Sputter Deposited Nanocarbon Film Electrodes for Electrochemical Analysis of Biomolecules

Abstract

Carbon-based electrode materials have been widely applied for the electrochemical analysis of biomolecules. In addition to traditional carbon electrodes such as glassy carbon and carbon paste, a wide variety of carbon materials such as nanocarbons and boron doped diamond (BDD) electrodes have been employed for electrochemical analysis and biosensors in the last 25 years. Of the carbon electrode materials, carbon films are practically advantageous because they can be fabricated reproducibly with a wide range of shapes and sizes. In this paper, we report the application of sputter deposited nanocarbon film electrodes for the electrochemical analysis of biomolecules. The pure nanocarbon film electrodes have been employed for detecting DNA methylation, and lipopolysaccharides (LPS). Nitrogen-containing carbon films show improved electrochemical activity for biomolecules and excellent biocompatibility with interferents such as proteins. Metal nanoparticle embedded or modified carbon film electrodes show excellent electrocatalytic performance with sugars.

1. Introduction

Carbon-based electrode materials have been widely applied for the electrochemical analysis of biomolecules.¹ Traditional materials such as glassy carbon (GC), highly oriented pyrolytic graphite (HOPG) and carbon paste electrodes have been used mainly for basic electrochemical studies. More recently, carbon pastes (or carbon inks) have been used in enzyme biosensors and biofuel cells for wearable devices mainly by using a screen printing process.^2–4 New carbon materials have been studied since the 1990s by many groups. Boron-doped diamond (BDD) electrodes have led to an increase in the range of analytes thanks to their wide potential window and low adsorption of biomolecules after an electrochemical reaction.^5–7 However, the substrate materials that can be used to form BDD are limited due to the high deposition temperature (around 700 °C) required by a plasma or thermal CVD process.

In contrast, nanocarbon materials such as carbon nanotubes (CNTs) and graphene nanosheets have extremely large surface areas and exhibit excellent electrochemical activity.^8,9 This large electrode surface found with nanocarbon materials is suitable for immobilizing biomolecules such as enzymes to construct electrochemical biosensors. However, this also increases capacitive and background currents, which makes the signal to noise (S/N) ratio worse. In addition, nanocarbon materials are not free-standing and require solid conductive substrates to immobilize nanocarbon materials on measurement electrodes.

Compared with such materials, carbon films have various advantages because they can be fabricated reproducibly into electrodes with a wide range of sizes and shapes including the integration of many electrodes. Traditionally, carbon film electrodes were fabricated by the pyrolysis of organic molecules using organic polymers such as polyimides and small molecules for example 3, 4, 9, 10-perylenetetracarboxylic dianhydride.¹⁰ Such films are relatively thick but show sufficient electrical conductivity and electrochemical activity. Then, vacuum processes such as various types of sputtering,^11–13 electron beam evaporation,¹⁴ and a filtered cathodic vacuum arc system¹⁵ have been used to form carbon films.

We have employed electron cyclotron resonance (ECR) sputtering and unbalanced magnetron (UBM) sputtering to fabricate nanocarbon film electrodes.^16,17 The film electrodes contain both sp² and sp³ bonds and the sp²/sp³ ratio can be controlled by changing the ion acceleration voltage.¹² The sp³ ratio can be increased up to 50 % by UBM sputtering, which realizes a wide potential window.¹⁷ The film surface is very flat (average roughness <0.1 nm), which helps to suppress the adsorption of biomolecules. In this paper, we focus on an analysis of biomolecules using nanocarbon film electrodes,¹⁸ which have advantages such as the ability to detect small biomolecules, the suppression of fouling with biomolecules, application to DNA analysis and biosensors, and the electrocatalytic detection of sugars by modifying the carbon film surfaces with metal nanoparticles.¹⁹

2. Nanocarbon Film Electrodes for Direct Electrochemical Detection of Biomolecules

2.1 Sputter deposited pure nanocarbon films

For electrochemical analysis, electrodes with fast electron transfer and a low background noise level are suitable for achieving high sensitivity and a low detection limit. Edge plane rich carbon film is suitable for fast electron transfer. Diao et al. fabricated a carbon film with high electrochemical activity since the film contained many nanosized graphene sheets.¹³ This structure was formed by using ECR sputtering equipment with electron irradiation by supplying a positive bias voltage between a target and a substrate. They used the films to measure electroactive molecules such as catechols and resorcinols.²⁰ In contrast, we developed nanocarbon film electrodes by using ECR and UBM sputtering with ion irradiation by applying a negative bias voltage between a target and a substrate,^16,17 which realized carbon film with a sub-nanometer surface flatness. The sp³ concentration in the films can be increased up to 50 %. Figure 1 shows the potential window of ECR sputter deposited nanocarbon films compared with those of GC, BDD and metal electrodes.¹⁶ The potential window of an ECR sputtered nanocarbon film is wider than that of GC electrodes. The positive potential limit of the film is slightly lower than that of a BDD electrode. Thanks to the atomic level flatness, the nanocarbon film suppresses the adsorption of oligonucleotides after electrochemical oxidation.¹⁶ In contrast, the magnitude of the current decreases significantly at a GC electrode due to the adsorption of analytes after electrochemical oxidation. On the basis of the above performance, ECR nanocarbon films have been employed to detect various biomolecules including glutathione and 5-hydroxytryptamine (5-HT) without fouling the electrode surface.²¹

