Physical Structure and Electrochemical Response of Diamond

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Functional Nanostructured Materials (including low-D carbon)

Physical Structure and Electrochemical Response of DiamondGraphite Nanoplatelets: From CVD Synthesis to Label-Free Biosensors Nuno M. F. Santos, Sónia Oliveira Pereira, António Jose S. Fernandes, Thiago L. Vasconcelos, Chung Man Fung, Braulio S. Archanjo, Carlos Achete, Sofia Teixeira, Rui Ferreira e Silva, and Florinda M. Costa ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00352 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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ACS Applied Materials & Interfaces

Article type: Article

Physical Structure and Electrochemical Response of Diamond-Graphite Nanoplatelets: From CVD Synthesis to Label-Free Biosensors Nuno F. Santos a,†, Sónia O. Pereira a, António J. S. Fernandes a, Thiago L. Vasconcelos b, Chung M. Fung c, Bráulio S. Archanjo b, Carlos A. Achete b, Sofia R. Teixeira d, Rui F. Silva e and Florinda M. Costa a a i3N

and Department of Physics, University of Aveiro, 3810-193 Aveiro, Portugal

b Materials c Centre

Metrology Division, INMETRO, 25250-020, Duque de Caxias - RJ, Brazil

for NanoHealth, College of engineering, Swansea University, Singleton Campus,

Swansea, SA2 8PP, UK d College

of Engineering, Swansea University, Bay Campus, Swansea, SA1 8QQ, UK

e CICECO

and Department of Materials and Ceramic Engineering, University of Aveiro,

3810-193 Aveiro, Portugal Abstract: Hybrid diamond-graphite nanoplatelet (DGNP) thin films are produced and applied to the label-free impedimetric biosensors for the first time, using avidin detection as a proof of concept. The DGNPs are synthesized by microwave plasma chemical vapor deposition through H2/CH4/N2 gas mixtures in a reproducible and rapid single-step process. The material building unit consists in an inner 2D-like nanodiamond with preferential vertical alignment covered by and covalently bound to nanocrystalline graphite grains, exhibiting {111}diamond||{0002}graphite epitaxy. The DGNP films’ morphostructural aspects are of interest for electrochemical transduction in general, and for faradaic impedimetric biosensors in particular, combining enhanced surface area for biorecognition element loading and facile faradaic charge transfer. Charge transfer rate constants in PBS/[Fe(CN)6]4- solution are shown to increase up to 5.6x10-3 cm.s-1 upon N2 addition to DGNP synthesis. For the impedimetric detection of avidin, biotin molecules are covalently bound as avidin specific recognition elements on (3-aminopropyl)triethoxysilane -functionalized DGNP surfaces. Avidin quantification is attained within the 10 to 1000 μg.mL-1 range following a logarithmic dependency. The limits of detection and of quantitation are 1.3 and 6.4 μg.mL-1 (19 and 93 nM), respectively, and 2.3 and 13.8 μg.mL-1 (33 and 200 nM) when considering the non-specific response of the sensors. Keywords: nanodiamond, nanographite, charge transfer, impedimetry, label-free, biosensors

†

Corresponding author. E-mail: [email protected]

1 ACS Paragon Plus Environment

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1. Introduction Label-free biosensors (LFB) are undeniably a contemporary topic. The prospect of having devices for diagnosis of certain biomarkers, at the point-of-care, is of extreme interest from the medical point of view. The full process of producing LFBs, from the electrode material itself to the fully functional sensing devices, is clearly a multi-disciplinary task involving knowledge from different backgrounds. Several physical, chemical, biological and engineering aspects need to be considered in order to produce reliable and portable sensors, characterized by fast responses, high sensitivities and reduced number of false-positive outputs, while maintaining low production cost and simplicity. A wide range of allotropic forms of carbon has been regarded as suitable material platforms for a plethora of electrochemistry-based processes and applications, including biosensing. Graphite, glassy carbon, amorphous diamond-like carbon and boron-doped diamond (BDD) are well-known and established electrode materials because of their low residual currents, readily renewable surface and significant overpotentials for O2 reduction and H2 evolution.1,2 BDD electrodes are very good options when biocompatibility restrictions or issues due to adsorption of chemical species are of upmost importance. Nevertheless, in the specific case of the impedimetric LFBs based on oxidative functionalization, the procedure is much more complicated. Diamond surfaces are less prone to oxidation at atmospheric conditions and thus recognition element loading can be rather lower when compared to sp2 carbon phases. Finally, BDD electrodes are also known to yield relatively low surface areas, which is detrimental to bioanalyte recognition element loading. Within the last decades, carbon nanotubes (CNTs) and graphene based- materials have been thoroughly studied,3–6 not only from a fundamental point of view, but also regarding its application in biosensing, being able to detect and quantify within the ng.mL-1 range of bioanalyte or even lower.7–10 The main advantages of CNTs and graphene over glassy carbon and diamond-like carbon arise from their unique charge transport characteristics, combined with low dimensionality, which is comparable to the size of most biomolecules.11 Some CNT and graphene-based electrodes also attain more facile charge transfer (CT) kinetics than graphite and glassy carbon,12 as well as a more uniform distribution of electrochemically active sites.6 Advantageously, all graphene volume is exposed to the surrounding environment making it very sensitive to adsorbed or linked species. It also has been shown that graphene offers a superior electrochemical performance compared to CNTs.6,13,14


