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Article

Fully Inkjet-Printed Biosensors Fabricated with a Highly Stable Ink Based on Carbon Nanotubes and Enzyme-Functionalized Nanoparticles



Mijal Mass 1 , Lionel S. Veiga 1, Octavio Garate 1, Gloria Longinotti 1, Ana Moya 2,†, Eloi Ramón 2 , Rosa Villa 2,3, Gabriel Ybarra 1,* and Gemma Gabriel 2,3,*



1 INTI-Micro y Nanotecnologías, Instituto Nacional de Tecnología Industrial (INTI), San Martín, Buenos Aires B1650WAB, Argentina; mmass@inti.gob.ar (M.M.); lveiga@inti.gob.ar (L.S.V.); ogarate@inti.gob.ar (O.G.); glonginotti@inti.gob.ar (G.L.)

2 Institut de Microelectrònica de Barcelona, IMB-CNM (CSIC), Campus Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain; ana.moya@eurecat.org (A.M.); eloi.ramon@imb-cnm.csic.es (E.R.); rosa.villa@imb-cnm.csic.es (R.V.)

3 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain * Correspondence: gybarra@inti.gob.ar (G.Y.); gemma.gabriel@imb-cnm.csic.es (G.G.) † Present Addresses: Eurecat, Centre Tecnològic de Catalunya, Functional Printing & Embedded Devices Unit,

08302 Mataró, Spain.



Citation: Mass, M.; Veiga, L.S.; Garate, O.; Longinotti, G.; Moya, A.; Ramón, E.; Villa, R.; Ybarra, G.; Gabriel, G. Fully Inkjet-Printed Biosensors Fabricated with a Highly Stable Ink Based on Carbon Nanotubes and Enzyme-Functionalized Nanoparticles. Nanomaterials 2021, 11, 1645. https://doi.org/10.3390/ nano11071645

Academic Editor: Piersandro Pallavicini

Received: 3 June 2021 Accepted: 17 June 2021 Published: 23 June 2021



Abstract: Enzyme inks can be inkjet printed to fabricate enzymatic biosensors. However, inks containing enzymes present a low shelf life because enzymes in suspension rapidly lose their catalytic activity. Other major problems of printing these inks are the non-specific adsorption of enzymes onto the chamber walls and stability loss during printing as a result of thermal and/or mechanical stress. It is well known that the catalytic activity can be preserved for significantly longer periods of time and to harsher operational conditions when enzymes are immobilized onto adequate surfaces. Therefore, in this work, horseradish peroxidase was covalently immobilized onto silica nanoparticles. Then, the nanoparticles were mixed into an aqueous ink containing single walled carbon nanotubes. Electrodes printed with this specially formulated ink were characterized, and enzyme electrodes were printed. To test the performance of the enzyme electrodes, a complete amperometric hydrogen peroxide biosensor was fabricated by inkjet printing. The electrochemical response of the printed electrodes was evaluated by cyclic voltammetry in solutions containing redox species, such as hexacyanoferrate (III/II) ions or hydroquinone. The response of the enzyme electrodes was studied for the amperometric determination of hydrogen peroxide. Three months after the ink preparation, the printed enzyme electrodes were found to still exhibit similar sensitivity, demonstrating that catalytic activity is preserved in the proposed ink. Thus, enzyme electrodes can be successfully printed employing highly stable formulation using nanoparticles as carriers.



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Keywords: biosensors; electrochemical detection; inkjet printing; carbon nanotubes; carbon-ink electrodes; silica nanoparticles



Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).



1. Introduction

Printed electronics are expected to have a great impact on the manufacturing of functional electronic devices [1–7], especially in the biomedical field, where the fabrication of low-cost single-use sensors and biosensors have led to interesting applications, such as personalized medicine [8–12]. Inkjet printing (IJP) is one of the most used printing technologies for sensor production because it allows low production costs and high massproduction of devices with the desired reproducibility [13–15]. A variety of functional materials can be digitally deposited by IJP in microscale dimensions at low temperatures on a wide variety of substrates. Furthermore, as a non-contact, mask-less deposition approach, it reduces fabrication time and costs, and permits customized geometries [16–19].



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Recent research has also demonstrated that inkjet printing is a reliable solution for the fabrication of sensors and biosensors [20–23], making the technology advantageous for the development of green electronics [24] and reliable, easy-to-use diagnostic tests.

Specifically in the biosensing field, IJP benefits from its versatility and high resolution for the development of prototypes. However, IJP of biomolecules presents some challenges, especially regarding molecular stability. In this respect, non-specific adsorption of biomolecules onto the ink chamber walls and maintaining stability during printing, where the molecules suffer thermal and/or mechanical stress, have been reported as two major issues for IJP of biomolecules [21,25].

A range of additives have been employed in order to maintain the stability of biomolecules [26] with varying results. Although it is expected that some enzymes immobilized on a surface might exhibit a lower catalytic activity, it is well known that surface-immobilization often leads to improved stability, since their aggregation is no longer possible. Furthermore, as indicated by Rodrigues et al. [27], enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted, thus decreasing its activity. Finally, as has been reported by Hoarau et al., methods of enzyme attachment can be fine-tuned so that activity and stability can be greatly enhanced [28].

On the other hand, carbon-based nanomaterials, especially carbon nanotubes (CNTs) and graphene, have been recognized as convenient materials for the construction of biosensors, since they can be employed as solid supports for immobilization of biomolecules and also provide a high electronic conductivity [29]. The high surface to volume ratio of CNTs enables an increase in the number of immobilized biomolecules, as well as reducing the time response of the biosensors [30]. In order to immobilize enzymes onto CNTs, they are usually oxidized to produce carboxylic groups that can act as anchoring points. For instance, a usual method to immobilize proteins onto CNTs is via the carbodiimide crosslinking reaction between carboxylic groups formed on the CNTs and free amino groups of enzymes [31]. However, it is well known that the oxidation of CNTs has a negative impact on their conductive and mechanical properties. Therefore, alternative methods of immobilization of biomolecules, which do not affect CNT electronic properties, are of interest.

The use of particulated carriers such as polystyrene microspheres, onto which biomolecules can be immobilized, has been proposed as an alternative to direct linking to CNTs for the preparation of inks for printed enzyme electrodes [32]. This approach allows enzyme immobilization without significantly affecting the electrical and electrochemical behavior of CNTs. However, inks containing microspheres are not suitable for IJP, since particles with a size higher than 200 nm cause nozzle clogging. This problem might be solved by the use of an adequate nanocarrier and, in this sense, silica nanoparticles seem a convenient material of choice. Silica nanoparticles can be prepared by the Stöber method with a low dispersity and a controlled size between 9 and 800 nm [33], amenable to IJP. Furthermore, the surface of silica particles can be easily functionalized to provide amino or carboxylic groups onto which enzymes can be covalently linked [34].

In this paper, we propose a general approach to increase the stability of biomolecules in inks and during the printing by immobilizing enzymes onto silica nanoparticles. The incorporation of these nanoparticles in a single walled carbon nanotube (SWCNT) conductive ink allows a simple and general procedure for the preparation of IJP enzyme electrodes. Our results show that this approach could be used to immobilize a considerable quantity of biomolecules without significant loss of their biological activity. As a proof a concept, we present the results for the preparation of an enzyme electrode prepared with horseradish peroxidase (HRP).