Figure 1.

Comparison of potential windows of GC, ECR nanocarbon film and BDD electrodes in 0.05 M H₂SO₄ solution. Scan rate = 100 mV/s. Reproduced with permission from Ref. 16. Copyright, 2006 American Chemical Society.

The above performance of ECR sputtered nanocarbon film is particularly advantageous when measuring biomolecules with a large molecular weight since large biomolecules often adsorb on the electrode surface and slow the electron transfer between the analytes and electrode. We succeeded in detecting one base mismatch of oligonucleotides (9 mer) by direct electrochemical measurement with background-subtracted differential pulse voltammetry (DPV).²² We also used the film to distinguish between cytosine (C) and methylated cytosine (mC) by utilizing the difference between the oxidation potentials of C and mC, although the oxidation potential of C is higher than that of other nucleobases including guanine (G), adenine (A) and thymine (T).²³ Figure 2 shows background subtracted square wave voltammograms (SWV) of CpG oligonucleotides at an ECR nanocarbon film electrode in 50 mM (M = mol dm⁻³) pH 5.0 acetate buffer containing 0.3 M NaNO₃. The oxidation peak of mC increases and that of C decreases as the mC number increases. This indicates that we could recognize between oligonucleotides with one base methylation difference by direct electrochemical oxidation. We also succeeded in detecting one mC difference in 24 mer retinoblastoma oligonucleotides between 5′-AATGGTTCAC CTCGAACACC CAGG-3′) and 6(5′-AATGGTTCACCT(mC)GAACACC CAGG-3′) with a simple SWV measurement.²⁴

Figure 2.

Background subtracted square wave voltammograms of oligonucleotide with different numbers of mC in 50 mM acetate buffer (pH 5.0) containing 0.3 M NaNO₃. Oligonucleotide concentration: 3 µM. Reproduced with permission from Ref. 23. Copyright 2008, American Chemical Society.

2.2 Doped and surface terminated nanocarbon films

The electrochemical performance of nanocarbon films can be modified by introducing surface terminated groups containing different atoms as shown in Fig. 3. With CF₄ plasma treatment, nanocarbon film with a hydrophobic surface can be obtained, which significantly suppresses the response of hydrophilic redox species.²⁵ The capacitive current is reduced by CF₄ plasma treatment since surface oxygen-containing groups are removed during treatment. Nitrogen doped carbon materials have been studied by many groups due to their low overpotential for oxygen reduction reactions (ORR),^26,27 because this electrochemical performance is highly suitable for application to platinum-free fuel cell electrodes. For basic electrochemical studies and electroanalytical applications, various fabrication processes have been utilized for carbon films containing nitrogen, which include pulsed laser-arc deposition,^28,29 sputtering processes^30,31 and chemical vapor deposition (CVD).³² Swain’s group reported that the sp² concentration increases and the sp³ concentration decreases with increasing nitrogen concentration and the electron transfer of redox species such as Fe(CN)₆^3−/4− improves.²⁸ A ΔE decrease in redox species such as Fe(CN)₆^3−/4− and an overpotential decrease in ORR were observed at nitrogen-doped carbon film electrodes.^19,31 We applied doped or surface terminated carbon film for the direct electrochemical detection of biomolecules. Table 1 shows examples of such carbon film electrode for detecting small molecules and ions. Bicontinuous microemulsions (BMEs) in which water and oil phases coexist bicontinuously on a microscopic scale, can dissolve hydrophilic, lipophilic, and amphiphilic compounds simultaneously. A BME was first used for electrochemical measurement by Rusling et al.³³ Recently, Kunitake, Kuraya and the authors employed fluorinated carbon (F-carbon) films for the selective detection of α-tocopherol against L-ascorbic acid since only the oil phase in which the α-tocopherol dissolved covers the electrode surface, and L-ascorbic acid existing in the aqueous phase is not oxidized on the electrode.³⁴ This method was then successfully used to detect lipophilic antioxidants including α-tocopherol in olive oil.³⁵ More recently, F-carbon film was modified with BME hydrogel to realize a stand-alone electrochemical system.³⁶

Figure 3.

