and energy harvestors. 9,10 For electrochemical sensor applications, although carbon-based materials (e.g., graphene and carbon nanotubes) and conductive polymers have recently received considerable attention, elemental metals remain the dominant material class for such applications due to their superb electrical conductivity, outstanding mechanical stability, and versatile chemical functionality. 11 As illustrated in Fig. 1a, the dominant fabrication approach for solidstate electrochemical sensors centers on subtractive techniques that involve thin film deposition, photolithographic patterning, and etching. In spite of their extensive use in sensor device fabrication, including in large-scale manufacturing, these techniques have several inherent limitations from the perspectives of process complexity and cost. 12,13 The deposition of metal thin films is typically performed using techniques such as electroplating and vacuum deposition. Electroplating is a relatively slow process performed in solution, and the plated film may not be uniform, resulting in substandard metal layers for thickness-sensitive applications. 14 Vacuum deposition, in the form of either chemical vapor deposition (CVD) or physical vapor deposition (PVD), produces high quality films with precisely controlled thicknesses. However, the widespread commercial adoption of these techniques for low-volume production applications (e.g., sensors and micro-electro-mechanical systems) is often limited by the high capital cost of the deposition, patterning and etching systems. 15 The subtractive nature of these processes is associated with the etching step, where materials in the unwanted areas are selectively removed and the liberated material converted to waste. In the case of designs having low pattern densities, more of the deposited metal is wasted than actually used. Although materials can be recovered from the waste stream, the recycling process may cost more than the extraction or synthesis of the material from raw stock. 16 Recently, fabricating sensors by printing has attracted significant interest due to its simple implementation and scalability to highvolume manufacturing. A number of printing technologies, including screen-printing, have been used to fabricate sensors and electronic devices. 17,18 The advantages of screen-printing to produce conductive and functional structures for electrochemical sensors applications have been comprehensively reviewed in several recent publications. [19][20][21] In this article, we focus on a printing method that is one of the leading techniques in additive manufacturing: inkjet printing. Inkjet printing is a technique to fabricate a structure by depositing thin material layers from precursor inks in prescribed locations and in specific patterns by precision layering. As illustrated in Fig. 1a, compared to conventional subtractive fabrication methods, inkjet printing significantly reduces the generation of hazardous/environmentally-sensitive waste and the expense associated with waste treatment. As the variety of substrates for printed sensors continues to expand from those that are rigid and bulky to alternatives that are flexible and stretchable to satisfy the demand for sensors in biomedical, 22,23 automobile, 24 agricultural, 25 and wearable electronics 26 applications, inkjet printing has become a leading solution to fabricate widely-deployable and disposable sensors. Electrochemical sensors, being a simple but versatile and widelyused category within the sensor family, have made a steady transition to widespread adoption of inkjet-printing as a primary method of fabrication. 27 The metal electrode materials typically used in electrochemical sensors are noble metals such as gold (Au), platinum (Pt), and silver (Ag) due to their high electrical conductivity and chemical stability. 11 Coincidentally, these metals are among the few elemental metals that have also been rendered into inks suitable for inkjetprinting. The most common inkjet-printing inks are based on metal nanoparticles. 1 These inks consist of colloidal nanoparticle suspensions that are stabilized using capping agents to prevent nanoparticle agglomeration. Metal nanoparticles can be sintered into bulk structures with conductivities close to bulk metal after the capping agents are thermally removed. The most common sintering technique involves conventional thermal annealing; however, in cases where thermal damage to the substrate is a concern, the sintering process can be performed using low-temperature methods such as chemical treatment and photonic radiation. A leading alternative to the nanoparticle inks is metallo-organic decomposition (MOD) inks. 28 MOD inks use soluble metal-containing compounds as the precursor for the metal. MOD inks are typically reduced to metals by thermolysis. Recently, a new class of inks made of inorganic metal salts was developed. 29 These inorganic salt inks can be reduced to metals by low-temperature Ar plasma. Several metals that are not currently available in nanoparticle and MOD inks (e.g., bismuth, tin, and lead) can be printed using this process and their potential in electrochemical sensing has been demonstrated. A general illustration of a representative inkjet-printed 3-electrode electrochemical sensor is shown in Fig. 1b. Some metallic electrode materials have electrocatalyic activity that can be directly employed as the electrochemical sensing mechanism. 30,31 However, in practice, functional materials are typically deposited on the electrode surface to enable the detection of a larger variety of substances. For electrochemical sensors, the electrodes can be functionalized by direct material growth on the electrode, such as electrode oxidation, 32 chlorination, 33 and sulfurization, 34 or by depositing materials on the electrode surface by drop casting, 35,36 inkjet printing. 33 electrochemical plating. [37][38][39][40] As shown in Fig. 1b, a wide range of materials including inorganic compounds, 41 polymers, 37 and biomolecules 42 can be deposited as functional materials depending on the requirements of the particular application.
As illustrated in Fig. 1b, inkjet-printed electrochemical sensors have been utilized in many applications including pH measurement, 38 gas sensing, 42 ion detection, 43 and biomolecule analysis. 44 In most cases, the use of additive manufacturing to fabricate sensors on low-cost, large area polymer substrates significantly reduces the cost per sensor. Moreover, metals printed using inkjet printing tend to be more porous than their conventionally fabricated counterparts. 45 In some cases, the porous electrodes improve sensor performance by promoting electrode-analyte interactions at the enlarged interface. 29,37,40 Metallic Inks for Inkjet Printing Depending on the precursor for the metal and the type of mixture it forms in an appropriate solvent, metal inks for inkjet printing generally fall into one of two groups: colloidal suspensions of metal nanoparticles and solutions of metal-containing compounds. Inks from both groups may also contain additives such as dispersants, surfactants, thickeners, and stabilizers to improve the jettability, stability, wettability, and adhesion of the ink. 1 Nanoparticle inks.-Nanoparticle inks are colloidal liquids consisting of metal nanoparticles that are suspended either in water or organic solvents such as toluene or ethylene glycol (EG). Metal nanoparticles can be synthesized by physical methods such as milling and mechanical grinding; however, the size of the nanoparticles obtained by these methods tend to lack uniformity. 46 Chemical methods, such as the reduction of metal salts, are more commonly used in metal nanoparticle synthesis because of their reliability in controlling particle size. 47 The typical chemical reduction process involves a metal containing precursor and a reducing agent that are mixed in a solution. The ionized metal precursor obtains electrons from the reducing agent, initiating reduction, which ultimately leads to the formation of metal nuclei. The surfaces of the nuclei are energetically unstable. 48 Under these conditions, reduction of the metal precursor will continue and the resulting metal ions will bind to the surfaces of nuclei. The metal nuclei will continue to grow in size until reaching a critical radius, at which they become energetically stable and nanoparticle growth ceases. 49 The radius of the average nucleus is only a few nanometers during the first few seconds of the nucleation process, quickly growing to ∼100 nm within two minutes. 50 Typically, metal nanoparticle growth is terminated before reaching a critical radium by adding an organic stabilizer to the suspension. As shown in Fig. 2a, the stabilizers diffuse through the colloid and attach to the surface of the nanoparticles, thus inhibiting the particles from absorbing more metal ions. Metal nanoparticles are intentionally kept small for two reasons. First, the size of the nanoparticles must be 50 nm or less to prevent print nozzle clogging and to improve printing fidelity. 51,52 Second, due to a phenomenon known as melting-point depression, smaller particles have a lower melting point than bulk material which leads to a lower required sintering temperature for the nanoparticles to form continuous, densified structures after printing. 53 For example, Ag nanoparticles with diameters less than 5 nm can be melted at ∼300°C, while the melting temperature of bulk Ag is 960°C.