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Nevertheless, CNTs and graphene-based materials present some drawbacks. CNTs need to be physically separated due to their metallic or semiconductor nature. Moreover, difficulties in controlling the roughness and density of CNT films and composites hinder the application of standard lithography techniques. Also, the rapid, facile and low cost graphene synthesis and transfer is still a developing area. Finally, vertically aligned CNTs forests or graphene/graphene oxide sheets are generally recognized as the most promising for electrochemical applications, because of their appropriate morphology. However, it is not straightforward to reproducibly translate the CT characteristics of individual nanobuilding blocks to macroelectrodes containing many of those sub-units. Hence, the exploration of other nanostructured carbon materials for the detection of bioanalytes is still required. Diamond-graphite nanoplatelets (DGNP) are 2D-like sp2/sp3 carbon hybrids with preferential vertical alignment, structurally composed of an inner diamond nanoplatelet covered by conductive nanographite. These materials were previously shown to be highly biocompatible and to maintain the structural integrity upon prolonged exposure to biological solutions.15 Also, an improvement in nanographite crystallinity and a decreasing trend in electrical resistivity, down to c.a. 10-5 Ω.m, were observed upon increasing N2 concentration of the microwave plasma chemical vapor deposition (MPCVD) feed gas. Therefore, it is important to evaluate the CT standard rate constants in physiological solution and check if and how the N2 gas content during synthesis affects it. These aspects are of interest for application in biosensing, envisaging a reliable and efficient bioanalyte transduction. The suitability of these electrodes in the development of reliable faradaic impedimetric biosensors is demonstrated, using the avidin-biotin biorecognition system on (3aminopropyl)triethoxysilane (APTES) -functionalized DGNP electrodes and appropriate controls to validate the specific response of the sensors.

2. Materials and Methods 2.1 Reagents Non-reacting insulating varnish (Lacomit) was purchased from Agar Scientific. Potassium hexacyanoferrate(III) (K3[Fe(CN)6]) and potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6].3H2O) were obtained from Merck. Phosphate Buffer Saline (PBS) tablets

(pH

=

7.4,

10

mM)

were

purchased

from

Fisher

Bioreagent.

N-(3-

dimethylaminopropyl)-N’-ethylcarbodiimide hydrochlorine (EDC), N-hydroxysuccinimide 3 ACS Paragon Plus Environment


(NHS, 98%) and (3-aminopropyl)triethoxysilane (APTES) were obtained from Merck. Dbiotin and avidin (from eggs) were purchased from Fisher Scientific, anti-E.coli serotype O/K Polyclonal from Thermofisher Scientific and anti-hCG from Ig Innovations. Iron(II) sulfate heptahydrate (FeSO4.7H2O) purchased from Panreac Aplichem. N,N-dimethylformamide (DMF, ≥99.8% purity) and bovine serum albumine (BSA) were purchased from Alfa Aesar. Deionized (DI) water was obtained from a MilliQ water purification system. All reagents were used as received. 2.2 Synthesis, Morphology, Structure and Surface Chemistry The DGNP electrodes were fabricated as follows. The substrates consisted of 1 cm2 Si squares (Siegert) pre-coated with a 3 μm-thick nanocrystalline diamond (NCD) MPCVD film. This interlayer film prevents electrode contamination with Si impurities arising from the harsh and dense plasma conditions used inside the reactor. In order to achieve the temperatures needed to synthesize the DGNPs, above 1000 ºC, the Si/NCD substrates were placed on a graphite microwave susceptor over a Mo holder in thermal contact with the cooled dish, inside the MPCVD reactor (ASTeX AX 6350, 6 kW). The synthesis took place during 25 min at a pressure of 90 Torr, a MW power of 2.5 kW, a fixed H2/CH4 flow ratio of 6:1 and variable (014.5 vol%) N2 content. Afterwards, CH4/N2 supply was cut off and the samples were subjected to a H2 plasma treatment for 5 minutes (amorphous carbon etching step) followed by a cooling ramp to room temperature in H2 plasma for 10 minutes. The films’ morphology was accessed through a Hitachi SU70 scanning electron microscopy (SEM) instrument. XPS measurements were performed in an ultra-high vacuum system with a base pressure of 2x10–10 mbar, equipped with a hemispherical electron energy analyzer (SPECS Phoibos 150), a delay-line detector and a monochromatic AlKα (1486.74 eV) X-ray source. High-resolution spectra were recorded at normal emission takeoff angle and with a pass-energy of 20 eV, which provides an overall instrumental peak broadening of 0.5 eV. Regarding the C1s XPS fitting procedure, two Gauss-Lorentz shapes at c.a. 284.7 (sp2 bonded carbon) and 285.3 eV (sp3 bonded carbon) are initially fitted to the unmodified DGNPs spectrum. All spectra are corrected in energy by the sp2 carbon peak and in intensity by the system’s transmission factor. The full widths at half maximum (FWHM) of the remaining C1s peaks were constrained to a maximum value of twice the sp2 peak’s FWHM (1.7 eV). The transmission electron microscopy (TEM) measurements, including the electron diffraction analysis, were performed using a probe-corrected Titan 80−300 kV (FEI 4 ACS Paragon Plus Environment

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Company) working at 80 kV. The TEM is equipped with an electron energy-loss spectroscopy (EELS) system comprising a post-column energy filter (Tridiem Gatan Image Filter). Highresolution scanning transmission electron microscopy (STEM) images were acquired using a high angle annular dark field (HAADF) detector in high camera length conditions. The crosssectional sample for TEM analysis was prepared using a dual beam microscope (Helios Nanolab 650; FEI Company). 2.3 Preparation of Biotin-Functionalized Electrodes After electrical contact placement with high-quality silver ink (Agar Scientific) and a baking step at 130 ºC for 5 min, the active area of the electrodes were defined by PTFE rings having 0.50±0.02 cm in diameter. All remaining contact and substrate regions were coated with a non-reacting, electrochemically inactive insulating varnish. The samples were then washed with isopropanol and DI water. The surface functionalization of DGNP electrodes was carried out in three steps: i) Hydroxylation of electrodes’ surface via Fenton reaction: 43 mg of FeSO4.7H2O was slowly added to an aqueous solution of H2O2 (6.6 mL of H2O2 (30% v/v) in 18.4 mL DI water). After 5 min to calm down the violent bubbling, the substrates were dipped into the stirred solution during 1h. Afterwards, the hydroxylated substrates were thoroughly rinsed with DI water and dried with a gentle N2 flow. ii) Preparation of amine-terminated surface electrodes: APTES (0.1 vol%) was added to a mixture of Ethanol:DI H2O (7:3 v/v) and 50 μL was dropped onto the surface of the freshly prepared hydroxylated electrodes. After 1h the amine-terminated electrodes were rinsed with DI water, dried with a gentle N2 flow and baked at 120ºC during 20 minutes for improved APTES adhesion. iii) Biotin immobilization: solutions of biotin, EDC and NHS were prepared in PBS (10 mM, pH 7.4). The biotin solution contains 0.5% of DMF to increase biotin solubility. A mixture of biotin (25 μL, 5 mM), NHS (12.5 μL, 0.2 M) and EDC (12.5 μL, 0.1 M) was prepared and immediately dropped onto the amine-terminated electrodes. After 2h, the electrodes were rinsed with PBS and dried with a gentle N2 flow. Control samples were also produced using anti-hCG and anti-E.coli (500 μg.mL-1) as recognition elements instead of biotin. 2.4 Electrochemical Methods and Biosensor Response The electrochemical cell was set up at low area/volume ratio conditions (0.2 cm2 to 75 mL of electrolyte solution) in a 5 ACS Paragon Plus Environment