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2. Materials and Methods 2.1. Synthesis of SiO2 Nanoparticles

Spherical SiO2 nanoparticles (SNPs) were prepared by a sol-gel process by controlled hydrolysis of tetraethyl orthosilicate (TEOS) in batch synthesis at room temperature. Firstly, 13.5 mL of deionized H2O, 24.5 mL of anhydrous EtOH and 1.22 mL of concentrated NH3 were placed together in a glass flask. Then 830 µL of TEOS were quickly added under vigorous magnetic stirring (800 rpm) and the flask was closed to avoid reagents evaporation. After 15 min, the solution turned into a pale blue-white solution indicating the presence of colloidal SiO2. The solution was left covered overnight on a rotary mixer. To remove by-products and solvents after the synthesis of the silica particles, they were centrifuged at 3000 rpm for 30 min (Mikro 1200, Hettich, Tuttlingen, Germany) and gently resuspended in 10 mL deionized water. After repeating the washing process three times, hydrodynamic size was measured by dynamic light scattering (DLS) with a DynaProNanostar from Wyatt Technology (Santa Barbara, CA, USA). Finally, the particles were dried until a solid was obtained, and then they were placed in an oven at 120 ◦C and under vacuum for 48 h. After this stage, they were allowed to cool and then resuspended in H2O. The solid was placed in a FalconTM type tube and H2O was added until total resuspension was achieved. Finally, they were centrifuged again at 6000 rpm for 20 min with the addition of absolute EtOH twice. All chemicals were of reagent grade and used without further purification. The size and distribution were analyzed using field emission scanning electron microscopy (Quanta 250, FEI, Waltham, MA, USA).

2.2. Preparation of SNP-HRP 2.2.1. Functionalization of SNPs

Firstly, the dried silica nanoparticles were treated at 120 ◦C for 24 h to ensure the consolidation of the inorganic structure. Then, the particles were resuspended in 1.5 mL of EtOH and 200 µL of APTES was added. The reaction was allowed to stir overnight to obtain NH2 modified silica particles. Subsequently, they were centrifuged at 14,000 rpm for 5 min and washed with EtOH and three times with dimethylformamide (DMF). The SiO2-NH2 NPs were modified into carboxylic groups. Although several methods of covalent enzyme immobilization may be used, our experience shows that enzymes conjugated using carbodiimide present a higher activity. Thus, amino groups in the NPs were changed to carboxylic groups in order to link them to amino residues in the enzyme forming an amide covalent link. The NH2-modified particles dispersed in DMF were added to a previously prepared solution of 5 mL of 1% anhydrous succinic and 1.9 mL of pyridine. The mixture was stirred overnight. The nanoparticles were washed twice in DMF and resuspended in 5% HCl for 5 min. Finally, the particles were centrifuged at 8000 rpm for 10 min and resuspended in deionized water until the pH of deionized water was reached. The NPs were resuspended in 2 mL of 0.1 M phosphate buffer pH 7.0.

2.2.2. HRP Immobilization onto SiO2 Particles

HRP was immobilized by coupling the amine groups of the enzyme with the carboxylic groups of the SNPs. Firstly, the carboxylic groups were activated by adding 500 µL of a mixture of 0.1 M EDC and 25 mM NHS (in 0.1 M phosphate buffer pH 7.0) to 500 µL of COOH-modified particles. The mixture was incubated for 60 min with low vortexing. It was washed twice with 500 µL of 0.1 M phosphate buffer solution of pH 7.0 and subsequently 80 µL of HRP (0.002 g/80 µL in phosphate buffer) was added. They were left under stirring for 2 h at room temperature. Then, the particles were centrifuged for 20 min at 3000 rpm to avoid enzyme denaturation. The supernatant was removed and gently resuspended in 500 µL of phosphate buffer. The above process was repeated 5 times. Finally, the SNPs were resuspended in 4 mL of phosphate buffer and stored in a refrigerator.



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2.3. SWCNT Aqueous Ink

Single-walled carbon nanotubes (SWCNT) carboxylic acid functionalized with 90% carbon basis, with a diameter of 4–5 nm and a length of 0.5–1.5 µm (bundle dimensions, Sigma-Aldrich, St. Louis, MO, USA), sodium dodecyl sulfate (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) and deionized water (conductivity less than 1 µS cm−1) were used for the preparation of the aqueous ink. Two inks were formulated with different concentrations of SWCNT: 7.5 mg/mL and 5 mg/mL. The first (SWCNT-7.5) was formulated with 11.2 mg of SWCNT mixed with 8 mg of sodium dodecyl sulfate (SDS), which acts as a surfactant, in an Eppendorf® tube with 1.5 mL of deionized water at room temperature; while the second ink (SWCNT-5) was formulated with 7.1 mg of SWCNT mixed with 5.3 mg of SDS in 1.5 mL of deionized water. These mixtures were sonicated for 30 min and then centrifuged at 18,000 rpm for 10 min. The supernatant was used. The inks were allowed to stabilize for at least 12 h and then sonicated it again for 30 min before use. Both inks were prepared to be printed with IJP.



2.4. SWCNT-SNP-HPR Bio-Inks

An aqueous bio-ink was formulated by incorporating silica nanoparticles (SNP, with a mean diameter of 66 nm ± 8 nm) with covalently immobilized HRP to the SWCNT ink prepared as previously described, fulfilling the necessary rheological conditions for IJP. In order to find an adequate formulation with good electrochemical response and easy printing, several inks were prepared combining the two carbon nanotubes inks (SWCNT-7.5 and SWCNT-5) with different concentrations of the enzyme-functionalized silica nanoparticles (SNP-HRP) (Table 1).



Table 1. Electrodes printed with different combinations of SWCNT and SNP-HRP concentrations and number of layers, including the estimated masses of SWCNT and SNP-HRP deposited on the electrodes.



SWCNT Concentration

(mg/mL)



SNP-HRP Concentration

(mg/mL)



7.5



0



5



0



7.5



1



5



1



5



5



Number of Layers

6 12 18

6 12 18

6 12

6 12

6 12



SWCNT Mass (µg)

1.5 3.0 4.5

1.0 2.0 3.0

1.5 3.0

1.0 2.0

1.0 2.0



SNP-HRP Mass (µg)

0 0 0

0 0 0

0.2 0.4

0.2 0.4

1.0 2.0



2.5. IJP Electrodes Process

A three-electrode electrochemical cell was designed so that the dimensions were compatible with the electrodes used for a previously reported portable multipotentiostat [34] (Supplementary Information Figure S1). Printing patterns were made using the Electronic Design Automation (EDA) layout software and imported with the Dimatix Bitmap editor software. For the development of the conductive path, the working electrodes (WE) with a geometric surface area (GSA) of 0.78 mm2 and counter electrode (CE) with a GSA 5.25 mm2, a commercially low curing Au nanoparticle ink were used (Drycure Au-JB 1010B, C-INK Co., Okayama, Japan). For the reference electrode (RE) with a GSA 2.55 mm2, a silver nanoparticle ink (DGP-40LT-15C from the firm ANP, Sejong, Korea) was used. The passivation and protective layer of the electrodes was done using a dielectric PriElex®



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SU-8 ink (MicroChem, Westborough, MA, USA). Finally, a circular area with a diameter of 1 mm was printed onto the WE with the carbon nanotubes water-based ink. All inks show drop-on-demand (DoD) inkjet compatible specifications.

The manufacturing process was performed without the need of temperature and humidity control in a standard laboratory environment. All electrodes were fabricated on a 125 µm thick polyethylene terephthalate (PET) substrate (Melinex ST504, DuPont Teijin Films, Chester, VA, USA), without any extra surface treatment, using a DoD Dimatix Materials Printer (DMP 2831 from Fujifilm Dimatix, Santa Clara, CA, USA) and three disposable and fillable cartridges containing 16 individually addressable nozzles each with a diameter of 21.5 µm and 10 pL nominal droplet volume (DMC-11610 from Dimatix Fujifilm, Lebanon, NH, USA). Each cartridge was filled with the above commented inks. Several samples of WEs were manufactured, combining different concentrations of carbon nanotubes and functionalized nanoparticles and also different numbers of layers. In the case of WEs printed with bio-ink (SWCNT-SNP-HRP), the reduction of printing times was prioritized so they were only manufactured with 12 and six layers. The details of these combinations can be seen in Table 1.