The most common metal nanoparticle inks are comprised of Ag. 56 These inks have been widely used for research and commercial applications because of their excellent electrical conductivity and resistance to oxidation. The most common precursor for Ag nanoparticle formation is silver nitrate (AgNO 3 ) due to its high solubility in a wide range of solvents. AgNO 3 can be reduced by monoethanolamine, 57 D-glucose, 58 or sodium hydroxide (NaOH) 59 at moderate temperatures to form Ag nanoparticles in the presence of stabilizers such as polyvinyl pyrrolidone (PVP) 60 or Arabic gum. 58,59 By proper regulation of temperature and pH, the size of the Ag nanoparticles can be controlled in a range from 30 to 120 nm. 61 Alternatively, Ag nanoparticles can be synthesized by UV-induced reactions 62 using ethanol as the solvent and methylmethacrylate as the stabilizer. 63 Other metals that have been rendered into nanoparticle inks include Au, Copper (Cu), and nickel (Ni). A major issue associated with Cu and Ni nanoparticle inks is the inherent tendency of the nanoparticles to oxidize under ambient conditions. Oxidation results in two negative consequences: (1) it increases the temperature required for sintering, and (2) it reduces the electrical conductivity. One method to address the oxidation problem is to coat the nanoparticles with a dense capping agent to enhance oxidation resistance 64 ; another solution is to coat the nanoparticles with oxidation resistant metals such as Ag by electroless plating. 65 As-printed structures fabricated using nanoparticle inks are generally not conductive because the nanoparticles are encapsulated by insulating organic stabilizing agents. A post-print sintering process is therefore required to drive off the stabilizing agents and to densify the nanoparticles to form a bulk, homogenous structure. 48 As shown in Fig. 2a, the sintering process typically involves some form of thermal annealing. In most cases, the resistivity of printed structures decreases with increasing sintering temperature. For Ag nanoparticle inks, the lowest resistivities achieved by thermal sintering ranges from 2X to 10X that of bulk Ag for sintering temperatures from 200°C to 250°C. 47,[66][67][68] The high temperature, post-printing activation process limits the selection of viable substrates to materials that can withstand high temperatures, such as silicon, glass, and temperature-tolerant polymers like polyimide (PI) and polyarylate. 69 For printed flexible electronics, these polymers are suitable for some applications, but widespread adoption is limited by their mechanical rigidity or expense. Use of some of the more common low-cost, flexible polymers such as polyethylene terephthalate (PET), polyvinyl alcohol (PVA), and polyethylene naphthalate (PEN) is limited because their glass transition temperatures are below 150°C. To enable printing conductive structures on these materials, low-temperature curing techniques such as chemical, 70,71 electrical, 72,73 plasma, 74 photonics 75-77 and microwave 78 sintering have been developed. Chemical sintering involves the removal of non-conducting, stabilizing encapsulants through dissolution or chemical reaction. For example, ethanol and water have been used to remove dodecylamine from Ag nanoparticles. 79,80 After a solvent treatment from 1 min to 2 h, the activated ink achieves a resistivity of 45X to 1400X that of bulk Ag. Further improvement requires thermal annealing. Electrical activation of metal nanoparticle inks is based on localized Joule heating. To first order, the printed structures behave as high resistance resistors due to their high electrical resistivities and small crosssectional areas. When a properly selected electric current passes through the as-printed structures, heat is generated, causing the stabilizing agent to thermally decompose. Concurrently, the uncoated metal nanoparticles begin to coalesce, eventually forming a percolated conductive network. 81 For example, following a preactivation step at 150°C for 1 h, a Ag nanoparticle ink reached a resistivity 3X bulk Ag after passing a 500 mA current through the structures. 82 Plasma sintering utilizes the active species in plasma to remove the stabilizing polymers by chain scission. The decomposed polymer molecules are volatile and are removed by the plasma flow. A resistivity of approximately 4X that of bulk Ag was achieved for a Ag nanoparticle ink subjected to low-pressure argon (Ar) plasma sintering. 74 Photonic sintering utilizes photons of different wavelengths to selectively heat the printed structure. At a certain wavelength, the ink is highly absorptive to photon energy while the substrate material is nearly transparent. 69 Depending on the optical properties of both the ink and substrate, ultra-violet (UV), 83 infra-red (IR), 84 and visible light 85 have been successfully used to sinter nanoparticle inks. Resisitivities ranging from 3.3X to 8X that of bulk Ag having been achieved by photon activation.
MOD inks.-MOD inks use soluble MOD compounds as metalcontaining precursors. Unlike colloidal nanoparticle inks, MOD inks (see Fig. 2b) are particle-free solutions which eliminates the risk of particle agglomeration and subsequent print nozzle clogging. The inspiration for MOD inks finds its origins in metal-organic chemical vapor deposition (MOCVD). Many MOD inks are synthesized from modified versions of the same precursors used in MOCVD. 86 The structure of MOD compounds consist of a metal core and supporting ligands bonded by coordinate covalent bonds. 86 The supporting ligands can be liberated through a thermally induced reaction, and the remaining metal core will form nuclei that can be fused together by thermal sintering. MOD compounds commonly used in printing are metal carboxylates synthesized in different carboxylic acids such as citric acid, 87 neodecanoic acid, 88 and lactic acid. 89 The use of long chain organic carboxyl groups reduces the weight percentage of the metal; therefore, MOD inks typically have low metal loading.
The most widely-used MOD inks are Cu, 87,89 Au, 90 and Ag 28,83 based on their high conductivity. Cu is typically reduced in an inert or reducing atmosphere to prevent oxidation. 87,89 A study showed that a Cu(I) based MOD ink can be instantly reduced when heated in air. 91 However, Cu (+1) is not stable, especially in solution, and the reported Cu (+1) compound disproportionates instead of decomposes, generating gas phase Cu (+2) byproducts. Au is more resistant to oxidation than Cu, thus Au inks can be reduced in air. A gold carboxylate-based MOD ink that can be sintered in air has been reported. 90 Ag MOD inks are the most frequently used MOD inks because they cost less than Au inks and have a higher oxidation resistance than Cu inks. Other reported MOD inks include Pt, 92 palladium (Pd), 93 and (Al) 93 inks, but all use MOCVD precursors that rely on specific chemical reactions to synthesize.