three-electrode configuration, where the DGNP, platinum and Ag/AgCl (1 M KCl) (CH Instruments) are the working, counter and reference electrodes, respectively. The [Fe(CN)6]4-/3redox pair in 10 mM PBS was employed and the cyclic voltammetry (CV), chronocoulometry and AC experiments are run through a Versastat3 electrochemical station. Electrolyte solution was thoroughly bubbled with N2 prior to experiments. In order to determine the sensing response of the biotin-functionalized electrodes, several concentrations of avidin were prepared from a stock solution of 1 mg.mL-1 in 10 mM PBS. The functionalized electrodes are incubated during 45 minutes with a 50-μL drop of avidin solution having the desired concentration. Afterwards the substrates are rinsed with PBS and dried with a gentle N2 flow. Finally, electrochemical impedance spectroscopy (EIS) measurements are performed within the 1 kHz to 1 Hz range (logarithmic distribution of 10 points per decade) applying a 5 mV AC perturbation. All EIS measurements in this work are performed at the open circuit potential (c.a. 0.2 V vs. Ag/AgCl), unless specified otherwise. A mixed kinetic-diffusion (Rs[CPE[RCTW]]) equivalent circuit is used to model the cell, where Rs is the series ohmic resistance, CPE is a constant phase element modeling non-uniformities in the double layer, RCT is the faradaic CT resistance and W is the Warburg impedance. The equivalent circuits are fitted through least square regression method. The fittings were validated for total error below 2%. 3. Results and Discussion 3.1. Morphology and Crystalline Structure The MPCVD synthesis of DGNPs is simple, rapid (25 minutes) and reproducible over all substrate area. A representative SEM image of an unmodified DGNP surface is shown in Figure 1a. The morphology is composed of 2D, platelet-like structures protruding perpendicularly from the surface. The platelets are densely packed, separated by steep voids and coated with rounded nanosized grains. In order to fully understand the formation of these peculiar hybrid nanocarbons, TEM and EELS measurements were performed on a thin specimen produced by focused ion beam technique, cut perpendicularly with respect to the substrate (the vertical z direction). EELS measurements were performed along the cross-section of the nanoplatelet base unit, revealing an inner blade-like diamond core onto which the outer nanographite is bound, see Figure 1b. Regarding the near-edge structure of carbon K-edge, the graphitic sp2 peaking at c.a. 285 eV 6 ACS Paragon Plus Environment

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and the sp3 characteristic peak at c.a. 295 eV and dip at c.a. 305 eV further distinguish the two carbon allotropes (Figure 1c). In the low-loss region, the nanographite material is characterized to generate absorptions at c.a. 6 eV (π plasmon) and a broad absorption centered at c.a. 26 eV (   plasmon peaks), whereas diamond generates no absorption until c.a. 5 eV (energy band gap) and a strong broad absorption centered at c.a. 33 eV (   plasmon peaks). Therefore, energy windows at 5±2 eV and 36±2 eV were used to produce the energyfiltered TEM images, revealing the nanographite and diamond structures in brighter contrast (as seen in Figure 1d and Figure 1e, respectively), perfectly illustrating the hybrid structure arrangement. The STEM images made using an annular dark field (ADF) detector outline the crystallinity of the sp3/sp2 nanocarbon hybrid material and allow for a better understanding of the growth mechanism. Figure 1f shows the inner diamond nanoplatelet set to the [211] zone axis, where it is possible to observe the surrounding nanographite {0002} lattice fringes (interplanar spacing of 0.335 nm) parallel to {111} planes of the diamond nanoplatelet. This orientation was therefore used to determinate the epitaxial relation between both materials, which is {111}

diamond

||{0002}

graphite,

as also seen in references,16,17 suggesting a covalent

bonding type between the nanocarbons. The diamond platelets are 5 to 10 nm in thickness and are exposed in some edge regions where the nanographite coating gets progressively thinner. The growth takes place along the diamond {111} planes nearly parallel to the z (vertical) axis, as seen on Figure 1g, with the diamond platelets at the [011] zone axis. Crystal twinning is clearly observed, typical of diamond {111} facets. The Σ = 𝟑 twin boundary has the lowest defect energy in diamond,17,18 showing twinning mirror (111) plane (k1) and the twinning direction (1) [211], as shown by FFT (Figure 1h) and nanobeam diffraction pattern (Figure 1i). The conjugate twinning plane k2 = (111) in the matrix coordinate system (k'2 = (111) in the twin coordinate system), and the conjugate twinning direction 2 = [211] ('2 = [211]), are other twining elements.



b)

a)

b) b)

b) +

+

+

50 nm

10

+

(000¯ 2)graf

1 µm [211]

20

30

40

280

290

Electron energy loss, eV Electron Energyloss, Loss (eV) Electron energy eV

300

10

e) 100 nm 30 20

f)

h)