2.6. Fabrication of Inkjet Printed Electrode

Optimization of the IJP process was performed as previously reported [35]. The manufacture of the electrodes was carried out following the sequence of steps as shown in Figure 1, in which SWCNT illustrates the strategy used for the printed electrodes with SWCNT ink and SWCNT-SNP-HRP the WE printed with bio-ink. This second strategy assumed that the enzymes functionalized in the bio-ink could not be exposed to temperatures higher than 37 ◦C, as this could affect the enzyme activity. As a first step (Figure 1-1), we printed the WE, CE and conductive paths with the Au nanoparticles ink, setting a drop spacing (DS) of 15 µm, which is equivalent to a printing resolution of 1693 dpi (dots per inch). Subsequently, the cartridge containing the Au ink was replaced with one containing Ag nanoparticle ink to print the pseudo reference electrodes (pRE) (Figure 1-2). In this case, the printing was done with a DS of 40 µm (resolution of 635 dpi). They were then subjected to thermal drying of 90 ◦C for 10 min and then to a sintering process in an oven 30 min at 120 ◦C (Figure 1-3). This step is where the final electrical properties of the inks are achieved. The pRE was chlorinated by cyclic voltammetry (CV) in 0.1 M HCl, scanning potential from 0 to 0.2 V against Ag/AgCl commercial RE (Metrohm, Herisau, Switzerland Germany) and Pt CE (Metrohm) at 20 mV/s to obtain a stable pRE (Figure 1-4) [36]. For the WE printed with the SWCNT ink, the next step was the printing of the WE area of 1 mm in diameter with the CNT water-based ink with a DS of 15 µm (1693 dpi) (Figure 1-5.A). Subsequently, they were subjected to a drying process of 20 min at 120 ◦C and then sintering at 140 ◦C for another 20 min (Figure 1-6.A). Afterwards, the PriElex® SU8 dielectric ink was printed with a DS of 15 µm (1693 dpi) as a protective layer for the conductive paths and delimiting the active area for the WE and the contact pads of the electrodes (Figure 1-7.A). In this case, the curing was carried out first on a hot plate at 100 ◦C to evaporate the solvents and then with UV lamp exposure for 30 s, to generate the polymerization of the ink by cross-linking (Figure 1-8.A). For the electrodes printed with the bio-ink (SWCNT-SNP-HRP), after the stage of shaping the conductive paths of the electrodes, the dielectric layer was also printed with PriElex® SU8, in the same way as previously described (Figure 1-5.B,6.B). In the last step, the bio-ink was printed on the active area of WE also with a DS of 15 µm (Figure 1-7.B), but in this case the curing was carried out at room temperature for 24 h (Figure 1-8.B). This curing strategy assumed that the bio-ink enzymes could not be subjected to higher than room temperature in order to avoid damage and losing enzymatic activity in the electrodes.



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FFigiguurree22sshhoowwssoopptitcicaallmmicicrroossccooppyyimimaaggeessooffddififffeerreennttWWEE::(a(a))aaAAuueelelecctrtrooddee, ,(b(b))aa 1122-l-alayyeerrSSWWCCNNTT(7(7.5.5mmgg//mmLL)) eelleeccttrrooddee,,aanndd((cc))aa1122-l-alayyeerrSSWWCCNNTT-S-SNNPP-H-HRRPPeelelecctrtoroddee (w(witihth55mmgg//mmLLaanndd55mmgg//mmLL ccoonncceennttrraattiioonn ooff SSWWCCNNTT aannddSSNNPP--HHRRPP,,rreessppeecctitviveelyly).).TThheessee aarreetytyppicicaallimimaaggeessrreeppreresesenntatatitviveeoof fththeeoobbtatainineeddeelelecctrtoroddese.s.BBooththAAuuaannddSSWWCCNNTTpprrinintetedd eelelecctrtrooddeesspprreesseennteteddaahhoommooggeenneeoouussssuurrffaacceeinintthheeoopptticicaallmmiiccrroossccooppyyimimaaggeess..HHoowweevveerr,, ininFFigiguurree22cc, ,pprirnintitningglilnineesscacannbbeeobosbesrevrvededononthteheSWSWCCNNT-TS-NSNP-PH-HRRPPeleelcetcrtordoed.e.TThhee cchhaarraaccteterrisistitcicggooldldccoololorrisissseeeenninintthheeAAuueelleeccttrrooddee,,wwhhilielebbootthhSSWWCCNNTTeelelecctrtrooddeesspprreesseennt t aaddaarrkkccoololorrccoommppleletetelylyccoovveerrininggththeeuunnddeerlrylyininggAAuususurfrafacece. .TThheeSSWWCCNNTTeelelecctrtrooddeeinin FFigiguurree22bbisisddaarrkkeerrtthhaanntthheeSSWWCCNNTT-S-SNNPP-H-HRRPPoonnee, ,pprorobbaabblylydduueetotoththeeddififfefererennt tmmaassssooff SSWWCCNNTTddeeppoossitietedd((33µµggvvss. .22µµgg))..AAsstthheennuummbbeerrooffpprriinntteeddllaayyeerrss ooff SSWWCCNNTTssiinnccrreeaasseess, ,

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totoththeepprreesseenncceeooffnnaannootutubbeess(S(SuupppplelemmeenntataryryInInfoformrmaatitoionnFFigiguurereSS22).).



FFigiguurree22.. OOppttiiccaall mmiiccrroossccooppyyiimmaaggeessooffpprirnintetdedeleelcetcrtordodese:s(:a()a1)-l1a-ylaeyreAruA, u(b, )(b12) -1la2y-learySeWr SCWNCTN(7T.5 (7Hm.5Rgm/Pm)g.L/mofLSWofCSNWTC)N, aTn)d, a(nc)d1(2c-)la1y2e-lraSyWerCSWNTC-NSNT-PS-NHPR-PH(R5Pm(5g/mmgL/omfLSWofCSNWTCaNnTda5nmdg5/mmLg/omf SLNoPf SNP-HRP).

TThheeeelelecctrtrooddeesswweerereddeesisgignneeddtotohhaavveeaannaarereaaoof f00.7.788mmmm22aannddaaddiiaammeetteerrooff11000000µµmm.. TThheeaacctutuaallmmeeaassuurereddddiaiammeetetersrswweerere11006644µµmm, ,11112266µµmmaanndd11112299µµmmfoforrAAuu, ,SSWWCCNNTTaanndd SSWWCCNNTT-S-SNNPP-H-HRRPPeelelecctrtoroddeess, ,reressppeecctitviveelyly. .TThheererefoforer,e,ththeeddimimeennsisoionns,s,aasswweellllaassththeeaarereaa ooffthteheeleectlerocdtreosd, ewse, rewcelroesectlootsheosteoofththoeseoroigfintahledeosriiggni,nwalhidchescigonnfi, rwmhtihcehcocmonpfairtimbilitthye ocfotmhepmatiabtielritiaylsofanthdethmeapterirniatilns ganpdrotcheessp. rFiinntainllgy, pitrcoacnesbs.e Fsieneanlltyh,aitttchaeninbseulsaeteinngthlaaytetrhse eifnfescutliavteinlygcloavyeerrssthefefeccotnivdeulycticvoevpearsthtshaencdodndeluimctiitvseapnaatphps raonxdimdaetelimciritcsulaanr aarpepar. oximate circuTlhare arreesais.tance along the Au printed paths with a length of 13.5 mm was measured and anThaeverreasgisetarensciestanlocnegvathlueeAouf 6p3r±int1e2dΩpa(sthtasnwdaitrhd adelevniagttihono)fw1a3s.5ombtmainweda.sTmheraesfuorred, naengdligainblaevoehrmagiec rfaelslisswtaenrceetvoableuexopfe6c3te±d1d2uΩe (tsotathnedcaornddduecvtiivaetiponat)hws.aTsyopbictailnceudrr.eTnhtevraelfuoerse, wneergelignibthle orhdmericorfablleslowwe1re0 µtoAb, esoeIxRpedcrtoepdsdinuethteoctohneducocntidveucptaivthespwaethres.bTelyopwic1almcVur.rent valuTehs ewSeWreCinNtThse coorndceernotrrabteiolonwu1s0edµ Ain, tshoeIRindkrsohpasdinatmheacjoorndefufecctitvoenpaththespwrienrteabielliotyw. W1 hmeVn.concentrations higher than 7.5 mg/mL were used, the nozzles clogged during the

first imThpereSsWsioCnNs.TsElceocntrcoednetrsactioounldusbeedpirninttheedinuksisnhgaSdWaCmNaTjoirnekfsfewctitohnathcoenpcreintraabtiiloitny.