As shown in Fig. 2b, activation of MOD inks is often achieved by thermal annealing. The MOD compound undergoes thermolysis that cause the chemical bonds between the metal core and organic ligands to break. The organic ligands further break down into small molar mass molecules that are volatile. The metal atoms nucleate and grow into a continuous conductive structure. The annealing temperature, which depends on the specific MOD compound, ranges from 150°C to 580°C. 28,83,[87][88][89][90][91][94][95][96][97] The resistivity of printed Ag structures after annealing ranges from 1.2X to 10X that of bulk Ag. 28,83,88,96,97 Alternatively, MOD inks can be reduced at lower temperatures by reaction with a reducing agent. Typically, the reducing agent is printed directly onto the printed features in a process called reactive inkjet-printing (RIJ). 98 Recently, researchers have developed a reactive ink that contains both a MOD precursor and a reducing agent. 99 The reduction reaction is relatively slow at room temperature but significantly accelerates when the ink is mildly heated. The reactive ink is comprised of silver ammonia and diethanolamine (DEA). At above 50°C, DEA decomposes and the byproduct is able to reduce Ag cations to elemental Ag. The lowest resistivity achieved is 4X that of bulk Ag after the ink is heated at 70°C for 1 h and washed in water to remove the byproducts and residual reactants.
Inorganic metal salt inks.-Inorganic metal salt inks are solution-based as well; however, instead of containing an MOD compound, they utilize more readily available inorganic metal compounds. Inorganic metal salts are more thermally stable than MOD compounds; therefore, the inks generally have a longer shelf life. However, this also means that inorganic metal salt inks require thermal annealing at much higher temperatures than MOD inks. Most metal halides, sulfates, and nitrates only fully decompose into metals at temperatures higher than 800°C, at which practical polymer substrates will thermally degrade or decompose. The most common inorganic metal salt ink is composed of Ag because its precursor AgNO 3 is thermally unstable and decomposes to Ag at temperatures higher than 400°C. One of the earliest reported inorganic Ag inks used AgNO 3 mixed in deionized water and dimethyl sulfoxide (DMSO). 100 The ink was printed on PI and then cured in air at 300°C. The resulting resistivity of the printed Ag was 10X bulk Ag resistivity, mainly because the decomposition of AgNO 3 was incomplete at the curing temperature. 100 To enable printing metal using inorganic metal salt inks on temperature-sensitive substrates, alternative metal salt reduction methods have been studied. EG vapor was used as a reducing gas to convert an inkjet-printed aqueous AgNO 3 ink to metallic Ag on a PI substrate. 101 The printed ink was heated to 250°C to melt the AgNO 3 and prevent the EG vapor from condensation. The resistivity achieved was 40X that of bulk Ag. In another study, Ar plasma was used to reduce inkjet-printed aqueous AgNO 3 ink to metallic Ag. 102 The as printed ink was treated in a low-pressure capacitivelycoupled plasma at 75°C for 5 min and the resistivity of the printed Ag was close to bulk Ag resistivity. As indicated in Fig. 2c, a recent study shows that with modified ink constituents and plasma settings, the plasma approach can be applied to a large variety of metal salt inks including HAuCl 4 , PdCl 2 , H 2 PtCl 6 , CuSO 4 , BiCl 3 , PbCl 2 , and SnCl 2 , enabling inkjet printing of a wide range of metals including Au, Pd, Pt, Cu, Bi, Pb, and Sn. 29 The resistivity of these printed metals range from 2X to 10X their respective bulk metal resistivies. The variety of metals that inorganic metal salt inks offer is well aligned with the material requirements associated with electrochemical sensors.

Metallic Electrode Functionalization
Many metals are well suited for chemical sensing because of their high electrocatalyic activity and affinity for biomolecules. For example, Pd can be directly used to measure hydrogen gas (H 2 ), 30 and Ag can be directly used to measure hydrogen peroxide (H 2 O 2 ). 31 To broaden the range of applications of inkjet-printed metals in electrochemical sensing, electrodes are functionalized by depositing or growing other materials such as oxides, polymers and biomolecules.
Chemical functionalization.-Chemical functionalization of inkjet-printed electrodes is typically performed by direct modification of the metallic electrode material or deposition of a functional material onto the electrode surface. Direct modification of the electrode is typically used when the metallic electrode material can be used as the precursor for the desired functional material. For example, palladium/palladium oxide (Pd/PdO) has shown its effectiveness in pH sensing. 32,93 On a Pd/PdO sensing electrode, the oxidized surface undergoes the following ion-to-electron transduction 32 : This phenomenon is used as the sensing mechanism for the measurement of pH. For example, in a study involving Pd inkjet printing of an MOD ink, 47 the Pd structure was annealed in air to reduce the MOD compound to Pd through thermolysis and to sinter the newly formed Pd particles. At 200°C, the as-formed Pd was oxidized by O 2 from air. 103 The resulting PdO passivated the surface and prevented the printed Pd structure from complete oxidation. As a result of the annealing step, a pseudo bi-layer electrode consisting of a functionalized Pd/PdO layer in the near surface region sitting atop a conductive Pd layer was formed. The authors proposed that the formation of PdO could be controlled by performing the annealing step in an oxidizing atmosphere mixed with an inert gas. Chemical modification of metal structures can also be performed in a solution. Silver/silver chloride (Ag/AgCl) electrodes are commonly used as reference electrodes in electrochemical measurements. In many printed three-electrode electrochemical cell designs, Ag/AgCl electrodes are fabricated by screen printing 104 because the Ag/AgCl slurry contains coarse particles that can clog print nozzles and thus are not suitable for conventional inkjet printing systems. Due to its poor solubility, it is not feasible to create AgCl inks suitable for inkjet printing. However, AgCl can be chemically produced on a printed Ag structure. As shown in Fig. 3a, AgCl can be formed on an inkjet-printed Ag electrode by direct exposure to a drop cast, sodium hypochlorite (NaOCl) solution. 41 After 30 s of retention, the excessive NaOCl could simply be rinsed off by deionized water. In another study, ferric chloride (FeCl 3 ) was deposited by inkjet printing onto a printed Ag reference electrode to chlorinate the electrode. 33 The as-formed Ag/AgCl electrode was subsequently coated with a PVC reference membrane that incorporated a lipophilic salt to reduce electrode response to varying chloride anion concentrations. Besides chemical treatment, AgCl can also be formed on Ag by electrochemical chlorination. 105 In an inkjetprinted pH sensor design, AgCl was produced on an Ag surface by cyclic voltammetry in HCl to fabricate a stable Ag/AgCl reference electrode. The sensitivity of the inkjet-printed Ag/AgCl electrode was in good agreement with the results obtained with a much larger commercial Ag/AgCl electrode. Electrochemical deposition can also be used for electrode sulfurization. To fabricate a sulfide-selective electrode for ion selective sensors, a two-step inkjet printing method was developed. 34 The electrode was first prepared by printing Ag structures using a nanoparticle ink followed by thermal sintering at 140°C. The working electrode area was then delimited by printing an insulating SU-8 layer on top of the structure. Subsequently the electrode was electrically configured to serve as the working Functionalization through material deposition does not rely on the printed electrode material as a precursor for functional material formation. Instead, the printed electrode serves as a conductive structure and the electrode material is selected based on electrical conductivity, adhesion strength to both the functional coating and underlying substrate, and environmental compatibility. The functional material can be deposited onto the electrode by numerous methods including drop casting, 35,36 electrodepostion, 37-40 and inkjet printing. 33,107 Physical functionalization does not rely on a specific chemical reaction; therefore, it can be performed on a wide range of electrode materials.