Å 2.06 } 1 {11

(11¯ 3)

K2 K`2

40

FFT η η`

η K1

d) (0¯ 2¯ 2)

(1¯ 11) ¯

(¯ 111)t

(¯ 1¯ 11)t

(022)t

(111)

i) Nanographite

Nanodiamond

2 nm

Diffraction

[01¯ 1]

Figure 1. a) Representative SEM micrograph of the DGNP surface. b) Representative STEM image of a nanoplatelet cross-section cut. Crosses mark selected regions for EELS analysis. c) Low-loss and high-loss EELS spectra taken at the inner thin platelet and outer coating regions (blue and red lines, respectively). (d-e) Energy-filtered TEM images formed using electron energy loss of d) 5±2 eV and e) 36±2 eV. (f-i) STEM-ADF images of a DGNP cross-section: f) Sample orientation at nanodiamond-[211] and nanographite- [1010] zone axis, as shown in the electron diffraction pattern in the inset. g) The nanoplatelet is positioned with the [011] direction of the nanodiamond parallel to the electron beam, where the diamond twinning defects are clearly visible. These were measured via fast Fourier transform (FFT) on image h) and a nanobeam diffraction on image i). Green lines and blue arrows on image g) depict the twining planes and directions, respectively. This growth habit was previously explained by a depletion of adsorbed hydrocarbons by atomic hydrogen on the (111) lateral facets of the diamond platelet, in harsh and high temperature plasma conditions during CVD growth.19,20 This leads to a sustained crystal growth taking place preferentially along {111} planes, because the growth rate is hampered in the orthogonal directions through methane radical starvation. Herein, the formation of nanographite crystals on the lateral platelet facets is seemingly facilitated. This is probably related to the large CH4/H2 ratio used in this work relatively to other report were the platelets were only composed of diamond crystals.16,19 Since, in this case, there is a lower (higher) 8 ACS Paragon Plus Environment

280

290

c) Electron Energy Loss(e

g)

f)

310

d) b)

~3.35 Å {0002}

GGraphite raphene Diamond Diamond

c)

50 nm

a)

(1¯ 11) ¯

(0¯ 22)

Intensity(arb.units)

a) a)

Intensity, arb.units units Intensity, arb. Intensity(arb.units)

a)

(0002)graf

1 2 3 4 5 6 7 8 9 10 11 12 13 14 (¯ 13¯ 1) 15 16 17 (¯ 111) 18 19 (¯ 113) ¯ 20 21 22 23 24 25 26 27 28 29 530 nm 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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concentration of H (CHx) radicals, the adsorbed hydrocarbons are not so easily depleted. The smaller H radical concentration also means that the sp3-terminated {111} lateral facets are not so efficiently stabilized, favoring the nucleation of nanographite. Finally, the presence of CN species arising from the addition of N2 to the growth atmosphere is known to favor nanographite phase development. 15,21,22 The peculiar morphology of these nanocarbon hybrids provides desirable characteristics for application in electrochemical transduction. This is so regarding the improvement of both the available surface area for bioreceptors immobilization and the superior electrochemical activity of edge defect sites when compared to pristine basal graphitic planes,23 notwithstanding the significant contribution of the latter.24 3.2. Assessment of Electrochemical Charge Transfer Standard Rate Constants Using [Fe(CN)6]3-/4- Redox Probe The knowledge of charge transfer (CT) standard rate constants (k0) is important because of its intimate relation with the CT resistance (RCT), which in turn constitutes the bioanalyte quantification parameter commonly used in faradaic impedimetric biosensors. The equivalent electrical circuit that describes the response of an electrode to an AC perturbation in a particular frequency range is, though not exclusively, dependent on k0. If the rates are high enough, reversibility holds and CT is very facile. Therefore, the electrochemical impedance spectroscopy (EIS) response is controlled solely by the diffusion of [Fe(CN)6]4-/3towards the electrode (Warburg impedance, W), by the double layer capacitance (Cdl) and by the series ohmic resistance (Rs) at the usable frequencies (f), usually within 10-1 to 104 Hz; direct quantification of RCT is hampered because kinetic information cannot be directly retrieved. On the other hand, the more sluggish the CT process is, then more significant overpotentials are needed to drive the reaction; hence RCT is larger and a defined semi-circle is distinguishable in the Nyquist plot. The EIS response of the functionalized DGNP electrodes is composed of regions of electrode kinetics and ion diffusional control, as seen in subsection 3.3 and subsection 3.4. Thus a mixed kinetic-diffusion equivalent circuit, Rs[CPE[RCTW]], is used to model the cell (see Figure 2 for circuit schematics). Therefore, in order to accurately get a measure of RCT, it cannot be sufficiently large so that the vast majority of the charge transits through the constant phase element (CPE) branch of the



circuit; if it is, the EIS response is obviously dominated by the CPE and several complications arise. That limit is semi-quantitatively set by the condition 25 𝒌𝟎𝒍𝒊𝒎 ≥

𝑹𝑻𝑪𝒅𝒍𝒘 𝑭𝟐𝑪𝑨

(1)