oWf 7h.e5nmcgo/nmceLn.trHatoiownesvheirg, haeftrerthaanfe7w.5(fmrogm/m5Ltow8er)eduaysesdo,fthuesen,otzhzelensoczlzolgesgecdlodgugeridnganthde

tfhierspt riminpthresasdiohnasd. Etolecbteroddisecsacrodueldd. bIenpcrointreadstu,spinrignStaWbiCliNtyTgirnekastlwy itmhparcoovnecdenwthraetniotnheof

S7W.5CmNgT/mcLon. Hceonwtreavtieorn, awftaesr alofwewer(tfhroamn 5 tmog8/)mdaLy, sboefinugsep,othsseibnloeztzoleps rcilnotgfgoerdaatnldeatshte 3pmrinotnhtehasdkeheapdintgo tbhee ndoiszczalredsewd.orInkincgo.ntIrnasat,ddpirtiinotna,binliktys pgrepatalryedimwpirtohvtehdiswSWheCnNthTe cSoWncCenNtTratcioonncewnetraetiloensswseanssliotiwvertothpanrin5timngg/pmaLra, mbeeitnegrspsouscshiblaes two apvreinfotrfmor, vatollteaagsets3 amppolniethdstokteheepninogzztlhe,eetnco.,zwzlheischwhoardkitnogb.eIcnaraedfudlilytiocnh,osiennksfopr rmeporaerecdonwceinthtrattheids SSWWCCNNTT incoknsc(eSnutpraptlieomnewntearrey lIensfsorsmenastitoinveFitgourperiSn3t)i.nTghpeafirnamalemtearsssoufcdhepasoswiteadveSfWorCmN, Tvoclotaugldes be estimated using the concentration of SWCNT, drop spacing and the volume of ink



Nanomaterials 2021, 11, 1645



applied to the nozzle, etc., which had to be carefully chosen for more concentrated SWCNT inks (Supplementary Information Figure S3). The final mass of deposited SWCNT could be estimated using the concentration of SWCNT, drop spacing and8 otfh1e5 volume of ink deposited per layer, as reported in Table 1. Since the final response of the SWCNT electrodes depended on the amount of deposited SWCNT, electrodes printed with 12 layers and a SWCNT concentration of 7.5 mg/mL were equivalent to those printed wdeitpho1si8teldaypeerrslwayiethr, aascroenpcoerntetrdaitnioTnabolfe51m. Sgin/mceLt.hTehfienraelforersep, oanbseetotefrthperiSnWtaCbNiliTtyecleocutrldodbees odbetpaeinneddedwiotnh tahSeWamCNouTnctoonfcdeneptroastiitoendoSfW5 CmNg/Tm, eLleactttrhoedeesxpperninsteeodfwthiethn1e2edlaoyfeprsrianntidnga mSWorCeNlaTyecrosn. centration of 7.5 mg/mL were equivalent to those printed with 18 layers with a conAclelnptrrianttieodneolefc5trmodge/smwLe.reTehleercetfroorceh,eambiceatltleyr tpersitnedtaibnilditiyffecroeunltdsboleuotibotnasininedorwdeitrhtoa dSeWteCrmNiTnecotnhceeinr tcraaptiaocnitoafn5cem, gth/emeLleacttrtohceheexmpiecnaslelyoaf cthtieveneaerdeao,fapnrdintthinegemleocrtreolcahyeemrsi.cal reversAibllilpitryinttoewdaerldesctrreoddoexs pwroerbeese,lesuctcrhocahsehmexicaaclylyantoesfeterdratine (dIIiIf/fIeIr)eanntdsohlyudtiroonqsuiinnoonred.er to deFtierrsmtlyin, ethteheciarpcaacpitaivcietabnechea,vthioereolfecthtreoeclheecmtroicdaellsywaactsivsteuadrieead, banydCtVh.eFeigleucrtero3cahsehmoiwcasl trheevevrsoilbtailmitymtoogwraamrdss roebdtoaxinperdobiens,pshuochspahsahtee-xbaucfyfaenreodfersraaltinee(II(IP/BIIS))ansodluhtyidonroqfourinaonAe.u electrFoidrsetalyn,dthSeWcaCpNacTitpivrienbteedhaevleioctrroofdtehseoenleactAroud. eAs cwapasacsittuivdeiebdebhyavCioVr. cFaignubree o3bassehrovweds ftohre avlolltealmecmtroodgreasm. As osbitganinifeidcainntpihnocsrpeahsaetei-nbutfhfeerecdapsaalciintiev(ePBcuS)rrseonluttiwonithforthaeAnuuemlebcetrrodoef parnindteSdWClaNyeTrsprisinotebdseerlveecdtroadneds ocanna bAeua. sAcricbaepdacbitoivthe btoehtahveiohrigchanerbreooubgshenrvesesd ofof rthalel e(rasbeFcicSeulssuhlyieusergoarctpcfphrucbtrateerpaoresrcconoceelnetedsttdrifeStlmvvveoeoor4eessscipe)vl.sd.a,yneatosAlia.atscclcamnunasaTroonddmiypigfbhcra1cnesneraac8eoIiartfininr-esantolvcfcitaowbbvoaeoiyometrendpeaemtcrayiarcenhbisatunSceeay(3trcdrWridr2roiriaic0ebaesncCo,nealacts7ndstNuev.7efFrevsTob0Tiiisrssgconhaea.tpauteindgthsclrhrdevcieobmtenaao1fentvcl4ineSultacao2hd4rarepl0waoe)unsa,etµseoc)hoelcieFmafiatogwcoinccspvathfamdreyetweoperr−scedrae(p1uir2dcela38oerlifsilt2-rucoss,aale0ica.rgrnfsn,,uTyiehct6c7tssehne-hwsp7r,eeeceo0e1isdavtbc2Sshhapatt-Woniaib(latgvuSfihdecnCheueiltetoelhsp1Naynsdwe4op.nuuT2fleaTc)rme0clesfhmeaaapbtµcescphroeetFeiraonerfwnocbttocstdiaeottemlfthraleoadplyvsiepn−nr2saoIaecieenesnflesudoclfteoooatremShca,pbbrdWtmevest6arlaeeoc-aCahri,rridyntatvaNi1iieegetoegv2irsTdhdnoee-s, evlaelcutreoodfesspwecaisfic2.c6aFpagc−i1t,aninceacocfotrhdeasnecSeWwCitNhTperleevcitoroudsersepwoarsts2.s6taFtign−g1ty, ipnicaaclcvoardluaenscerawnigteh fproremvi2outos 4re5pForgt−s1 s[3ta7t]i.ng typical values range from 2 to 45 F g−1 [37].