Surface termination with different functional groups on nanocarbon film.

Table 1. Direct electrochemical detection of small molecules and ions at doped or surface terminated carbon film electrodes.

Carbon film structures	Electroanalytical application
CF4 plasma treated UBM carbon films	*Selective detection of α-tocopherol (Vitamin E) against L-ascorbic acid in bicontinuous microemulsion (BME) solution.34,35 *Selective detection of vitamin E after modification with BME gel36
Nitrogen-doped carbon film deposited by unbalanced magnetron (UBM) sputtering31 (1) Sputter deposited carbon film obtained by introducing Ar/N2 gas (2) Pure carbon film annealed with NH3 at 300 °C	Improved electrochemical activity for detecting NADH and L-ascorbic acid.31
Nitrogen-incorporated tetrahedral amorphous carbon (ta-C:N) thin-film grown by laser-arc physical vapor deposition	HPLC–EC analysis of estrogenic compounds with high sensitivity and low detection limit37
Nitrogen-doped diamond-like carbon film by DC magnetron sputtering	Detection of Pb2+ by CV38
Boron-doped and undoped diamond-like carbon films by femtosecond pulsed laser ablation	Detection of heavy metal ions such as Cd2+, Pb2+, Ni22+ and Hg2+ by square wave anodic stripping voltammetry39
N-doped diamond-like carbon thin film by ablation of a high purity graphite target	In situ spectroelectrochemical studies of electrochemical polymerization of aniline40
N-doped carbon film deposited by UBM sputtering and then treated with NH3 or N2 plasma.	Low adsorption of protein (bovine serum albumin) and improved redox behavior of Fe(CN)63−/4− in BSA-containing solution41
	Low adsorption of serotonin and improved electrochemical stability42

Nitrogen-containing (N-carbon) films show improved electrocatalytic activity for biomolecules. Films containing mainly a pyridine structure exhibited higher activity than other nitrogen-containing structures. The peak potentials of L-ascorbic acid and NADH decrease compared with those of pure and graphite nitrogen rich carbon film as shown in Figs. 4a and 4b.³¹ The negative peak shift is significant for L-ascorbic acid compared with NADH because N-carbon film has a pyridine structure that might interact with the anionic charge of L-ascorbic acid. N-carbon films show excellent biocompatibility due to their hydrophilic surface and improved electrocatalytic activity. Swain et al. fabricated a nitrogen-incorporated tetrahedral amorphous carbon (ta-C:N) thin film by laser-arc physical vapor deposition and applied for analysis of estrogenic compounds with high sensitivity and low detection limit.³⁷

Figure 4.

Cyclic voltammograms of 1: pure UBM, 2: graphite-like nitrogen rich UBM (N = 4 %) and 3: pyridine structure-rich UBM (N = 4 %) for (a) 0.1 mM NADH in 0.1 M phosphate buffer (pH 8.2) at a scan rate of 0.01 V/s and (b) 1.0 mM L-ascorbic acid in 1.0 M NaNO₃ at a scan rate of 0.1 V/s. Reproduced with permission from Ref. 31. Copyright 2019, The Royal Society of Chemistry.

The nitrogen doped diamond-like carbon (DLC) film was fabricated by direct current (DC) magnetron sputtering and applied for detecting Pb²⁺ by CV measurement.³⁸ B. Khadro and N. Jaffrezic-Renault et al. fabricated boron doped and undoped DLC films by femtosecond pulsed laser ablation and applied for anodic stripping voltammetric (SWASV) technique. By SWASV, low detection limits of 1, 1, 2 and 1 µg/l were obtained for Cd²⁺, Pb²⁺, Ni²⁺ and Hg²⁺, respectively.³⁹ An optically transparent electrode can be fabricated by depositing very thin carbon film. Very thin nitrogen doped DLC film was applied for infrared attenuated total reflection spectroelectrochemistry. The electropolymerization of polyaniline was monitored spectroscopically at the nitrogen doped DLC film.⁴⁰