,

where R is the universal gas constant (8.314 J.K−1.mol−1), T = 298 K, F is the Faraday constant (96485.33 C.mol-1), w = 2πf (rad.s-1) is the angular frequency of the AC perturbation, C (mol.cm-3) is the concentration of [Fe(CN)6]4-/3- species and A is the electrode active area (cm2). Low capacitances thus favor meeting the criterion of Equation 1 over a wider range of k0. Estimations of capacitance per unit area are performed through combined chronocoulometry and voltammetry measurements (see Figure S1) and refined via EIS measurements (see Figure S2 and Table S1). An adequate capacitance value from these calculations is 125 μF.cm-2; Therefore, if C = 5 mM and T = 298 K, then 𝒌𝟎𝒍𝒊𝒎> ≅ 4x10-4 cm.s-1 to 4x10-5 cm.s-1 at 10 Hz to 1 Hz, respectively, which constitutes a typical frequency range dominated by CT kinetics. The DGNP electrodes’ k0 can be determined via cyclic voltammetry (CV) using the potential difference between anodic and cathodic peaks (𝜟𝑬𝒑 = 𝑬𝒑, 𝒂𝒏𝒐𝒅𝒊𝒄 ― 𝜟𝑬𝒑, 𝒄𝒂𝒕𝒉𝒐𝒅𝒊𝒄) and its departure from reversibility (𝜟𝑬𝒑~ 58 mV for a 𝑛 = 1 process). Those overpotentials are related to electrode kinetics and k0 can be retrieved in the basis of the Nicholson’s method, in which the dimensionless function Ψ(ν) is estimated via a logarithmic relation with ΔEp(ν), where 𝝂 (V.s-1) is the CV scan rate.25,26 Assuming a CT coefficient of 0.5 and a similar diffusion coefficient (D) for the [Fe(CN)6]4-/3- ions in 10 mM PBS (6.67x10-6 cm2.s-1), the k0 is retrievable through 𝒌𝒐

𝜳=

(

𝟏 𝝅𝑭𝑫𝒗 𝟐 𝑹𝑻

(2)

.

)

The derived k0 values are (1.97±0.23)x10-3 (0% N2), (3.34±0.11)x10-3 (4.1% N2), (3.35±0.20)x10-3 (11.3% N2) and (5.63±0.06)x10-3 cm.s-1 (14.5% N2). This trend is qualitatively similar to the electrical conductivity of the DGNP with increasing N2 content of the MPCVD process, as reported elsewhere.15


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b) 0.24

D G N P  0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .0

0 .5

fff 

750 4

500

RCT ()

( )

500

-1/2

1/2

6 -0 .0 4

250

8

0

750

14.5 % N 2

k00% =

300 0

k

200

-3

(2 .0 9  0 .0 3 ) x 1 0 (c m .s -3

14.5%

= (4 .6 5  0 .1 0 ) x 1 0 (c m .s

Z ( Re )

-0 .0 4

(V

500

250

0 .0 0

-1

-1

)

)

100 2 .0 0

2 .2 5

2 .5 0



2 .7 5

3 .0 0

0 % N2 1 4 .5 % N 2

400

RCT ()

Figure 2. a) CV dependence on the scan rate for DGNP samples grown at 14.5, 11.3, 4.1 and  0% N2 (top tok bottom). b) 𝜳 functions obtained via the Nicholson method. Inset: k  Corresponding ΔEp. c) and d) Nyquist plots as function of the DC bias for the DGNP0% DGNP14.5% samples, respectively, and e) corresponding k0 estimations. The equivalent circuit  In these experiments, C = 5 mM [Fe(CN) ]4- in 10 mM PBS and the model is also shown. 6 active areas are as determined by chronocoulometry (see Figure S1a). In b) and e), points are mean values and error bars are statistical standard deviations from independent triplicate measurements. 0

0% = (2 .0 9

300

0

200

14.5%

= (4 .6 5

-3

0 .0 3 ) x 1 0 (c m .s

-3

0 .1 0 ) x 1 0 (c m .s

-1

-1

)

)

100

2 .0 0

2 .2 5

2 .5 0

2 .7 5

3 .0 0

A 3-fold improvement in CT rate constants is observed upon increasing N2 concentration up to 14.5 vol%, in which case it is one (two) order(s) of magnitude higher than the limit set by Equation 1 at 10 (1) Hz. Therefore, these results validate the DGNP electrodes for faradaic impedimetric sensing using [Fe(CN)6]4-/3- redox probe. In fact, the k0 of DGNP films synthesized at 14.5 vol % N2 is lower than the reported for BDD electrodes (10-1 to 10-2 cm.s-1)27 and comparable to ridge-like nanodiamond (apparent rate of c.a. 7x10-3 cm.s1)28

but higher than oxygen terminated BDD (3x10-4 cm.s-1)2, graphite (1.76x10-3 cm.s-1),

some types of glassy-carbon (3.22x10-5 cm.s-1), some types of CNTs (5.09x10-6 cm.s-1) and nanoporous carbon (3.30x10-3 cm.s-1).29 It is also six orders of magnitude larger compared to the electrochemically inert graphene basal planes (< 10-9 cm.s-1), but lower than the measured from edge-planes of graphene sheets (c.a. 10-2 cm.s-1).12,30 11 ACS Paragon Plus Environment

500

Z ( Re )

0 % N2

E-E

-Z

500

0 .0 0

250

750

E-E

Z ( 500 Re )

8

-0 .0 8

0

1/2

-0 .0 4

750

)

0

(V

-0 .0 8

250

)

( )

im

im

-Z

250

7

-0 .0 8

400

14.5 % N 2

500

6

1/2

0  V -1/2 .s .s1/2 Z (ν50-1/2 ,V 0 .0 0 250 Re )

1 kHz

500

1 kHz

( 

750

-1 /2

250

e)

1 Hz

750

5

-1/2

0

d) 0 % N2

4

-1/2, VV-1/2 .s .s1/2 ν ff

2

Potential, V vs. Ag/AgCl

c)

5 03 0

1/2

-0 .1

1

im

-1

2

-Z

-1

0 .2 5 0 V .s

750

)

0 .0 7 5 V .s

( 

-1

- (0.073  0.04 0)

0.08

1 .0

im

0 .0 5 0 V .s

()

-1

750

0.12

-Z

1 0 0 A

-1

0 .0 2 5 V .s

0 % N2

1 kHz

0 .0 1 5 V .s



0 % ( ) = (0.069  0.00 8) 

0.16

- (0.154  0.036 )

- (0.1 25  0 .024 )

1 Hz

D G N P 



4 .1 % ( ) = (0.1 17  0 .004 ) 

1 Hz

1 .5

- (0.2 03  0 .012 )



1 1 .3 % ( ) = (0.1 21  0.007 ) 

(V

D G N P 

14.5% 11.3% 4.1 % 0%

0.20

2 .0



1 4 .5 % ( ) = (0.197  0 .002 ) 