FFiigguurree 33.. EEleleccttrroocchheemmiiccaallrreessppoonnssee ooff pprriinntteedd eelleeccttrrooddeess.. ((aa)) CCVVss iinn PPBBSS bbuuffffeerr ooff ppHH 77..44 oobbttaaiinneedd ffoorr AAuu aanndd 66--,, 1122--,, aanndd 18-layer SWCNT printed electrooddeess pprreeppaarreeddwwiitthhaaccoonncceennttrraattiioonnooff77.5.5mmgg//mL. (b) CVs obtained for Au and 6-, 112-, aanndd 18-layerr SSWWCCNNTTpprirnintetdedeleelcetcrtordoedsepsrpepreapreadrewditwhiathcoanccoenctreantitorantioofn7.o5fm7g.5/mgi/nma isnolautsionluotifo1n0omf M10hmexMachyaenxaocfeyrarnatoefe(IrIrIa/tIeI) (aInIId/I0I).1aMndK0N.1OM3. (Kc)NCOVs3.o(bct)aCinVeds foobrtAaiunepdrinfoterdAeulecptrroindtee,da 1e2le-lcatyreordoef, 7a.51m2-gla/ymerL ocof n7c.e5nmtragt/imonLocfoSnWceCnNtrTatpirointeodf eSlWecCtroNdTe panridntaed12-ellaeycetrroSdWeCaNnTd-SaN1P2--HlaRyPerprSiWnteCdNeTle-cStNroPd-eHwRiPth p5rmingte/dmLeleacntdro5dme gw/imthL 5comncge/nmtrLatiaonndof5SWmgC/NmTLacnodnScNenPt-rHatRioPninoaf SsoWluCtiNonT oafn4dmSNMPh-HydRrPoqiunianosnoeluitnioPnBoSfo4f mpHM7h.4y.dArlolqvuoilntaomnemiongPraBmS sofwpeHre 7a.c4q.uAirlel dvoalttaamscmanogrartaemosf w0.0e5reVacsq−u1.ired at a scan rate of 0.05 V s−1.

Secondly, the electrochemical reversibility of the electrodes was assessed using hexacyanSoefecrornadtely(,IIIt/hIeI)ealnedctrhoycdhreomquicianlonreevaesrrseibdiolixtyproofbetsh.eFiegluecretr3obdeshs owwassthaessCeVssseodbtuasininegd hfoexraacyAaunoafnedrrtahtere(eIISIW/IIC) NanTdphryindtreodqueliencotnroedaess,rweditohx6p, r1o2baens.dF1ig8ulareye3rbs, sihnoawssoltuhteioCnVosf o1b0tmainMedhefxoarcyaaAnoufearnradteth(IrIeIe/ISI)W. ICt cNanTbpersineetendthealet cthtreoodxeisd,ow-rietdhu6c,ti1o2n aonf dhe1x8aclyaayneorsfe, rirnatae s(oIIlIu/tIioI)nwoafs1m0 omstMly hinehxiabciyteadnoinferarsa-tperi(nIItIe/dII)A. uIt eclaenctrboedseese, nsinthcaetntohesiogxniidfioca-rnetdauncotidoinc oofr hceaxthaocydaicnopfeearrkasteca(nIIbI/eII)obwsearsvemdo. sTtlhyeininhiitbiaitleedlecintroacsh-pemrinictaedl reAsupoenlseectcroudleds,bseingcreantloy siimgnpirfoicvaendt baynocdleiacnoirncgatheodsuicrfpaecaekosfctahne bAeuoeblseecrtvroede. sT,haepinroitcieaslseklencotrwonchaesmsiucraflarceesapcotnivsaectoiounld[3b5e].gArefatetlryaicmtivparotivnegdthbey gcloeladnsinugrfathce,saunrofadciec oafntdhceaAthuoedlieccptreoadkessc, oaupldrobcesosbktnaionwedn; ahsoswuervfaecre, tahcetiovbasteiornve[d35p]e. aAkftpeorteancttiivaal tdinifgfetrheencgeooldf 2s1u4rfmacVe,waansodraicthaenrdhicgahth. oIndicconpteraaksst, cwouellld-dbeefionbetdaivnoeldta; mhomweetvriecr,ptehaekosbwseerrveeodbptaeiankedpoftoernStiWalCdNiffTesreenleccetroofd2e1s4. mPoVtewnatisarlaptheaekr hdiigffhe.rIenncoesntorfa9s1t,,w10e1ll-adnedfi1n7e9dmvoVltwamerme oetbrtiacinpedakws hweenre6-o,b1t2a-inaned f1o8r-lSaWyeCr NelTecsteroledcetrsowderse.

Puosteedn,tinadl piceaatkindgitfhfearteenlceecstroofn9t1r,a1n0s1fearnrdea1c7ti9omnsVprweeserentoebdtaaingeredawterhedneg6r-e, e12o-f aenledct1r8o-clhayemer-

eilceacltreovdeersswibeilrietyusfoedr ,ain6d-liacyaetirnSgWthCatNeTlecetlreocntrotrdaen.sfAers rceaanctbioenssepenre, speenatkedcuargrreenatteinr cdreegarseees

with the amount of SWCNT deposited on the electrode, since the peak current peaks for a

18-layer electrode was more than twice as much as the 6-layer SWCNT one.

This considerable increase in peak current observed with the increasing amount

of SWCNT can be attributed to an increase in the electroactive area of the electrode.

The electroactive area A could be determined by recording CVs at different scan rates

(Supplementary Information, Figure S5a,b). As expected, the current peak followed a linear



Nanomaterials 2021, 11, 1645



9 of 15



dependence (Supplementary Information Figure S5c) on the square root of the scan rate as expressed by the classic Randles-Sevcik Equation (1) [38]:



ip = 0.4463 n3/2F3/2 AC



D



1/2

v1/2



RT



(1)



where ip is the current peak, n the number of electrons transferred in the redox event, F the Faraday constant, D the diffusion coefficient, C the concentration of the analyte, ν the scan rate, R the gas constant and T the temperature. Electroactive areas of 1.8, 2.6 and 4.1 mm2 were determined for 6-, 12- and 18-layer SWCNT-7.5 printed electrodes. Therefore, SWCNT presented an electroactive area higher than the geometrical area.

This result might be connected to the high roughness of the electrode surface [39]. In order to study this hypothesis, SEM, FIB cross-sections and confocal images of Au and SWCNT printed electrodes were acquired to evaluate the thickness and roughness of these electrodes.

Confocal images in Figure 4 show average thickness and roughness (standard deviation) values for 1-layer Au; 6, 12 and 18-layer SWCNT-5 and SWCNT-7.5 printed electrodes, whose values are detailed in Table 2.



Table 2. Average thickness and roughness of electrodes printed with different combinations of SWCNT concentrations and number of layers and a layer of Au printed.



Printed Electrode



Concentration of SWCNT [mg/mL]



Au + SWCNT



5



Au + SWCNT



7.5



Au



0



Number of Layers

6 12 18

6 12 18

1



Thickness (ave) [nm]

1100 1148 1452

1118 1429 2107

230



Roughness (sd) [nm]

315 340 342

261 290 384

65



Figure 5a shows the SEM image of the surface of a SWCNT printed electrode, in which the fibrous nature of the CNT can be seen. The cross section made by FIB (Figure 5b) shows the compact layer formed by the printing of Au nanoparticle ink (orange shaded), forming a uniform film of 430 nm thickness with a low roughness. On the contrary, SWCNT printed films with a thickness of 1.4 µm (blue shaded) show a rough surface with the presence of voids in the bulk of the stack layers.

Considering that confocal images were taken on dried electrodes, the increased roughness and electroactive area observed for SWCNT electrodes suggest that SWCNT can penetrate in the solution beyond the diffusion layer and were able to collect redox species from a higher volume than that allowed for planar electrodes under semi-infinite diffusion conditions. Thus, the high values obtained for the electroactive area suggests that SWCNT were not confined to the electrode surface and were able to react with redox species beyond the diffusion layer as usual for planar electrodes.

Figure 3c shows the CVs obtained for an Au printed electrode and a 12-layer SWCNT printed electrode in a solution containing 4 mM hydroquinone in a PBS buffer of pH 7.4. For Au electrodes used without further treatment, the oxidation of hydroquinone was mostly inhibited. Since direct reaction on the underlying Au substrate was mostly absent, the electrochemical response can be attributed to SWCNT, whose electrocatalytic properties to the oxidation of hydroquinone have been previously reported [31]. It is worth noting that the addition of silica nanoparticles did not inhibit the oxidation of hydroquinone by SWCNT. As can be seen in Figure 3c, the voltammogram for SWCNT-SNP-HRP electrodes presented a lower peak current for the oxidation of hydroquinone, possibly as a consequence of a lower electroactive area as the silica nanoparticles are dielectric.