One of the drawbacks of electrochemical measurement compared with other analytical techniques such as optical measurements is fouling. This is because large biomolecules such as proteins foul the electrode surface and slow the electron transfer of the analytes. Protein adsorption is a serious problem when measuring biological fluids such as blood and serum. Figure 5 shows voltammograms of 1 mM Fe(CN)₆^3−/4− with and without the inclusion of 100 mg/ml bovine serum albumin (BSA).⁴¹ The ΔE value for NH₃ plasma treated film is smaller than those for pure and H₂O plasma treated film, which is consistent with the results of previous reports.^28,30 With BSA, the ΔE value for pure carbon film increased significantly from 0.162 V to 0.843 V, suggesting that BSA adsorbed on the electrode surface. In contrast, the ΔE values for NH₃ plasma treated and H₂O plasma treated film electrodes are still small and the ΔE increase for an NH₃ plasma treated film electrode is only 7 mV, suggesting that BSA adsorption is suppressed. However, the peak heights at both film electrodes become smaller despite the very small ΔE increase. This might be due not only to BSA adsorption but also to a diffusion coefficient decrease in a solution containing a high concentration of BSA.⁴¹ The NH₃ plasma treated carbon film also shows high stability for measuring hydroxytryptamine (5-HT) compared with pure film and plasma treated films with other gases.⁴² As described above, carbon films terminated with other atoms can extend the application of biomolecules by controlling the surface conditions of film electrodes.

Figure 5.

Cyclic voltammograms for 1 mM Fe(CN)₆^3−/4− at (a) pure carbon, (b) H₂O plasma treated carbon and (c) NH₃ plasma treated carbon film electrodes before (1) and after (2) adding 100 mg/ml bovine serum albumin in 1 M KCl. Scan rate, 0.1 V/s.

2.3 Biosensor applications

Carbon films are suitable for application as the base electrodes of biosensors. This is because carbon film can be fabricated with any shape and size by using a conventional lithographic technique and fabrication processes such as sputtering are common and easily available. Although such equipment is expensive, the sputter deposited film electrodes have been used for commercial sensors due to high throughput of film fabrication. The low capacitive current of our nanocarbon films is advantageous for achieving a low detection limit since the S/N ratio can be reduced due to the low background current. We have employed ECR and UBM sputtered nanocarbon films as electrodes for various biosensors. An ECR nanocarbon film electrode has a higher S/N ratio than a GC electrode, and this enabled the ECR electrode to realize the quantitative measurement of adenine dinucleotide phosphate (NADPH) at a concentration of less than 100 nM. The detection limit of NADH was 10 nM at the ECR nanocarbon film electrode. In contrast, the detection limit at GC electrode was only 250 nM.

By the modification of GABase, which contains two main enzymes, γ-aminobutyrate ketoglutarate aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSDH), the enzyme modified ECR carbon film realized a low detection limit of 30 nM for detecting γ-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the brain, by the following reaction.⁴³

  

\begin{align*} &\text{GABA} + \text{$\alpha$-ketoglutarate}\notag\\ &\quad\xrightarrow{\text{GABA-T}}\text{succinic semialdehyde (SSA)} + \text{glutamic acid} \end{align*}

  

\begin{equation*} \text{SSA} + \text{NADP} + \text{H$_{2}$O}\xrightarrow{\text{SSDH}}\text{succinate} + \text{NADPH} \end{equation*}

Electrochemical assessments of the DNA methylation status in genomic DNA have been developed by using a combined bisulfate restriction analysis and nanocarbon film electrodes fabricated by UBM sputtering.^44,45 We used a cadmium (Cd) nanoparticle labeled detection probe combined with anodic stripping voltammetry⁴⁴ and alkaline phosphatase labeled probe DNA for the voltammetric detection of p-aminophenol (p-AP), which is a product obtained by the enzymatic reaction of p-aminophenyl phosphate (p-APP).⁴⁵ Figure 6 shows the detection principle of the latter report. By the bisulfate treatment of genomic DNA, cytosine (C) was converted to uracil (U). In contrast, methylated cytosine (mC) was not converted to U. After PCR, only the 5′-ACGT-3′ region of methylated DNA was cleaved by HpyCH4IV, which is a restriction enzyme. The PCR product was then biotinylated and immobilized on the substrate with a biotin and avidin bond.

Figure 6.

Detection principle of electrochemical DNA methylation analysis. (1) Bisulfate treatment of genomic DNA, (2) PCR and cleavage by HpyCH4IV, which is a restriction enzyme. (3) Immobilization of biotinylated PCR product onto a streptavidin surface. The product is then denatured and hybridized with a biotinylated probe. (4) Biotinylated probe is labeled with streptavidin modified alkaline phosphatase. Finally, an electrochemical signal is obtained to detect p-AP, which is formed by the enzymatic reaction of p-APP. Reproduced with permission from Ref. 45. Copyright 2017, American Chemical Society.

The product is denatured and then hybridized with a biotinylated probe, which is labeled with streptavidin modified alkaline phosphatase. Finally, an electrochemical signal is obtained to oxidize p-AP, which is formed by the enzymatic reaction of p-APP.⁴⁵ The nanocarbon film enables us to detect p-AP quantitatively thanks to both the large oxidation current derived from the π–π interaction between aromatic p-AP and the graphene-like structure of our nanocarbon, and the low capacitive current made possible by the flat angstrom-level surface.