G rowth g a s N 2 c onte nt :

1/2

D G N P 

E-E

a)

1 Hz

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ep, V

Page 11 of 26

250


In this method one assumes a cell’s CT coefficient (α) of 0.5. It also has been shown that this still constitutes an accurate method for determining kinetic parameters if the α parameter falls between 0.3 and 0.7.25 To estimate this coefficient, the DC potential is scanned in the vicinity of the half-wave potential (E1/2) superimposed by a 5 mV-amplitude AC perturbation at 10 Hz. The dependence of the impedance phase angle (ϕ) on experimental parameters and constants is 25 𝟏

𝒄𝒐𝒕 ∅ = 𝟏 +

(𝟐𝑫𝒘)𝟐 𝟎

𝒌

𝟏

(3)

[𝒆𝜶𝜣(𝟏 + 𝒆 ―𝜣)],

𝒏𝑭

where 𝜣 = 𝑹𝑻 (𝑬𝑫𝑪 ― 𝑬𝟏). This function reaches a maximum at 𝟐

𝑹𝑻

∆𝑬𝜶 = 𝑬(∅𝒎𝒂𝒙) ― 𝑬𝟏 = 𝒏𝑭𝒍𝒏 𝟐

(𝟏 ―𝜶 𝜶),

(4)

and thus 𝜶 = 0.5 if 𝑬(∅𝒎𝒂𝒙) = 𝑬𝟏. The measured ∆𝑬𝜶 are within the -5 to -15 mV 𝟐

range for all samples (see Figure S3 for the DGNP14.5% electrode); Therefore, α deviates from 0.5 up to a maximum of 0.56, and thus the Nicholson’s approximation of Equation 2 is proven to be valid for the DGNP electrodes. Finally, in order to counter-proof the Nicholson’s method, one can analyse the variation of total impedance with the DC bias (see Figure 2c and Figure 2d for the DGNP electrodes synthesized at 0 and 14.5 vol% N2, respectively) and isolate the RCT values through equivalent circuit fitting. The dependence of RCT on the 𝜣 and α parameters is 25 𝑹𝑻

𝑹𝑪𝑻 = 𝒌𝟎𝒏𝑭𝟐𝑨𝑪

[

𝟏 + 𝒆𝜣 𝒆

(𝟏 ― 𝜶)𝜣

]=

𝑹𝑻 𝜦 𝟎

𝒌 𝒏𝑭𝟐𝑨𝑪

(5)

,

from where the k0 can be estimated (Figure 2e). The results are similar to the Nicholson’s method, (2.09±0.03)x10-3 cm.s-1 (0% N2) and (4.65±0.10)x10-3 cm.s-1 (14.5% N2), despite the latter being about 17% lower than the Nicholson’s counterpart. 3.3. Sensor Functionalization with Biotin Avidin is a tetrameric protein able to bind four molecules of biotin via a non-covalent interaction with an extremely high affinity constant of 1015 L.mol-1.31,32 The avidin specific recognition element, biotin (also known as vitamin H), was linked via amide bond between the carboxylic acid group of biotin and an amine group on the surface of the electrode. EDC 12 ACS Paragon Plus Environment

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and NHS chemistry were employed to promote and improve the efficiency of the reaction, respectively. Regarding the preparation of the electrodes’ surface with amine groups, the DGNP surfaces were hydroxylated through the Fenton reaction and further modified with APTES. This organosilane forms covalent bonds with hydroxyl groups, yielding amine surface termination (electrode-C-O-Si-NH2, see Figure 3 for detailed schematics). The functionalization steps were followed via XPS, CV and EIS measurements.

Figure 3. Schematic representation of the DGNP thin films functionalization process. The overall XPS spectra of each functionalization step are shown in Figure 4a. Two Gauss-Lorentz shapes at ~284.7 and ~285.3 eV are suffice to describe the C1s spectrum of the unmodified DGNP surfaces (black curve in Figure 4c). The peak position and relative separation are consistent with sp2 and sp3 (C-C, C-Hx, C ― N) carbon bonding.33,34 Note that despite oxidized carbon surfaces being also characterized by sp3 orbital hybridization, its contribution in the XPS experiments are differentiated from C-C, C-Hx and C ― N ones for convenience. No carbon-oxygen bond peaks are discernible, and although small amounts of oxygen are present (O=C at 532.6 eV, see Figure 4a and Figure 4d), their contribution to the C1s region is completely masked by the sp2 and sp3 photoelectrons. The sp3 intensity hardly originates from C ― N, for no N was detected on the unmodified surface, as seen in Figure 4b. Hence, the sp3 peak for the unmodified sample arises (i) from locally exposed inner diamond platelets (sp3 C) and/or (ii) from the surface hydrogenation (C-Hx) characteristic of 13 ACS Paragon Plus Environment