NNaannoommaatteerriiaallss22002211,,1111,,1x64F5OR PEER REVIEW



1100 ooff 1156



FFiigguurree44.. CCoonnffooccaall rreelliieeff iimmaaggeess aanndd pprroofifilloommeettrryyooffWWEEpprriinntteeddwwiitthh((aa))66--,,((bb))1122--,, ((cc)) 1188--llaayyeerr ooff SSWWCCNNTT--55iinnkk((lleefftt))aanndd ((dd))66--,,((ee))1122--,,((ff))1188--llaayyeerrooffSSWWCCNNTT--77.5.5ininkk((rrigighht)t.).

IFnigcuornec5luassiohno,wtshitshevoSlEtaMmmimeatrgiec oafnathlyesissursfhaocwe softhaaSt WSWCNCNT Tprpinritnedtedeleecletrcotrdoed, eins pwrhesicehntthae hfiibgrhoucaspnaactiutarencoef,thweitChNvTalcuaens btyepsieceanl. fTohrethcrisosms asetecrtiioaln. mWaidteh bryegFaIrBd(Ftoigtuhree e5lbe)ctsrhocohwesmtihcael creovmeprsaicbtililtayy, ethr efoorxmideadtiobnyotfhheexparcinytainnogfeorfraAteu(InI)aannodpahrytdicrloeqiunikno(noerawnagse mshoasdtelyd)i,nhfoibrmiteindginaaus-npirfoinrtmedfiAlmu eolfec4t3ro0dnems. Itnhiccoknntersasstw, SiWthCaNlTo-w7.5ropurgeshennetsesd. aOgnotohde ecloencttrraorcyh,eSmWicCalNrTespporinnsteedfofrilbmosthwrietdhoax tphriocbkense.ss of 1.4 µ m (blue shaded) show a rough surfaScienwceitbhotthhe6p- raensden1c2e-loafyveoriSdWs iCnNthTe sbhuolkwoefdtahegsotoadckelleacyterrosc.hemical response, these numbers of layers were maintained during the fabrication of enzyme electrodes with enzymes immobilized onto nanoparticles. SiO2 nanoparticles were chosen because they can be easily prepared with a high degree of quality by the Störber sol-gel process and with



Nanomaterials 2021, 11, 1645



11 of 15



Nanomaterials 202a1,s1i1z,ex FaOmRePnEaEbRlReEtVoIEIWJP (Supplementary Information Figure S6a). Besides, the surface of



11 o



silica nanoparticles can be functionalized to create anchoring points for biomolecules.



Figure 5. F2i-glauyreer 5A.u2-WlaEyeprrAinutedWwEipthri1n2t-eldayweritohf 172.5-lmayge/mr oLf o7f.5SWmgC/NmTL(ao)fSSEWMCimNaTg(eao) fStEhMe tiompavgieewofotfhietstsouprface and

(b) the covrrieeswpoonfditisngsucrrfoascsesaecntdio(nb.)Athuelacyoerrrsesapreonshdaidnegdcrinososrasencgteioann.dAthuelaSyWerCsNaTres sohnaeds eadreinshoardaendgienabnlduet.he

SWCNTs ones are shaded in blue. Considering that confocal images were taken on dried electrodes, the increa

The enzymreo-uimghmneosbsilainzdedelseiclitcroaanctainvoepaarertaicolbeseSrvWedCNfoTr SinWkCeNleTcterloedctersodweesrseutgegsetsetdthat SWC for the detectioncaonfpheyndetrroagteninptehreoxsiodlue,tiuonsinbgeyhoyndrtohqeudinifofunseioans laayredr oaxndmwederiaetoabr.leTthoecollect re reduction of hysdpreocgieesnfproemroxaihdieg,hceartavloyluzemdebthyaHn RthPa,tisalalocwcoemdpfoarnpieladnbayr etlheectoroxdideastuionndeorfsemi-infi hydroquinone, dwifhfiucshioanctcsoansdaitnioenlse.cTtrhouns,dtohneohri[g3h1]v.Talhueesfoorbmtaeidneodxifdoirztehdesepleecctireosaccatinvebearea sugg electrochemicalltyhdateSteWctCedNeTitwheerrebynoCtVcoonrfibnyedchtrootnhoeaemlepcetrroodmeesturyrfiafctehaenedlewcteroredaebploetteonrteiaalct with re is set at a convenspieenctievsablueyeo. nCdatthhoeddiicffluinseioanr plaoylearriazsautisounacl uforvr epslapnraerseenletcitnrocdreeass.ing current values with increasiFnigguhreyd3crosgheonwsptehreoCxiVdseocbotnaicneendtrfaotrioann,AauspcrainntebdeesleecetnroidneFaingduare126-ala. yer SWC Figure 6b showsptrhinetecdurerleencttrtordanesiinenatssoolubttiaoinnecdonattaaincionngs4tamntMaphpyldierodqpuointeonnteiailnoaf P−B0S.2b3uVffer of pH before and afterFtohre Aadudeitlieocntroodf ehsydursoegdewn iptheroouxtidfuer.tShiemr itlraerartmeseunltts, wtheereoxoibdtaatiinoendopf rhinytdinrogquinone 6 and 12 layers, malobsetiltysilnighhibtliytehdi.gShinecrecduirrreecnttrveaaclutieosnwonerteheobutnadineerldyifnogr 1A2u-lsauybesrteraletectwroadsems.ostly abs

Figure 6c shthoewseltehcetrcouchrreemnitc-caol nrceesnptornatsieoncacnurvbee oabtttariinbuedtedfortotheSW6-lCayNeTr,ofwShWosCeNeTle-ctrocatal SNP-HRP (withp5rompger/tmiesL taondth5e moxgi/dmatiLoncoonfcehnytdrraotiqouninoofnSeWhCavNeTbaenedn SpNrePv-iHouRsPly, rreesppoerct-ed [31]. tively) at a meawsuorretmh ennottitnigmethoaft 6th0es afodrdietlieocntroofdseislicparinnatendop1adrtaicyleasnddid90ndoat yinshaifbtietrtthhee oxidation ink preparationh. yAdsrocaqnuibneonseeebny, tShWe CpNerTfo. rAmsacnacneboefsbeoenthinenFziygumree e3lce,ctthreodveosltawmams soigmrailmar.for SWC The obtained senSNsitPiv-HitRyPwealsecatrrooudneds p5r7e+se/n−ted3 µaAlocwme−r 2pemaMk c−u1r,raenvtafloure tchoemopxaidraabtiloentoofthheydroquin ones obtained upsionsgsibenlyzyams ea ecloencstreoqdueesncpereopfaareldowbyerotehleecrtrmoaecatnivs,esaurceha aassotxhyegseinlicpalansamnaoparticles (sttreo5rieilxoone5sindcucd+taeelar/tlsotlps−yiwdorp,eenter4phseroµaeeswferAhsancetitaartcgieerbmohngwepddbinlplouril−oyeaei.eiormtcn2snlseysAdete-prtmbISmcFbanoonseteiaiabrnMtcflorgrceishlsccseecaueace−oete.ndnt.ldormnr1yzhebEfo)acbyeioinc6lignlcaelmunthaczeahnhdhysse,ycleddiemeit6moccbrreer-aaasneoisilnehtpclvae,dheiawaonecebidteilntrclhwdgrseresriaiihotreicFsnf1atbetsdteoii2nhripgorvlealo-mcalionulossstodeatny-tlar,wwypetacbe,siasenubwreoaemt6ritirwhtndsfialdvhStomietiahte,eebWnrytlredostyeshbehvtaCxodreoweaAepsiiNtdecnlybrdheuupvaizTirunaeerotraeyeeinsrext-slomlievudioahhennntdilaocboiygeoykbttxwtppourseiaitaoifmitrpdsehisceendndraheadsmidoeselneepshabdfxanotfp.aoseaohgbtbirIsaswicrmenooir.nifylmitosaigptcaieczhdclanirortaaahilthionsraetin1esoaiflrtf,sotieemrtname.sancr3sdSertas0ohira[Wtntlfoe3,eaitasoenc1rrSCeniinh,tzWnas4(dNie.yIevlz0Im.ECnmTy6i)]t.Wnsm0NiyaeciApz.tnadeTiiyretdTlvdi-ahlmne7iredhytle.thceyeeri5ses--yecdpgtpdroarroenerolddsseqeeecusn,ttirottnowhe