As a result, the methylation ratio at a site-specific CpG site was estimated from the peak current of a cyclic voltammogram obtained from a PCR product solution in the 0.01 to 1 nM range.

Lipopolysaccharide (LPS) is a component of the outer membrane of gram-negative bacteria and consists of a lipid A and polysaccharide chain. Although bacterial cells have been employed to produce important pharmaceuticals, the products have the potential to be contaminated by LPS, which is a known endotoxin. Therefore, LPS should be removed from biological products because it triggers a wide variety of biological effects such as septic shock and coagulation in humans, resulting in acute septicemic disease, disseminated intravascular coagulation and multi-organ failure.^46–48 The limulus amoebocyte lysate (LAL)-based assay technique is the gold standard.⁴⁹ Although a LAL assay offers extremely high sensitivity and selectivity against LPS, it becomes relatively expensive and time-consuming when the LPS concentration is low. We proposed two types of electrochemical LPS biosensors based on nanocarbon film electrodes. Figure 7a shows the concept of our LPS detection process without using LAL reagent.⁵⁰ First, LPS is accumulated on the surface of microparticles modified with poly-ε-lysine (ε-PL), which has high affinity to LPS. Then a newly synthesized LPS probe (Fig. 7b), which consists of an LPS affinity peptide and a Zn complex, was captured on the LPS accumulated microparticles. The LPS and probe adsorbed microparticles are treated with acid solution, and eluted Zn²⁺ ions are successfully deposited on our nanocarbon film electrode. This is because nanocarbon film has wide potential window in the negative potential region, which is sufficient for reducing Zn²⁺ ions without interfering reactions. Finally, the deposited Zn, which is proportional to the LPS concentration, is detected by anodic stripping voltammetry (ASV) since ASV is a well-known technique for detecting metal ions down to the ppt level.⁵¹

Figure 7.

(a) Schematic illustration of a measurement with an LPS-affinity microparticle, a Zn-complex-based probe for LPS detection, and a nanocarbon film electrode: (1) An LPS sample was captured on LPS affinity microparticles, and then (2) an LPS probe consisting of Zn-complex parts and a LPS-affinity peptide was captured on the LPS-adsorbed microparticles (accumulation of the LPS probe). The adsorbed LPS probe was treated with an acid solution, and finally (3) the Zn²⁺-ion concentration of the treated solution was measured by ASV. (b) Chemical structure of the Zn-based LPS probe. (c) Voltammograms obtained by changing the LPS concentration. Reproduced with permission from Ref. 51. Copyright 2018, American Chemical Society.

Thanks to the wide potential window in the negative region, which is advantageous in terms of reducing Zn²⁺ to Zn and deposition on the nanocarbon film electrode, we achieved a detection limit of 0.2 ng/mL for LPS as shown in Fig. 7c.

We also developed another LPS sensor based on (ε-PL) and ferrocene labeled polymyxin B (FcPMB). After accumulating LPS and FcPMB on an ε-PL film modified F-nanocarbon film electrode, the adsorbed FcPMB provided an amplified response with Fe²⁺ ions, mediated by the redox reaction of captured Fc. However, Fe²⁺ is directly oxidized to Fe³⁺ on the nanocarbon film and the background current increased. In contrast, Fe²⁺ cannot be oxidized on the fluorinated carbon due to the hydrophobicity of the surface. Thus, we can detect Fc mediated current selectively without any interference from the direct oxidation current of Fe²⁺.⁵²

3. Carbon Film Electrodes Modified with Metal Nanoparticles

Carbon-based electrodes exhibit excellent electrochemical performance including a wide potential window and high stability. However, the electrocatalytic activity of carbon electrodes is usually low except for some doped carbons such as nitrogen-doped carbon alloy. Metal nanoparticles such as platinum (Pt) have been used to modify carbon materials for application as fuel cell electrodes. For electroanalytical applications, various carbon films modified with metal nanoparticles have been developed using various processes including the pyrolysis of a polymer and a metal complex composite,⁵³ electrodeposition,⁵⁴ ion implantation,⁵⁵ the use of a filtered cathodic vacuum arc,⁵⁶ and sputtering.¹⁹ Gold (Au), iridium (Ir) and bismuth modified boron-doped diamond electrodes have been employed to detect toxic metal ions such as arsenic, Pb²⁺ and Cd²⁺.^55,57,58 Carbon films modified with Pt nanoparticles are advantageous for detecting H₂O₂ and O₂, which can be combined with enzymatic reactions.¹⁹ Nickel (Ni) and copper (Cu) modified carbon film electrodes can be used to detect sugars such as glucose since these nanoparticles show excellent electrocatalytic activity in alkaline solution.¹⁹

We have developed carbon film electrodes modified with metal nanoparticles by employing a radio frequency (RF) and UBM sputtering process sometimes combined with electrodeposition. A schematic representation of the cross-sectional structures of our film electrodes is shown in Fig. 8. Currently, we are developing a process for fabricating a monometallic nanoparticle embedded carbon film, bimetallic alloy (nanoalloy) embedded carbon film, and metal heterodimer (consisting of attached two metal nanoparticles) modified carbon film electrodes. The structures and electrochemical performance of these electrodes are detailed in this section.