MPCVD growth.35,36 Since the C-Hx fingerprint overlaps strongly with the sp3 carbon one, both contribute to the 285.3 eV peak intensity assignments through fitting procedures. However, this sp3 carbon peak is not needed to properly fit the spectrum after the Fenton treatment (red line in Figure 4c). This is consistent with the replacement of superficial hydrogen by oxygen, indicating that the sp3 intensity of the unmodified DGNP C1s spectrum is most likely related to hydrogenation. After the Fenton reaction, the C1s spectrum is characterized by the appearance of two new strong peaks at approximately 286.2 and 288.3 eV. These account for [C-O/C-OH] and C=O/O=C-OH], respectively. A significant concentration of C-O/C-OH is achieved as seen in the C1s quantitative analysis of Figure 4f. This is accompanied by an improved wettability of the DGNP surfaces, noticeable by the naked eye, also suggesting the effectiveness of the hydroxylation procedure. The Si2p and Si2s peaks appear after APTES treatment, at energies consistent with the organic Si coordination of the organosilicate (102.1 and 152.5 eV, respectively, see Figure 4e). A doublet separated by ~0.8 eV constitutes the former. The appearance of nitrogen peaks at 399.8 and 401.4 eV in a 0.82/0.18 proportion is characteristic of protonated and deprotonated primary amines at pH 7.4, respectively, also confirming the APTES immobilization (see Figure 4b).37 After APTES treatment the C1s region signature remains fundamentally unaltered. Regarding the O1s region (Figure 4d), overlapped with the O=C peak, a third peak at 532.8 eV is needed to properly fit the spectra. This is consistent with OxSi coordination environment. Concomitantly with the C1s spectrum, a noticeable decrease in the relative intensity proportion of the O-C/H-O-C (531.6 eV) peak is observed after APTES treatment (Figure 4g). Finally, after incubation in a mixture of PBS/NHS/EDC/Biotin in 10 mM PBS, a new element fingerprint appears in the spectrum at ~130 eV (P2p, see Figure 4a), corresponding to phosphorous contamination from incubating media. A new N1s peak at 398.2 eV also appears (Figure 4b), along with a pronounced dominance of the C-C/C-N/C-H peak in the C1s region (Figure 4c). This is consistent with the C-(NH) segment of the amide bond from biotin.38 In addition, the formation of the new amide bond (N-C=O) and the imidazole group from biotin (N-(C=O)-N) notoriously contributes to the peak at 401.4 eV (Figure 4b). Altogether, these considerations explain the changes in the N1s spectrum suggesting a significant amount of covalently bound biotin.


Page 14 of 26

525

535

530 530 530

280 525 525 525

540

535

285

530 530 530

285 530 530

530 285 530

280 525 525 525

525 280 525

525 525 280

535 4

5x10 540

4x10

525

4

4

540

535

535

3x10

4

2x10

4

1x10

4

530 540

290 0 540 540 540

290 540 540 540 290 540 540 540

540 530

535 525

530

535

530

525 535

540

a)

540 530

535 525

540 530

535 525

290 540 540

540 540 285 535 535

290 540 540

535 535

285

540 540 290

540 540 285 535 535

530 530 530

535 535 530 530

535

280 525 525

530 530 280 525 525

290 540 540

525 525

530 530

280 525 525

290 540 540

535 535 530 530

530 530 525 525 280

525 525

f) 285

530 530

U nm odifie d F e nton AP TE S B iotin

525 525 280

0 .2

C = C ,C -C ,C -H ,C -N

C -O ,C -O H

400 530 P 2p

530

525

525 395

540

535

405

5304 0 0

535

535 535 525 2 0 0

530

400 530 530 540

525

395 525 525 0 535

C = O ,O = C -O H

O x . C . T ota l

525

O1s 540

d)525

535 535 280 530 530530 535

540285 540 535 530 535 530 405 540 540 535 535 535 540 530 525

525 525

525 525 400 530 530 530 540

525 395 525 525 535

535 535

525 525

540

540

540285 0.6 535 535 540

535 530 530 535

530 525 530 280525

170

525 525

O -C ,H -O -C 170

530

525

395

540 405

395

530

535

535

530

405

400

395

400530

405 530

540 540

535 535

525 535

525395

540 540

165

O = C ,O -S i-O 165

400

530

525 525

530 530

S2p

90

525

395

90

100

90

530 530

170

100

540

405

535

525 525

100 170

525 525

h)

100 170

535 535

530 400

90

100

530 530

535

e)

170 energy (eV) 165 Binding

535 535

540405

395

100

535 535

540 540

530

530 100

Fe3s/Si2p 400

90 165

525 525

170

165

165

Binding energy (eV)

170

165

The surface is inevitably contaminated with S and Fe impurities after the Fenton reaction. The S2p doublet (Figure 4h) separated by ~1.12 eV at 169 eV clearly indicates the presence of a metal sulphate (FeSO4). Accordingly, the Fe2p1/2 and Fe2p3/2 multiplet signal peaking at ~724.8 and ~710.9 eV, respectively, accompanied by the corresponding satellites at higher binding energies, is indicative of ferrous and/or iron hydroxide compounds (Figure

ACS Paragon Plus Environment

540

530

400

525

Figure 4. a) Overall XPS spectra of the unmodified, post-Fenton, post-APTES and post-biotin electrode surfaces (from bottom to top: black, red, blue and green lines, respectively). (b-d) Normalized detailed spectra of the b) N1s, c) C1s and d) O1s regions. The fitted peaks are dashed lines and the sum in solid orange cures. f) C1s and g) O1s quantitative analysis based on the spectra of c) and d), respectively. The normalized e) Fe3s/Si2p and h) S2p regions of the spectra are also shown for the post-Fenton, post-APTES and post-biotin surfaces (from bottom to top: red, blue and green lines, respectively), along with the pertinent fitting.

15

535

405

395

Binding energy (eV) Binding energy (eV) 535 530 525

540 540

540

540

525

0.2

F e -O ,F e -O H

525

400

530

0.4

0.0

530

400

535

405

U nm odifie d F e nton AP T E S B iotin

g)

525

400

90

525 525

b)

530

400

535

405

90

Binding energy (eV) 540 535 530 525 540 535 530 525

525395

525

525 525

530 280525 530 525

535

405

100

535 535 530 530

535

540

525

530 530

N1s

405

100

540 540

540285 540 535 535

0.8

540

405

530 280 530 525 525 530

400530

525

540

540285 535 540 535 535 530 535 530 540 535

535

530

F e 3 s F e 3 p525 530 S 2p S 2s

540 540

1.0

540 540 290

0 .4

0 .0


540 405

530

525 535

530

525

0 .8

535 535

525 395

535

B inding e ne rg y (e V (eV) ) Binding energy

Binding energy (eV) Binding energy (eV) Binding energy (eV)

540 540 290

525

530 540

Binding energy (eV) 285

535

540

540

530

S i2 s S i2 p

285 290 535 530 525 540 535 530 525 540 535 530 525 540 285 535 530 525 280 540 535 530 525 535 530 540 525 525 535 535 530 535 530 525