It is worthennoztyimngesthimatmthobeilcioznedceonntrtoatnioannoopfaSrtNicPle-sH. RSiPO2handanaopsaigrtnicifilecsanwteirme cphaocsteonnbecause t the response ofcathnebbeieoasseinlysoprr.epItarwedaswoitbhsearhviegdh tdheagtreinekoffoqrumaluitlyatbioyntsheprSetpörabreerdsuols-ignegl process concentrations owfitShNaPs-iHzeRaPmleonwaebrlethtoanIJP1 (mSugp/pmleLmwenetraerynoInt feonrmouagtihontoFcigouvreerSt6hae).sBuersfaidcees, the sur electrode (SupploefmsielnictaarnyanInofpoarrmtiactlieosncaFnigbuerefuSn6cbt)io. nTahleizreefdotroe,ctrheeastee caonnchceonritnragtipoonisntosffSoNr bPi-omolecu HRP were not furtheTrhceoennsziydmeree-dim. Omnobtihliezeodthseilrichaannadn,oipf athrteiccleosnScWenCtrNatTioinnkwealescttoroodheisgwh,ere tested the electrochemtichael pdeertefoctrimonanocfe hofydthreogSeWn CpNerTo-xSiNdeP,-HusPiRngelhecytdrorodqeuwinaosniemapsaiaredre.dWoxhemnediator. electrodes withrceodnuccetinotnraotfiohnysdorof g5emn gp/ermoxLidoer, hciagtahleyrzeodf SbNy PH-RHPR,Piswacecroemupseadn,ierdepbeyattehde oxidatio measurements shhyodwroeqduainnoinnec,rewahsiinchg asicgtsnaalsuanntielleacstrtaobnled,orneoprro[3d1u].cTibhleefcourmrreendtovxailduiezewdasspecies can obtained. This reesleuclttrcoacnhebme iicnatellryprdeetetedctinedteremithseorf abycerCtVainobrlobcykacgheroonf othaemepleecrotrmocehtreymiicf atlhe electr activity of SWCNpoTt-eSnNtiPa-lHisPRsefitlmats, awhcoicnhvceonuielndtbevarleuvee.rsCedathafotdericrelpineeaatredpmoleaarsizuarteimonenctus,rves pre possibly due toinacrreeacsoinnfiggcuurrraetniotnvailnuetshwe ifithlmincsrtreuascitnugreh.ydOrnogtehnepoetrhoexridheacnodn,ceenletrcatrtioodne, sas can be s printed with SNinP-FHigPuRreco6nac.eFnitgruartieon6bs lsohwoewrsththane 5cumrrge/nmt tLrainmsimenetdsiaotbetlayinreedspaotndaecdontostant app hydrogen peroxpidoetegnetniaelraotfin−g0.2a3reVprboedfourceibalnedelaefctterrocthheemadicdailtisoingnoafl haysdshroogwenn ipnerFoigxuidree. 6Sci.milar res



Nanomaterials 2021, 11, 1645



oxidation of carbon-based electrodes followed by enzyme immobilization [31,40]. Additionally, the stability of enzyme electrodes was also evaluated in similar fashion. Enzyme electrodes were prepared and calibration curves were obtained after 1, 30 and 60 days since preparation. As can be seen in Figure 6d, they exhibited a similar sensitivity. The results presented in Figure 6c,d show that both the bio-ink and the printed enzym1e2 of 15 electrodes were highly stable. Enzyme electrodes were printed presenting a similar sensitivity (55 +/− 4 µ A cm−2 mM−1) and a higher stability as previous approaches.



FiFgiugruere6.6(.a()aC) CVVs sanadnd(b(b) )chchrornoonaomampepreormometertircicucruvrevsesfofrorananapapplileidedppootetnentitailaol of f−−0.203.2V3 Vobotbatianiended for



foar6a-la6y-elary(edrot(tdeodttliendesl)inaensd) 1a2n-dlay1e2r-l(asyoelird (lsinoelisd) wlinitehs)prwinitehdpSrWinCteNdTS-SWNCPN-HTR-SPNwPi-tHhRaPcowncitehntaration



coonfc5enmtrga/timonLooff5SmWgC/mNLToafnSdW5CmNgT/amndL5omf SgN/mPL-HoRf SPN, rPe-sHpeRcPti,vreeslyp,ewctiitvheolyu,twainthdoaufttearntdhaeftaedrdthiteion of 7aSd.WHr4ad2CtaieOttNioo2aTnfts-o0oSc.aNfa0nH5fiPV2nr-OHaats2lRe−tcPooo1 .fna(6(c0fc-e.il)0nna5DaytrleeVacrptoiwseon−nn1ci.tdeho(necfnt5)r3caDmemtiegoopM/nfmecnoiLundfr3eaarnnemscndoeMtl5udotimfenioncganus/imrstoroyfeLlun4octtnmioodnHMnecn2eoOhsnfiytt24rydacmrtoooiMnnoqncuHheionyn2fOdtorSrn2aWoetcqiooCiunnnNicnPefTooBnnrStaerntaowidtfnioopSPnHNSBWfSP7o-o.CrH4fNtaRpwtTPHoa-, SsNcaPnreHspRePct(iv6-ellayy)eerlewcittrhod5ems gp/rimntLedanwdit5hm1gd/amyL(coopnencencitrrcalteiso)naonfdSW90CdNaTysan(fdulSlNciPr-cHleRs)P,arfteesrpeincktively)

preelepcatrraotdioens. prLiinnteeadr wriethgr1esdsiaoyn(oipsenshcoirwclnes)aasnda90ddotatyeds (fulinllec.ircCleusr)reanftterwinaks pmreepaasruarteidon.bLyinear



chrerognreosasmiopneriosmsheotrwy nata6s0as wdoitthteadnlainpep.liCeduprroetnent twiaal sofm−e0a.2s3uVreidn b0.y1 cMhrpohnoosapmhapterboumffeetrroyfaptH607.s4 with



anadn a4pmplMiedhypdortoeqnutiianloonfe.−(d0.)23DeVpeinnd0e.n1cMe opfhcousrprehnattedbenusfifteyr oonf pHH2O72.4coanncden4trmatiMonhfyodr rtohqrueeinone.



cSoW(mdneC)daDiNtsiueToprn-eSesdNnadP1s ,e-dHn3e0cRsecaPrnoibdfeelce6duc0rftorrdoeradnFyetisgsdauemfrnteeesr6aitcsby.ueroinendgHp12r, eO3p20acraeondndcuenn6d0treadrtasioyansmfeaofcrteotrnhdrbeieteiinoSgnWspCarsNedpTae-rsSecNdriPbue-nHddRfeoPrr



eslaemcterodes Figure 6c.



ItTishiws ofratcht ncoatninbgethfuarttthheercuonncdeenrtsrtaotoiodnboyf SsNtuPd-yHinRgP thhaedma osirgpnhiofilcoagnitciaml pdaecptoosnititohne and rethspicoknnseessooff tthhee mbiaoteserinasloorv. eIrtthweaeslecotbrsoedrev.eFdigtuhraet7ainskhofworsmthuelaStiEoMnsimpraegpeaorfedtheuesliencgtrode

surface modified with SWCNT-SNP-HPR ink with 7.5 mg/mL and 1 mg/mL concentration of SWCNT and SNP-HRP, respectively. It is possible to distinguish the typical structure that the SWCNTs forms and among them the nanoparticles. When the concentration of SNP-HRP was 5 mg/mL or higher, the top view of the electrode (Supplementary Information, Figure S6c) was completely covered with nanoparticles. In Figure 7b,c, cross sections of 6- and 12-layer of SWCNT-SNP-HPR electrodes are shown. The 2-layer Au, highlighted in orange, presented a thickness of approx. 900 nm, while the thickness of SWCNT-SNP-HPR films was approximately 220 nm and 900 nm for 6-layer (7.5 mg/mL of SWCNTs and 1 mg/mL of SNP-HRP) and 12-layer (5 mg/mL of SWCNTs and 5 mg/mL of SNP-HRP) respectively.