Drop casting is the most straightforward method of electrode functionalization. For example, cupric oxide (CuO) has been used as the functional material in a non-enzymatic glucose sensor. 36 CuO microparticles were prepared by oxidizing Cu nanowires dispersed in water using water as the oxidant. Drop casting was chosen to deposit the microparticles because the coarse particles can clog print nozzles. The CuO microparticle suspension was subsequently dropcasted onto an inkjet-printed and thermally sintered Au electrode. A Nafion solution in ethanol was later drop-casted to immobilize the Cu microparticles after drying at room temperature. It was found that the CuO modified Au electrode had a much higher sensitivity to glucose than the bare Au electrode. Drop casting is also commonly used to deposit organic polymer materials because the method does not require special ink formulations or chemical reactions. Such an approach was used in a resistance-based ammonia (NH 3 ) sensor. 35 An Ag ink was printed and thermally sintered to form an interdigitated electrode. A conductive polyaniline (PANI) mixed with EG as the functional material was drop-casted onto the Ag electrode. Upon exposure to NH 3 , The PANI:EG film undergoes deprotonation, resulting in a measurable increase in resistivity. This approach has been used to quantitatively detect ammonia. Printed electrodes can also be functionalized with ionic liquids. For example, in a paper-like electrochemical oxygen (O 2 ) sensor, 108 an inkjet-printed Au electrode was modified by a drop-cast ionic liquid 1-butyl-3methylimidazolium hexafluorophosphate (BMIMPF 6 ). The paper-like cellulose substrate is insoluble in BMIMPF 6 , ensuring a good stability of the Au electrode based sensor.
Unlike drop-casting, electrochemical deposition requires the use of conductive substrates, enabling selective functionalization when the process is performed on electrode surfaces. Iridium oxide (IrO x ) is a commonly used sensing material for pH measurement because of its stability in acidic and alkaline environments. 109 IrO x has successfully been electrodeposited onto inkjet-printed Pt electrodes in an electrolyte composed of iridium chloride (IrCl 3 ), oxalic acid (H 2 C 2 O 4 ), and potassium carbonate (K 2 CO 3 ). 38 The printed and electrodeposited electrode was tested for over one year and demonstrated excellent mechanical stability. Similar electrodeposition of IrO x has also been performed on inkjet-printed Au electrodes for pH measurement. 39 In addition to depositing new materials onto an electrode, electrochemical functionalization can also be used to modify the morphology of an electrode surface in order to enhance the sensitivity of a printed sensor. For example, in a carbon nanotube (CNT) gas sensor, 40 a porous Au layer was electrochemically deposited on an interdigitated electrode that was printed with a Ag nanoparticle ink. CNTs were then deposited onto the Au layer. The porous Au enlarged the Schottky contact area with the semiconducting CNTs, resulting in a higher sensitivity than its dense Au counterpart. The porosity of functional materials prepared by electrochemical deposition also applies to organic polymer materials. A study systematically compared the performance of printed Au-based ion sensors functionalized by poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) deposited on the electrodes by three different methods, namely electropolymerization, inkjet printing, and drop casting. 37 The electrode was fabricated by inkjet printing using an Au nanoparticle ink, followed by photonic sintering of the printed structures. The working electrode area was defined by printing an insulating hydrophobic styrene-isobutylenestyrene (SIBS) polymer. PEDOT:PSS was subsequently deposited onto the working electrode using the three different methods. Electropolymerization was performed in a three-electrode electrochemical cell wherein the printed Au electrode was used as the working electrode, a Pt wire was used as the counter electrode, and a Ag/AgCl electrode was used as the reference electrode. PEDOT:PSS was electrochemically deposited through galvanostatic polymerization in a EDOT and NaPSS electrolyte. Inkjet printing was performed using a commercial PEDOT:PSS ink. Of the three methods, electropolymerization produced the thickest and roughest PEDOT:PSS functional layer, resulting in the highest sensitivity among the three sensors.
Biochemical functionalization.-Biochemical functionalization is typically used in sensors designed to capitalize on an enzymatic reaction or to perform some sort of immunoassay. The coupling of enzymes with electrochemical sensors enables rapid detection of metabolites and proteins. One of the most widely-used biosensors is the glucose sensor. 110 In a paper-based glucose sensor design, 111 an inkjet-printed Au electrode was functionalized by glucose enzyme glucose oxidase (GOx). The Au electrode was fabricated using an Au nanoparticle ink and subsequently sintered at 210°C. The Au electrode was then submerged in a 3,4-ethylenedioxythiophene (EDOT) and GOx solution dissolved in a phosphate buffer. The GOx was entrapped on the Au electrode by electropolymerized PEDOT forming a PEDOT-GOx complex. PEDOT is conducting therefore the entrapped GOx maintains its electroactivity. The glucose sensor, which was evaluated in a chronoamperometric setup, showed excellent sensitivity to glucose. The sensor also passed a selectivity test by showing no response to mannose. An alternative and possibly more efficient approach to functionalizing electrodes with enzymes is printing. 112 In an effort to develop an antioxidant sensor, 112 the enzyme Tyrosinase (Tyr) for antioxidant detection in foods and beverages was deposited onto an Au working electrode by large-area rotogravure printing. The interdigitated electrode was inkjet printed using a Au nanoparticle ink and thermally sintered at 200°C. The Tyr was rendered into an ink and subsequently gravureprinted on the Au electrode. The fabricated sensor showed repeatable and reliable response to antioxidants found in wines and olive oils.