Figure 8.

Illustrations of cross-sectional structures of carbon film electrodes embedded with metal nanoparticles.

3.1 Monometallic nanoparticles embedded carbon film electrodes

Figure 8a shows the structure of carbon film electrodes modified with monometallic nanoparticles. Since the compatibility of carbon and metal is not good, metal nanoparticles embedded in the carbon film can be prepared with a one-step co-sputtering process, which is based on the work on the fabrication of carbon film embedded with magnetic cobalt (Co) nanoparticles reported by Hayashi and Hirono et al.⁵⁹ Since the process can be performed at low temperature, we can prepare the film electrodes on plastic substrates, which is advantageous for developing inexpensive sensor devices. Carbon film electrodes embedded with Pt and Ir nanoparticles exhibited excellent stability and a low detection limit for the detection of hydrogen peroxide and were successfully employed to detect glucose, acetylcholine and L-glutamic acid by combining them with enzymes.^60,61 The low detection limit is due to the high electrocatalytic activity of the nanoparticles, and the excellent stability is because the nanoparticles are partially embedded in the carbon films, which can prevent the detachment and aggregation of the nanoparticles. More recently, a carbon film electrode with embedded Pt nanoparticles was employed to detect geosmin, which causes a bad odor to emanate from freshwater lakes and rivers.⁶² We also developed a carbon film electrode embedded with Au nanoparticles and used it to detect As³⁺ by ASV. The effect of interfering ions such as Cu²⁺; can be eliminated by adding EDTA, and the obtained results are comparable to those obtained by using ICP-MS, which is a standard method.⁶³

3.2 Carbon film electrodes embedded with Ni/Cu nanoalloy

A nanoalloy comprises metal nanoparticles consisting of two or more metals and is expected to exhibit excellent electrocatalytic activity. We developed a Ni-Cu bimetallic nanoalloy embedded in carbon film as shown in Fig. 8b. Since we used UBM sputter equipment with a 3-target system, the Ni/Cu ratio in the nanoalloy and its concentration in the carbon film can be well controlled by changing the sputtering power of each target. We used our Ni/Cu nanoalloy embedded carbon film electrodes to detect urine D-mannitol which is a diagnostic marker of severe intestinal diseases, and where a low detection limit is required. Figures 9a and 9b show voltammograms of a carbon film embedded with Ni₆₄Cu₃₆ nanoalloy and a Ni₆₄Cu₃₆ alloy film for oxidizing 300 µM D-mannitol.⁶⁴ The electrocatalytic oxidation current of D-mannitol at the Ni₆₄Cu₃₆ alloy film is larger than that at the carbon film embedded with Ni₆₄Cu₃₆ nanoalloy due to the larger metal surface area. However, the carbon film embedded with Ni₆₄Cu₃₆ nanoalloy shows a much larger current density (dashed line in Fig. 9a) than the Ni₆₄Cu₃₆ alloy film when the metal surface area is normalized, suggesting the high electrocatalytic activity of nanoalloy embedded film. Figure 9c shows the dependence of the variation in oxidation signals on Ni/Cu ratio.⁶⁴ The carbon film embedded with nanoalloy exhibits a higher current than carbon films embedded with Ni or Cu monolithic nanoparticles. The film has been applied as a detector in high performance liquid chromatography (HPLC) for detecting erythritol, rhamnose, lactulose, sucrose, and sucralose. The detection limits with a nanoalloy (16 %) embedded carbon film electrode were 9, 25, 15, 21 and 77 nM for erythritol, rhamnose, lactulose, sucrose, sucralose, respectively. The obtained detection limits are about 1–2 orders of magnitude improved than those obtained with Au (by pulse amperometric detection) and Ni bulk electrodes, which is sufficient to detect diagnostic marker sugars in urine samples.⁶⁵

Figure 9.

CVs obtained with (black line) or without (gray line) 300 µM D-mannitol in 0.1 M NaOH solution using (a) carbon film embedded with Ni₆₄Cu₃₆ nanoalloy and (b) the Ni₇₀Cu₃₀ alloy film before (solid line) and after (dotted line) correction of the current by metal concentration. (c) Electrocatalytic oxidation currents after background subtraction with various compositions of nanoalloy embedded carbon films for 500 µM D-mannitol at 0.6 V during an anodic scan (n = 3). Potential scan rate: 0.1 V/s. Reproduced with permission from Ref. 64. Copyright 2016, The Royal Society of Chemistry.