To summarize, enzyme electrodes were successfully printed using a bio-ink containing SWCNT and silica nanoparticles bearing immobilized HRP. The number of printed layers greatly influenced the performance of the enzyme electrodes, and the quantity of SWCNTSNP-HPR deposited had to be optimized in order to obtain a high sensitivity, covering the electrode surface without forming thick films.



Nanomaterials 2021, 11, 1645



concentration of SNP-HRP was 5 mg/mL or higher, the top view of the electrode (Suppl. Info., Figure S6c) was completely covered with nanoparticles. In Figure 7b,c, cross sections of 6- and 12-layer of SWCNT-SNP-HPR electrodes are shown. The 2-layer Au, highlighted in orange, presented a thickness of approx. 900 nm, while the thickness of SWCNT-SNPHPR films was approximately 220 nm and 900 nm for 6-layer (7.5 mg/mL of SWCNT1s3aonfd15 1 mg/mL of SNP-HRP) and 12-layer (5 mg/mL of SWCNTs and 5 mg/mL of SNP-HRP) respectively.



FFiigguurree 77.. (a(a)T)TooppvvieiwewSESMEMimiamgaegs eosf soufrsfaucrefaocfe1o2-fla1y2e-rlaSyWerCSNWTC-SNNTP--SHNRPP-HprRinPtepdrienletecdtroedleec. t(rbo,cd)e. (bFnoa,cnc)uoFspoeacdrutiisocelndesbioefoanrmb(ebcar)moassc6-r-sloeascystei-osrenScWitmioCanNgiemTo-aSfgNeelPeo-cHftreRoldePcet(sr7op.5driemnstgep/dmrisnLhteoodwf SsinhWgoCwthNienTgdsitfahfneerdedn1iftfmelargye/enmrtsLlaaoynfedrSsSNiaOPn-2d SHiOR2Pn) apnrionpteadrtieclleecstrfoodre(ban) da 6(c-)laoyfe1r2S-lWayCerNSTW-SCNNPT-H-SRNPP-(H7.R5 Pm(g5/mmgL/moLf SoWf SCWNCTNs Tans dan1dm5gm/gm/mL Lof SoNf PS-NHPR-HP)RpPr)ipnrteindteedleecltercotdroedaen. dOr(acn) goef a1n2d-labyleure SsWhaCdeNsTc-oSrNrePs-pHonRdP t(o5 pmrign/temdLAouf aSnWd CSNWTCsNaTn-d 5SmNgP/-HmRLPoffilSmNsP, r-eHspRePc)tipvreilnyt.ed electrode. Orange and blue shades correspond to printed Au and SWCNT-SNP-HRP films, respectively.

To summarize, enzyme electrodes were successfully printed using a bio-ink 4c.oCnotanincliunsgioSnWsCNT and silica nanoparticles bearing immobilized HRP. The number of printEendzylamyerselegcrteraotdlyesinwfleureencseudcctehsesfuplelryfoprrminatnecde uosfinthgeaebnizoy-minek ecloenctraoindiensg, aSnWdCthNeT aqnudansitliitcyaonfaSnWopCaNrtTic-SleNs Pb-eHaPriRngdiempomsiotebdilihzaed HtoRbPe. oTphteimenizzeydmine eolredcetrotdoeosbrteatianinaehdigthe csaetnaslyittiivcitayc,tcivoivteyrifnogr atht eleealsetct3romdoenstuhrsf.acWe ewhitahvoeusthfoorwmnintghatht iHckRfPilmensz. ymes immobilized onto silica nanoparticles can be used in the formulation of ink to print enzyme electrodes. T4h. eCocantcalluystiiocnasctivity remains stable for a longer time allowing for a longer shelf life. AmpeErnozmyemtreicebleicotsreondseosrws wereeresufcuclelysspfurilnlytepdridnetmedounsitnragtianbgioth-ienkgrceoanttpaointeinngtiaSlWoCf tNhiTs atynpde osfilticeachnaonloogpyarftoicrletshebdeaersiinggn,imdemveolboiplimzeedntHaRnPd. mThaneuefnacztyumree oefledcitsrpoodseasblreetbaionseednstohres, tchaatnaklysttioc aflcetxivibitiylitfyoirnadt elesiagsnt ,3thmeosnmthalsl. aWmeouhnavt eofsmhoawtenriathl autseHdRaPndentzhyemauestoimmamtioobniloizf ethde monutlotilsailyicear nparninotpinagrtipcrleoscecsasn. bIne ucsoemdbinintahteiofnorwmiuthlastiomnpolef iannkdtoacpcreinsstiebnlezyemleecterloenctircos,dtehsi.s tTyphe ocfatdailgyittiacl apcrtoivdiutyctiroenmapinpsrostaacbhleisfaorvearyloantgtrearctimveeoapltlioowninfogr ftohre avollounmgeerpsrhoedluf clitfieo.n oAf mhipgehrloymsteatbrilce,binioksjeent sporrisntwederbeiofusellnysoprrsi.nted demonstrating the great potential of this type of technology for the design, development and manufacture of disposable Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nano11071645/s1, Figure S1: Design of printed sensors, Figure S2: Images of a WE printed with SWCNT-5 ink, Figure S3: Waveform and parameters used for printing, Figure S4: Plot of capacitive current vs. scan rate for Au and SWCNTs printed electrodes, Figure S5: CVs obtained for a printed electrode in solution of 10 mM hexacyanoferrate (III/II) and Randles-Sevcik plots, Figure S6: SEM images of SiO2 nanoparticles and WE printed with SWCNT-SNP-HRP inks.

Author Contributions: Conceptualization, M.M., G.Y. and G.G.; methodology, M.M.; validation, A.M., G.L. and G.Y.; formal analysis, M.M. and G.Y.; investigation, M.M., L.S.V. and O.G.; resources, E.R. and G.G.; writing—original draft preparation, M.M. and L.S.V.; writing—review and editing, G.Y. and G.G.; visualization, G.Y.; supervision, G.Y., G.G. and E.R.; project administration, G.G. and E.R.; funding acquisition, R.V., G.G. and E.R. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by EMHE program “Enhancing Mobility in Health and Environment” and I-COOP2019 financed by the State Agency “Consejo Superior de Investigaciones Científicas” CSIC (EMHE-CSIC), references– MHE-200037 and COOPA20377.

Data Availability Statement: Data is contained within this article and the Supplementary Information.

Acknowledgments: This work was supported by the Spanish government funded project by MICIU RTI2018–102070-B-C21 and RTI2018-096786-B-I00 (MINECO/FEDER, EU). Authors also want to thank the support of the SU-8 Unit of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) at the IMB-CNM (CSIC) of ICTS “NANBIOSIS”. This work has also made use of the Spanish ICTS Network MICRONANOFABS partially supported by MEINCOM. The authors also want to thank to Luciano Patrone and Liber Solé and Abert Guerrero, for the FIB/SEM images obtained at INTI-CMNB and IMB-CNM, respectively, and Marta Duch Llobera for the images obtained in the 3D optical profilometer of the clean room of the IMB-CNM. M.M. also wants to thank the support of the INTI-Micro and Nanotechnologies, especially to theDepartment of Microelectronic Prototyping and Printed Electronics and Liliana Fraigi. The graphical abstract made use of HRP repre-



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sentation by Manuel Almagro Rivas taken from https://commons.wikimedia.org/wiki/ (accessed on 22 June 2021) File:HRP-xray.png and by Evannovostro taken from https://www.shutterstock.com/es (accessed on 22 June 2021) ID: 780740077.

Conflicts of Interest: The authors declare no conflict of interest.

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