Another detection mechanism commonly used in biosensors involves the immunoassay technique. 113 Noble metals such as Au and Pt are used as the electrode material because of their chemical stability and biocompatibility. Immunoassay-based electrochemical sensors are typically functionalized with an antibody that serves as a capture agent. The capture antibody is immobilized on the metallic electrode by a thiol molecule. The antibody can bind with a targeting analyte (antigen) resulting in an electrical signal that can be quantitatively measured. Immunoassay functionalization has been widely used in printed disposable biosensors. 114 For example, amyloid-beta-42 (Aβ 42 ), which is a crucial causative agent of Alzheimer's disease, was measured using a printed Au based sensor. 29 The Au electrode was printed using an inorganic Au salt ink composed of HAuCl 4 . The inorganic salt ink was reduced by Ar plasma at 67°C. The Au electrode was exposed to 3-mercaptopropionic acid (MPA) to form a self-assembled monolayer (SAM). The carboxylic group of the SAM layer was further processed using Nhydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl carbodiimide (EDC) to produce an active ester group for crosslinking of the Aβ 42 antibody. The immobilization of the analyte was electrochemically measured using the redox probe ferricyanide. The biologically functionalized electrode showed a strong sensitivity to Aβ 42 antigen. Similarly, antigens can be captured on the electrode to detect the corresponding antibody. 114 C-reactive protein (CRP), which is a biomarker for inflammation, was measured using a printed Au based sensor fabricated on latex coated paper. 114 The Au electrode was printed using a nanoparticle ink and the printed structure was sintered using short-wave IR light. A MuOH:Biotin-PEG (85:15 mol%, 5 mM) (MBP) thiol solution was used to form a SAM on the Au electrodes. Streptavidin molecules were then anchored to the thiol-linkers to provide a homogenous binding layer for bio-CRP antigens. A bio-CRP antigen layer was formed by dropcasting onto the electrode area, followed by antigen immobilization. The response of the sensor was analyzed by impedance spectroscopy to determine the concentration of CRP antibodies. In some cases, the electrode is also functionalized with the analyte and electrochemical detection is realized by immobilizing a secondary antibody. As shown in Fig. 3b, an inkjet printed Au electrochemical array on PI was used to measure interleukin-6 (IL-6), a cancer biomarker. 106 An insulating poly(amic acid) ink was inkjet-printed to define the electrode and contact pad areas. The electrode was functionalized by using a MPA SAM, which was further processed by NHS and EDC. Bovine serum albumin (BSA) was used as the primary antibody to block nonspecific binding. The IL-6 standards were then drop-casted on the electrode followed by incubation at room temperature. The IL-6 protein was detected by measuring the biotinlabeled anti-IL-6 using an amperometric method. A similar functionalized Au electrode has been used in a microfluidic sensor for IL-6 and interleukin-8 (IL-8) with a higher sensitivity. 115 Protein immobilization-based electrode functionalization can be simplified by using a bioconjugated protein and thiol-linker. Without the use of a SAM, the selectivity of the electrode and time efficiency of the fabrication process can be greatly improved. 116 An inkjet-printed biosensor for the profiling of protein-nucleic acid interactions was fabricated using an inkjet-printed Au electrode that was functionalized with the bioconjugated heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1). 44 The hnRNP H1 was processed by N-succinimidyl S-acetylthioacetate (SATA) to form a thiol-link. Compared with traditional SAM methods that take 24-48 h to incubate, protein immobilization by this method takes only 1 h. The functionalized Au electrode was tested using different nucleic acid oligonucleotides by differential pulse voltammetry (DPV) in the presence of ferricyanide to quantitatively analyze protein-DNA binding interaction. Similar bioconjugation was also applied to DNA aptamer to functionalize a printed Au electrode for trace mercury detection in water and organic solvents. 43 The inkjet printed Au electrode was fabricated using a nanoparticle ink and thermally sintered at 200°C. The electrode functionalization was performed by drop-casting a DNA aptamer modified with a thiol linker activated by disulfide bond reduction using Tris(2carboxyethyl)phosphine hydrochloride (TCEP). The functionalized electrode was tested by impedance spectroscopy in a trace mercury solution and the results were noticeably different than those obtained from sensors exposed to arsenic, lead and cadmium solutions, indicating the electrode had a high specificity.
Conventional SAM-based electrode functionalization and bioconjugate chemistry-based functionalization were compared in an impedimetric study of DNA hybridization on paper-supported inkjet-printed Au elelctrodes. 117 The electrode was printed using an Au nanoparticle ink. The electrode was functionalized using two methods. The first method used subsequent layers of biotinylated SAM, streptavidin and biotinylated DNA probe. The second method used a bioconjugated thiol-DNA probe (HS-DNA) and backfilled with a SAM to fill the gaps between HS-DNA molecules to prevent further binding. 118 The binding capacity of the two differently functionalized electrodes was characterized by surface plasmon resonance (SPR). The SPR measurements showed that the bioconjugated HS-DNA functionalized electrode has a higher binding capacity toward the complementary DNA target. Electrochemical impedance spectroscopy (EIS) showed that the HS-DNA functionalized electrode has a lower resistance, therefore is well suited for use as a capacitive genosensor.

Inkjet-printed metal-based electrochemical sensors
Using the aforementioned inks and functional materials, a wide variety of electrochemical sensors including pH, gas, ion, H 2 O 2 and glucose, and biomolecule sensors have been successfully developed. Table I summarizes a representative collection of these sensors and includes the type of sensor, inks and sintering methods used in their fabrication, substrate materials, functional electrode materials, detection methods, and sensor performance.
pH.-Conventional pH sensors use glass electrodes that are fragile and costly. 41 The sensors require regular calibration to maintain accuracy. Many studies of pH sensors focused on calibration-free or single-use designs that require low-cost sensor materials and fabrication processes. 122 Inkjet printing has recently been used to fabricate pH sensors of this type because it is well suited for lowcost polymer substrate-based fabrication.
Inkjet-printed pH sensors are typically potentiometric based and require the sensing material to have a pH-dependent potential. In an early study of an inkjet-printed pH sensor, 111 PANI was used as the functional material as its pH sensitivity has been well studied. 123 The theoretical pH sensitivity of PANI is 59 mV/pH −1 . 111 The working electrode of the pH sensor was printed on paper using an Au nanoparticle ink. The Au electrode was functionalized by electropolymerized PANI using cyclic voltammetry. The reference electrode was printed with an Ag nanoparticle ink, and the Ag electrode was subsequently chlorinated to form Ag/AgCl by electrochemical deposition. The pH sensitivity of PANI on the printed working electrode was verified by comparing its performance with the sensitivity of a bare Au electrode. The sensitivity of the PANIfunctionalized electrode was 65 mV/pH −1 after correcting for the Cl − buffer. As expected, the bare Au electrode had no response to variations in pH. The PANI-coated printed electrode was further compared to a bulk Au plate coated with PANI, and showed similar sensitivity. The paper-based printed pH sensor was reliable for over a 5-hour period, although it was designed to be a short term, singleuse sensor.