3.3 Ni/Pd heterodimer modified carbon film electrodes

Heterodimers, also called dumbbell-like nanoparticles, have a structure consisting of two different metallic nanoparticles in partial contact.⁶⁶ Synergistic effects, such as charge transfer between components, alter their optical^67,68 and catalytic^69,70 properties, and these characteristics are successfully utilized for various applications such as electroanalysis^71–73 and energy conversion.^69,74–76

We developed a simple process for fabricating a vertically oriented metallic heterodimer array embedded in flat carbon film by utilizing co-sputtering and electrodeposition.⁷⁷ Figure 10a is a scheme of our heterodimer fabrication process. First, metal and carbon were co-sputtered and carbon film with partially embedded metal nanoparticles was obtained. Then, a second metal was electrochemically deposited solely on the metal nanoparticle surface by utilizing the overpotential difference between the carbon and metal surfaces. Using this process, we successfully prepared Ni(upper)/Pd(lower), Pt/Pd, Au/Pd, Ni/Au, Pt/Au and Pd/Au heterodimers on the carbon film since the deposition potential of each metal is 0.08 to 0.37 V higher than that of carbon.⁷⁷ We can deposit the second metal nanoparticles by selecting a potential between the deposition onset potentials of metal and carbon surfaces.

Figure 10.

(a) Fabrication process for vertically oriented metallic heterodimers semiembedded in carbon film (b) cross sectional HR-TEM image of Ni/Pd heterodimer modified carbon film. Cyclic voltammograms in 0.1 KOH aqueous solution obtained by using (gray) Ni/Pd heterodimer modified and black Ni nanoparticles modified carbon films at scan rates of (c) 1 and (d) 500 mV/s. Reproduced with permission from Ref. 77. Copyright 2022, American Chemical Society.

Figure 10b shows cross-sectional HR-TEM images of carbon film modified with Ni@Pd heterodimer. The second nanoparticles were clearly deposited on the surface. No apparent change was observed in the carbon film modified with Ni@Pd heterodimer after 65 min ultrasonication in ethanol. In contrast, most of the Ni nanoparticles were detached from the pure carbon film, suggesting that Ni nanoparticles bind tightly to the Pd nanoparticles embedded in the carbon film. Figures 10c and 10d show cyclic voltammograms in 0.1 M KOH aqueous solution obtained by using carbon films modified with Ni nanoparticles (black) and Ni/Pd heterodimer (gray) at scan rates of 1 and 500 mV/s respectively. With a low scan rate, both films show a similar redox reaction of Ni(OH)₂ oxidation to NiOOH. However, oxidation and reduction peaks were not observed for film modified with Ni nanoparticles at 500 mV/s. In contrast, clear redox peaks were still observed at the carbon film modified with Ni/Pd heterodimer. These results suggest that the redox reaction of Ni(OH)₂ on the surface of the Ni nanoparticles was significantly improved at the heterodimer structure, which is important as regards the electrocatalytic oxidation of sugars. In fact, a decrease in the onset potential (about 0.113 V) of glucose oxidation and a peak current increase were observed at the carbon film modified with Ni/Pd heterodimer compared with the carbon film modified with Ni nanoparticles.⁷⁷

We developed a much simpler system to improve the electrocatalytic performance of Ni nanoparticles by modifying the carbon film surface. Figure 11 compares the oxidation current of 300 µM maltopentalose obtained by linear sweep voltammetry in (a) pH 12.0 and (b) pH 12.7 aqueous solutions containing NaOH. Ni nanoparticles were formed on pure and N-carbon films. Similar to carbon film modified with Ni/Pd heterodimer, the redox reaction cycle of Ni(OH)₂ on N-carbon film increased compared with that of pure carbon film modified with Ni nanoparticles.⁷⁸ At both pH values, the oxidation currents for N-carbon film modified with Ni nanoparticles are significantly higher than those for pure carbon film modified with Ni nanoparticles thanks to the improved turnover of Ni(OH)₂. At pH 12.7, a significant decrease in onset potential for maltopentalose oxidation was observed for N-carbon film modified with Ni nanoparticles because negatively charged sugars at a higher pH could be attracted to a positively charged N-carbon surface.

Figure 11.