Another printed pH sensor design utilized a functional material grown from the electrode material. In this example, a PdO-based pH sensor was printed on PI and glass substrates as shown in Fig. 4a. 93 Electrodes were patterned using a Pd MOD ink. The sintering was carried out by two-step thermolysis. The as-printed ink was first heated to 120°C to evaporate the solvent, after which the temperature was increased to 200°C to reduce the solid-phase MOD compound to Pd. Thermolysis and solid phase sintering prevented Pd nanoparticle aggregation in the presence of the ink solvent and produced a smooth Pd film. Sintering was performed in air so that a pH-sensitive PdO layer would form on the electrode surface. The resistivity of the printed Pd film was measured to be 2.6 μΩ•m, which is sufficient for electrochemical measurement. The fabricated Pd-based electrode was paired with a reference electrode for pH measurements using potentiometric methods. The sensitivities achieved on glass and PI were 60.6 ± 0.1 and 57 ± 0.6 mV pH −1 , respectively. The sensor was later developed into an all-printed design by fabricating the Ag/AgCl reference electrode by inkjet printing. 41 The all-printed sensor was integrated with temperature sensors in a water quality monitoring system to detect free chlorine (Cl) in samples of tap water (Toronto), lake water (Lake Ontario), and swimming pool water (McMaster University). The measured pH was within 10% of that measured by a commercial pH meter. IrO x has become a new material of interest for printed pH sensors because it has a higher theoretical pH sensitivity than conventional pH sensing materials. 38,124 An IrO x -base pH sensor was fabricated on PI and integrated in a water quality monitoring system. 39 The pH sensor was comprised of a working electrode printed using an Au nanoparticle ink and a reference electrode printed using Ag nanoparticle ink. The working electrode was functionalized by electroplated IrO X using cyclic voltammetry. The sensitivity of the printed pH sensor was measured to be 67.5 mV pH −1 , which was slightly lower than the 70 mV pH −1 achieved by non-printing methods. However, the result is still within the theoretical 60-80 mV pH −1 range. Because IrO x has several possible oxidation states, its super-Nernstian sensitivity can vary significantly. 124 The  drift of the pH sensor was measured to be 10 μV/s at a pH of 8. The stability could be improved by replacing Ag with Ag/AgCl as the reference electrode since the latter has a higher potential stability. 125,126 It was found that temperature is a influencing factor in pH measurement and that pH could change with temperature by as much as 0.06/°C. 127 The temperature sensor in the water monitoring system served to compensate the pH reading to eliminate temperature-induced measurement errors. In later work by another group, the Ag reference electrode was replaced by Ag/AgCl. 38 The pH sensor was fabricated on a PEN substrate. The working electrode was printed using an aqueous Pt nanoparticle ink and sintered at 180°C. The water-based Pt ink provided a highly rough surface that improves the adhesion to the functional materials deposited on the electrode. Despite the rough surface, the Pt ink had a resistivity of 0.63 μΩ•m, which is ∼6X bulk Pt resistivity. The Pt electrode was functionalized with electrodeposited IrO x . The reference electrode was printed using an Ag nanoparticle ink and subsequently functionalized by electrodepositing AgCl. The printed pH sensor tested in a potentiometric setup had an average sensitivity of 71.3 mV pH −1 over a pH range of 2 to 11, which was in good agreement with theoretical values. The performance of the sensor was monitored for over a year and demonstrated good stability that was attributed to the roughness of the Pt layer.
Gas.-Electrochemical gas sensors are widely used in automobiles, household appliances, and industrial manufacturing for environmental protection and safety control. 128 Gas sensor electrodes are typically fabricated using conventional microfabrication processes such as vapor deposition as well as gravure and screen printing. 12 Inkjet printing, being a facile additive deposition method, has been recently used for gas sensor fabrication because it can further reduce the cost of disposable gas sensors fabricated on inexpensive polymer substrates.
Although most inkjet-printed gas sensors are chemiresistorbased, 35,40,129,130 several studies demonstrated the possibility of fabricating electrochemical gas sensors by inkjet printing. In a study to develop an inkjet-printed O 2 sensor, 108 an Au electrode-based sensor was printed on a paper-like cellulose membrane. The electrode was printed using Au nanoparticle ink. The percolated cellulose membrane had pores into which the Au nanoparticles could reside, resulting in a highly porous Au structure. The porosity is ideal for gas sensing because it promotes the interfacing between the electrode and the electrolyte. 131 The Au electrode was functionalized by BMIMPF 6 , an ionic liquid that can dissolve O 2 . 132 The O 2 sensor was measured using an amperometric method in a nitrogen (N 2 ) atmosphere with a trace amount of O 2 . The sensor exhibited a linear response to O 2 with a sensitivity over a wide range of O 2 concentrations (from 0.054 to 0.177 v/v %), The sensor also exhibited a low detection limit of 0.0075%.
For applications that involve dissolved O 2 , e.g., in water, printed metallic electrodes can be used without functionalization to electrochemically measure the concentration of dissolved O 2 . For the dissolved O 2 sensor printed on PEN 43 shown in Fig. 4b, an Au nanoparticle ink was inkjet-printed to form both the working electrode and counter electrode. The working electrode was delimited by inkjet printing an insulating SU8 capping layer. The counter electrode was printed using the same Ag nanoparticle ink and subsequently chlorinated to form an Ag/AgCl structure. Samples of different dissolved O 2 concentrations were prepared by bubbling N 2 and air mixtures through a water bath. Measurements were first made using a commercial dissolved O 2 probe, then by the printed dissolved O 2 sensor using an amperometric setup. The printed sensor exhibited a sensitivity of 0.03 μA•L•mg −1 for dissolved O 2 concentrations between 0 and 8 mg•l −1 . The detection limit of the sensor was 0.11 mg•l −1 . A similar approach was demonstrated in another study. 39 In this example, the dissolved O 2 sensor was integrated into a water quality monitoring system. The sensor, which was comprised of an Au working electrode, an Au counter electrode, and an Ag reference electrode, was inkjet-printed on a PI substrate using nanoparticle inks. The sensor exhibited a linear response with a sensitivity of 0.4 μA•L•mg −1 for dissolved O 2 concentrations between 0 and 9 mg•l −1 , which is seemingly an improvement over the results reported in Ref. 42. However, the active areas of the working electrodes in Ref. 34. are unclear; therefore, the sensitivity should not be used for comparison purposes. Nevertheless, the dissolved O 2 sensor in this study was tested under shocks of dissolved O 2 . A solution containing 8.91 mg•l −1 of dissolved O 2 was diluted using a shock solution that contained 0.2 mg•l −1 dissolved O 2 to achieve a mixed solution that contained 8.13 mg•l −1 dissolved O 2 . The printed sensor exhibited output values that were between 98.5% and 101.5% of the reading provided by a commercial sensor, showing the printed sensor had a high accuracy.
Ion.-Ion sensors have a wide spectrum of applications, ranging from the detection of ions in water and soil systems to clinical analysis of biological fluids. [133][134][135][136] The majority of printed ion sensors are based on ion selective membranes deposited by screen printing. 137,138 Screen printing is heavily used because ion selective membranes (ISM) are typically comprised of viscous high molecular weight polymers and plasticizers that are not well suited for inkjet printing. 139 Inkjet printing has mainly been employed to fabricate ion sensors that utilize ISM-free designs. For example, a free chlorine sensor based on an inkjet-printed Ag electrode was fabricated on a PI substrate. 119 Free chlorine species (i.e. HClO and ClO − ) are able to oxidize the Ag electrode to form silver chloride/ silver oxide (AgCl/Ag 2 O) compounds and these reactions can be analyzed by linear sweep voltammetry. The Ag electrode was printed using a nanoparticle ink and subsequently sintered at 200°C. The electrode was used for direct electrochemical measurement without functionalization. The test was performed in an electrochemical cell comprised of the printed Ag working electrode, a Ag/AgCl reference electrode and a Pt wire as the counter electrode. The sensitivity of the sensor was tested in a range from 1 to 100 ppm and measured to be 30 μC ppm −1 with a lowest detectable concentration of 0.4 ppm. The sensor showed good agreement with the standard N,N-diethylparaphenylenediamine (DPD) method used in a standard swimming pool water sample test. In another study, 34 the printed Ag electrode was electrochemically functionalized by a sulfide to be used as a sulfide-selective electrode. The Ag electrode was patterned using a nanoparticle ink and subsequently sintered at 140°C to achieve a low resistivity of 0.1 μΩ·m. The area of the working electrode was delimited by a patterned SU8 capping layer that was inkjet printed. The working electrode was electrochemically sulfurized using a potentiostat setup. The electrolyte consisted of a 0.1 M sulfide solution with the pH adjusted to 14 by addition of NaOH. The sulfide-selective electrode was paired with an Ag/AgCl reference electrode during testing. The measured Nernstian sensitivity was −29.4 mV·decade −1 for a sulfide concentration range of 0.03 to 50 mM. When tested in the same sample solution, the printed sensor gave a readout that was in good agreement with a commercial sulfide sensor.
Although ISM is not suitable for inkjet-printing, it can be deposited onto a printed electrode by drop casting. A ISM for potassium ion (K + ) containing valinomycin, potassium tetrakis [3,5bis(trifluoromethyl)-phenyl]borate (KTFPB), bis(2ethylhexyl) sebacate (DOS), and poly vinyl chloride (PVC) was drop-casted onto an inkjet-printed Au working electrode after functionalization with PEDOT:PSS. 37 The working electrode was printed using an Au nanoparticle ink. The functional PEDOT:PSS layer was deposited on the electrode using three different methods: electropolymerization, inkjet printing, and drop-casting. The reference electrode was printed using the Au ink as well. After being coated with PEDOT: PSS, the reference electrode was further coated by a PVC membrane containing a lipophilic salt tetrabutylammonium tetrabutylborate (TBA-TBB) to form a solid-contact reference electrode. The surface roughness of PEDOT:PSS deposited using the three different methods was measured by atomic force microscopy (AFM). It was found that the electropolymerized PEDOT:PSS layer had the highest roughness. The rough film showed its advantage for sensing applications during the poteniometric sensitivity measurement; the electrode functionalized with electropolymerized PEDOT had the highest sensitivity for two different K + concentration ranges, 62.3 mV•decade −1 and 58.2 mV•decade −1 from 10 −5 M to 10 −2 M and from 10 −5 M to 10 −1 M, respectively. The high sensitivity is likely due to the thick and loose structured PEDOT:PSS prepared by electropolymerization.
Ion sensors for heavy metals in water have also been demonstrated using inkjet printing. Using an inorganic metal salt ink, a bismuth (Bi) based lead ion (Pb 2+ ) sensor was fabricated by inkjet printing. 29 The ink was made of bismuth(III) chloride (BiCl 3 ) dissolved in ethanol and EG and reduced by Ar plasma after printing. The printed Bi electrode was used as the working electrode in an electrochemical cell comprised of an Au counter electrode and an Ag/AgCl reference electrode. The detection of trace Pb 2+ was achieved by electrochemical reduction on the Bi electrode surface to form a Pb-Bi complex, which can be subsequently converted back to Pb 2+ by voltammetric methods. Differential pulsed voltammetry (DPV) analysis showed that the sensor exhibited a sensitivity of 0.083 A•m −1 •mm −2 , with a detection limit of 1 × 10 −7 M. The sensitivity was >12X higher than that of a previously reported electrodeposited Bi electrode, likely due to the porous morphology of the printed electrode. In another printed heavy ion sensor study, 43 the printed electrode was functionalized using a DNA aptamer to detect mercury ion (Hg 2+ ) in water and organic solvents. The electrode was printed using an Au ink. A thiol group-modified DNA aptamer [5'ThioMC6-D/TT TCT TCT TTC TTC CCC CCT TGT TTG TTT 3'] was activated by disulfide bond reduction using TCEP. The sensor was tested in deionized water and DMSO solution with trace concentrations of Hg 2+ using potentioelectrochemical impedance spectroscopy. The detection limits (LOD) of the sensor in water and DMSO were 0.01 ppm and 0.005 ppm, respectively. Both LOD values are far below the minimal concentration for practical implementation defined by the EPA.
Glucose and H 2 O 2 .-Glucose sensors are commonly used in monitoring blood sugar levels for diabetes diagnosis. 140 To enable routine self-monitoring, disposable glucose sensors for use with biofluid alternatives to blood, such as tear and sweet are currently being developed. Several studies have demonstrated that screen printing 141 and inkjet printing 140,142 can be used to potentially lower the manufacturing cost of the single-use glucose sensors.
An early study to develop inkjet-printed glucose sensors used an enzyme-based detection design. 111 The working electrode was fabricated by printing an Au nanoparticle ink on multi-layer coated paper. After photonic sintering, the Au electrode was electrochemically functionalized by PEDOT doped with GOx. The reference electrode was printed using an Ag nanoparticle ink followed by electrochemical chlorination. The sensor was evaluated using a chronoamperometric method. The glucose sensor printed on paper possessed a distinct response to changes in glucose concentration. The enzymatic sensor also exhibited good selectivity by showing no current response when mannose was tested. However, a slight variation in the baseline current was observed when the sensor was tested in a blank phosphate-buffered saline (PBS) solution, meaning the sensor may need further optimization in order to be used for clinical applications. Enzymatic glucose detection is widely used because of its efficiency and selectivity. However, the catalytic activity of enzymes is strongly dependent on temperature and pH. 143 Recently, non-enzymatic glucose detection methods have been developed, including for sensors fabricated by inkjet-printing. 13 A CuO microparticle based glucose sensor shown in Fig. 4c was printed on PET for glucose analysis in tear fluid. 36 The working electrode was printed using an Au nanoparticle ink. The electrode was functionalized using a drop-cast CuO microparticle-based solution, followed by drop-casting of a Nafion solution to immobilize the microparticles. The reference electrode was printed using an Ag nanoparticle ink followed by chlorination. As shown in Fig. 4c, Cu 2+ can be oxidized by hydroxide ions (OH -) to form Cu 3+ , which is subsequently reduced back to Cu 2+ by glucose. The glucose sensor was measured using a chronoamperometric method over a range of glucose concentrations from 100 nM to 30 mM. Excellent linearity was observed between of 3 and 700 μM. Moreover, the sensitivity was measured to be 850 μA•mM −1 •cm −2 with a detection limit of 2.99 μM in a concentration range of 3 to 80 μM.
H 2 O 2 is a by-product of many biological oxidase reactions, and therefore can be used to indirectly measure biomarkers found in oxidative stress-associated diseases including cancer, cardiovascular disorders, and neurodegenerative diseases. 120 Glucose can also be indirectly measured by detection of H 2 O 2 generated through various glucose-glucose oxidase reactions. 144 To this end, an inkjet-printed H 2 O 2 sensor was fabricated by printing Ag onto a PVC substrate. 31 The working electrode was fabricated from a nanoparticle Ag ink that was chemically sintered using a hydrochloride acid (HCl) vapor at room temperature after printing. The counter and reference electrodes consisted of a Pt wire and a Ag/AgCl electrode, respectively. The sensor was tested using an amperometric method. The sensitivity was 0.287 mA•mM −1 •cm −2 over a concentration range from 0.1 to 6.8 mM. The sensor was later used to detect H 2 O 2 secreted from living cells and demonstrated credible performance. In another study, Ag nanoparticles were printed onto a printed CNT working electrode to enhance the electrocatalyic properties of the working electrode toward the reduction of H 2 O 2 . 145 This enzymefree sensor was tested in an alkaline solution with H 2 O 2 concentration from 1 μM to 700 μM and showed an accurate and linear response.