Linear sweep voltammograms in the presence (solid line) and absence (dotted line) of 300 mM maltopentalose in (a) pH 12.0 and (b) 12.7 aqueous solution containing NaOH and 1 M sodium acetate. Ni nanoparticles electrodeposited on pure carbon (1 and 2) and N-carbon (3 and 4) films were used as working electrodes. The scan rate was 1 mV/s. Reproduced with permission from Ref. 78. Copyright 2011, The Royal Society of Chemistry.

4. Conclusion

We introduced the fabrication and application of surface terminated and metal nanoparticles modified carbon film electrodes. These films were fabricated by ECR and UBM sputtering including the co-sputtering of metal and carbon and combined with various types of treatment such as plasma and electrochemical treatment. Pure and surface terminated carbon films have been used to detect biomolecules including small biochemical, oligonucleotides and LPS. Carbon film electrodes embedded with metal nanoparticles show excellent electrocatalytic activity and stability and were used for detecting biomolecules combined with enzymatic reactions, and the direct oxidation of sugars. The performance of the electrodes was successfully improved to allow the fabrication of nanoalloy, heterodimer on the carbon film and the deposition of nanoparticles on N-carbon film, which can be utilized for detecting sugars.

In the future, further improvements in electrochemical performance and more practical application will be realized by optimizing electrode structures and employing new carbons and nanomaterials.

Acknowledgments

The authors thank Dr. T. Kamata of AIST for his support with carbon film deposition. This work was supported by JSPS KAKENHI Grant number: 17H03081 and 20K21133.

CRediT Authorship Contribution Statement

Osamu Niwa: Conceptualization (Lead), Funding acquisition (Lead), Methodology (Lead), Project administration (Lead), Writing – original draft (Lead)

Saki Ohta: Conceptualization (Equal), Investigation (Equal), Writing – review & editing (Supporting)

Shunsuke Shiba: Conceptualization (Equal), Funding acquisition (Supporting), Investigation (Lead), Methodology (Supporting), Writing – review & editing (Supporting)

Dai Kato: Conceptualization (Equal), Funding acquisition (Supporting), Investigation (Equal), Writing – original draft (Supporting), Writing – review & editing (Equal)

Ryoji Kurita: Investigation (Equal), Writing – original draft (Supporting), Writing – review & editing (Equal)

Conflict of Interest

The authors declare no conflict of interest in the manuscript.

Funding

Japan Society for the Promotion of Science: 17H03081

Japan Society for the Promotion of Science: 20K21133

Footnotes

O. Niwa: ECSJ Fellow

S. Shiba, D. Kato, and R. Kurita: ECSJ Active Members

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Biographies

Osamu Niwa (Professor Saitama Institute of Technology)

Osamu Niwa was born in 1958. He graduated from Kyushu University in 1981 and received M.S. and ph. D degree also from Kyushu University in 1983 and 1990. He worked in research labs. in NTT from 1983 to 2004 and then worked in AIST from 2004 to 2015. In Oct. 2015, he joined in his current institute as a professor. His research fields are electroanalytical chemistry, biosensors and nanocarbon materials. Hobby: Keeping fished (Nishiki Goi), Plastic models, Trekking

Saki Ohta (Researcher, Asahi FR R&D Co., Ltd.)

Saki Ohta was born in 1996. She graduated from Saitama Institute of Technology in 2018. She received M.S. and Ph.D. degree of engineering from Saitama Institute of Technology in 2020 and 2023, respectively. She joined Asahi FR R&D Co., Ltd. in 2023. Her research fields are electrochemistry and surface science. Hobby: Driving a car, Whisky Tasting

Shunsuke Shiba (Researcher, NiSiNa materials Co., Ltd.)

Shunsuke Shiba was born in 1990. He received ph. D degree of engineering from University of Tsukuba in 2018. He worked as an assistant professor in Ehime University from 2019–2022. He joined NiSiNa materials Co., Ltd. in 2023 as a researcher and started to study as a JST PRESTO researcher from Oct. 2023. His research field is electrochemistry (Electrocatalysts). Hobby: Trip, Netflix.

Dai Kato (Group Leader, National Institute of Advanced Industrial Science and Technology)

Dai Kato was born in 1975. He graduated from Kumamoto University in 1998 and received Ph. D degree of engineering also from Kumamoto University in 2003. He joined his current institute in 2004 and started his group as a group leader from 2020. His research fields are electroanalytical chemistry, biosensors and carbon nanomaterials. Hobby: Camping

Ryoji Kurita (Group Leader, National Institute of Advanced Industrial Science and Technology)

Ryoji Kurita was born in 1973. He graduated from Nihon University in 1995 and received ph. D degree of engineering from Kyushu University in 2004. He joined his current institute in 2004 and started his group as a group leader from 2015. His research fields are electroanalytical chemistry, biosensors (immunoassay and DNA analysis). Hobby: Keep tropical fish, Camping