Biological macromolecules.-Biological macromolecules such as proteins and DNA are commonly detected using immunoassaying techniques. Immunoassays employ a variety of labels for antibody or antigen recognition. The most commonly used labels in immunoassays are enzymes and such processes collectively known as enzymelinked immunosorbent assays (ELISAs). Electrochemical ELISAs have been widely used as the principal sensing mechanism in printed biological sensors. 146 For example, the Au-based electrochemical sensor shown in Fig. 4d was inkjet-printed on PI for immunodetection of a cancer biomarker protein IL-6. 106 The working electrode was patterned using an Au nanoparticle ink and subsequently functionalized using a SAM. The immunoassay was performed using a sandwiched structure whereas an anti-human IL-6 antibody was used as the primary antibody and a horseradish peroxidase (HRP)-labeled anti-human IL-6 was used as the secondary antibody. The sensor exhibited a linear sensitivity of 11.4 nA•pg −1 •cm −2 over a range of 20 to 400 pg•ml −1 and a detection limit of 20 pg•ml −1 in calf serum. In a later study, 115 IL-6 sensing was realized in a microfluidic device that incorporated disposable inkjet-printed Au electrodes. The analytes were immobilized for analysis using magnetic beads bioconjugated with enzyme label HRP and secondary antibodies. The printed Au electrode was functionalized with primary antibodies using a SAM. The microfluidic sensor was measured using an amperometric method. After an 8 min assay, the sensor exhibited a sensitivity of 2.18 μA•cm −2 •(log(pg•ml −1 )) −1 .
Label-free immunoassays have also been used in inkjet-printed biosensors. Label-free immunoassays are typically performed using surface plasmon resonance (SPR). 147 In some cases, redox probe or impedimetric methods can be used to detect an unlabeled antibody or antigen. 148 A biosensor for the detection of amyloid-beta-42 (Aβ 42 ), a biomarker of Alzheimer's disease was fabricated using an inorganic Au salt-based ink. 29 The working electrode was patterned using the Au salt-based ink and subsequently reduced to Au by an Ar plasma. The Au electrode was functionalized by a SAM followed by Aβ 42 antibody and antigen immobilization. The concentration of Aβ 42 was determined by measuring the faradaic current generated by the redox reaction of ferricyanide using DPV. The sensitivity of the printed electrode was compared with a similar electrode prepared by conventional magnetron sputtering. The printed electrode exhibited a sensitivity that was 10X higher than the sputtered electrode, likely due to the porous morphology of the surface structure. In another study, 114 an impedimetric method was used to measure an Au-based CRP sensor inkjet-printed on a paper substrate. The Au electrode was printed using a nanoparticle ink and subsequently sintered using a photonic drier. The Au electrode was functionalized using a SAM and streptavidin, prior to CRP antigen and antibody immobilization. The process was verified using SPR measurements. The CRP immunosensor was characterized using impedimetric analysis. It was shown that the real capacitance of the sensor systematically varied for each consecutive layer growth. The impedimetric method is highly versatile in that can be used for detecting CRP in miniaturized diagnostic devices.
The immobilization of proteins is can be achieved by depositing a biotinylated SAM on an electrode surface followed by bioconjugation of the proteins. Alternatively, thiol linkers can be bioconjugated to a protein prior to electrode functionalization. This method is more time-efficient than alternative methods and significantly simplified the alignment of the immobilized biomolecules. In a paper describing the development of inkjet-printed sensor for profiling protein-nucleic acid interactions, 44 a thiol-liner SATA was bioconjugated to the hnRNP H1 protein and subsequently used to functionalize an inkjet-printed Au electrode using an inorganic salt ink. The interaction between the hnRNP H1 protein and specific DNA strands was tested using a redox probe ferricyanide. The DPV results showed that hnRNP H1 had minor affinity to AAAAA oligonucleotides. The affinity to AGGGA and AGGGC oligonucleotides is moderate and to AGGGG oligonucleotides is the highest. The high selectivity of the sensor enabled a biosensor platform to discriminate minor structural differences in the G-tract nucleic acids. In another DNA hybridization sensor study, the bioconjugation method was systematically compared with the conventional biotinylated SAM method. 117 The electrode for the DNA hybridization sensor was fabricated using an Au nanoparticle ink. The electrode was functionalized using two different methods: the first method involved the sequential deposition of a biotinylated SAM, streptavidin and biotinylated DNA; the second method used a thiolfunctionalized DNA and subsequent backfill with a 11-mercapto-1undecanol (MUOH) SAM. The binding capacity and selectivity of both methods were assessed using SPR analysis. It was found that the electrode functionalized with the thiol-DNA probe had a higher binding capacity. The impedimetric analysis showed that the hybridization enables detection in pM range with both electrodes.
Inkjet-printed biosensors have also shown promise in bacteria detection. An Au electrode that was inkjet-printed using a nanoparticle ink and cured using photonic sintering was integrated in a microfluidic device fabricated using UV-nanoimprint lithography. 121 The device was used to detect salmonella which is a pathogen associated with contaminated food or water. An antibody-conjugated magnetic bead was exposed to salmonella in a PBS buffer solution to form a MB-Ab 1 -SA complex. The secondary antibody was labeled using alkaline phosphatase (ALP). A magnet was placed under the Au working electrode to capture the labeled salmonella complex. The reaction solution was comprised of L -ascorbic acid 2-phosphat (AAP) and tris(2-carboxyethyl)phosphine (TCEP). The ALP on the secondary antibody functioned as a catalyst to convert AAP to ascorbic acid (AA). The electroactive AA was electrochemically detected using amperometric methods. Salmonella solutions of different concentrations were tested and the results showed high accuracy and repeatability.

Summary
Inkjet printing as additive fabrication technology has advanced to the point where it is now a viable alternative to conventional subtractive techniques and other forms of subtractive printing (i.e., screen printing) to fabricate elemental metal-based electrochemical sensors. Inkjet printing is a particularly attractive option in applications that require low-cost, mechanically-flexible, or disposable sensors. Advancements in ink technologies have greatly expanded the portfolio of available materials from Ag and Au to a much wider range that include several metals (i.e., Bi, Pd and Pt) that are fundamentally important in electrochemical sensing. Equally important, numerous studies have shown that inkjet-printed metals are compatible with the same functionalization techniques routinely used in conventional electrochemical sensors. The inherent roughness and/or porosity of inkjet printed metals is a distinct advantage for electrochemical sensing as it increases the active surface area of a sensing electrode without any increase in lateral surface area. Advancements in post print sintering processes have greatly expanded the variety of polymer substrates available for use in electrochemical sensors, in particular polymers that exhibit a high degree of temperature sensitivity. Further advancements in ink technology, in particular the development of inks for metals where no ink currently exists as well as inks that require benign post print processing steps, should ensure further expansion of inkjet printing as a primary fabrication method for electrochemical sensors.