1 .0

0 .6

530

c) 280

535 535 535

525

540

400 540

C 1s

540 N 1 s405535

525

540 285 535 530 525 280 540 535 530 525 535 5308 0540 525 535 530 525 1535 000 0 600 535 530 525

290 540 540 540

535

525

O 1s

530

525

525

540 405

O A ug e r F e A ug e r F e 2 p

530

C1s

530

Normalized intensity (arb. units)

285

6 x530 10

535


540

540

535


540

Normalized units) Normalizedintensity intensity(arb. (arb. units)

530

525

525

Normalized Normalized intensity intensity (arb. (arb. units) units)

540

530

N orm a liz e d c ontribution

530

N orm a liz e d c ontribution

1 2 535 3 4 5 6 7 535 8 9 10 11 12 13 535 535 14 535 15 16 17 18 535 535 19 535 20 21 22 23 24 25 535 535 26 27 28 29 30 535 535 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

535


540

Normalized intensity (arb. units) Intens ity(arb. (a rb.units) units ) Normalized intensity (arb. units) Normalized intensity Normalized intensity (arb. units)

Page 15 of 26

170

530

400


4e).39 The samples used for avidin detection were therefore repeatedly rinsed in DI water prior to APTES immobilization. Rinsing the sensors in DI water immediately before APTES immobilization also ensures the stabilization of –OH terminated surfaces increasing the process efficiency.40 An efficient hydroxylation is an important factor because the APTES immobilization process itself is predictably more efficient in densely hydroxylated surfaces due to the superior availability of binding sites. The CV and EIS follow up of the functionalization procedure are plotted in Figure 5a and Figure 5b, respectively.

a)

= 2 .0 x 1 0

-4

1 .0 x 1 0

-4

0.1 V .s


-1

b)

400

Unmodified Fenton APTES Biotin

f = 1 kHz to 1 Hz 5 mV perturbation

-Zim, 

Current, A

300

0 .0

200

100

-1 .0 x 1 0

-4

0

-0 .1

0 .0

0 .1

0 .2

0 .3

0 .4

100

0 .5

200

300

F e nton

0.015 V .s 0.025 V .s

-1

0.050 V .s

-1

0.075 V .s

-1

0.100 V .s

-1

0.14 0.12 0.10 4

6 

-1/2

8 1/2

ν-1/2, VV-1/2.s.s1/2

0 .4

( )

AP T E S

F e nton AP TE S B iotin

0.16

0 .6 -1

500

0.18

d)

B iotin

,V ΔEpE,pV

c)

400

Zre, 


1 0 0 A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

0 .2



- (0 .0 4 3  0 .0 0 8 )



- (0 .1 2 0  0 .0 1 0 )

F e nton ( ) = (0 .0 5 4  0 .0 0 1 ) 

A P T E S ( ) = (0 .0 7 8  0 .0 0 2 ) 



0 .0 -0 .2

0 .0

0 .2

0 .4

0 .6

B iotin ( ) = (0 .0 4 1  0 .0 0 3 ) 

4




6

-1/2

1/2

ν-1/2 ,VV-1/2.s.s1/2

- (0 .0 1 8  0 .0 1 1 )

8

Figure 5. DGNP functionalization follow-up via a) CV and b) EIS Nyquist plots at 0.2 V vs. Ag/AgCl. c) CV dependence on the scan rate for DGNP14.5% samples after biotin immobilization, APTES treatment and Fenton reaction. d) 𝜳 functions obtained through the Nicholson method for k0 estimation. Inset: Corresponding ΔEp. Points are mean values and error bars are statistical standard deviations from independent triplicate measurements. In these experiments, C = 5 mM [Fe(CN)6]4- in 10 mM PBS. Both ΔEp in voltammograms and RCT in EIS are lower for the unmodified sample (in black in Figure 5a and Figure 5b, respectively) compared to any of the subsequent 16 ACS Paragon Plus Environment

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functionalization stages, as expected due to additional CT barriers created upon DGNP surface modifications. Along with this progressive degradation of kinetics, a transition to an irreversible regime eventually begins and the CV peak currents are also lowered, as seen in Figure 5a. Indeed, after hydroxylation via Fenton reaction (in red in Figure 5a and Figure 5b), faradaic peak currents decrease sharply and both ΔEp in voltammograms and RCT in EIS increase noticeably, which is consistent with the well-known deterioration of CT kinetics after oxidation of carbon surfaces.2,26 However, after APTES treatment, CV peak currents are increased and both the ΔEp and RCT are decreased (in blue in Figure 5a and Figure 5b), contrarily to some reports in the literature,40,41 but in agreement with others.42–44 In order to clarify this matter, rate constants were also determined via the Nicholson method for the different functionalization steps (see Figure 5c and Figure 5d). The calculated rate constants are 1.02x10-3 cm.s-1, 1.48x10-3 and 7.72x10-4 cm.s-1, for the hydroxylated, APTES-treated and biotin-functionalized surfaces (red circles, blue lozenges and green triangles in Figure 5d), respectively. The improved k0 of APTES-treated electrodes when compared to OH-terminated ones is concomitant with the lowering of ΔEp and RCT. As noted in the above paragraph, intuitively one would expect an opposite behavior because of the additional DGNP-electrolyte CT barrier created upon organosilane binding, which should slower the kinetics and increase ΔEp and RCT, but this is not necessarily true in all cases as shown herein. This is a result of the superficial polarization provided by amine terminations after APTES treatment, which tend to protonate at pH 7.4 as inferred via XPS (see Figure 4b and related discussion), improving CT kinetics through electrostatic interactions with negatively charged redox ions 8,24,45. After biotin immobilization, CV peak currents are decreased and both ΔEp and RCT increase noticeable (in green in Figure 5a and Figure 5b), i.e. k0 decreases (green triangles in Figure 5d) due to inhibited CT as a result of biotin binding to the amine groups of APTES. The k0 is diminished by almost 50%, from 1.48x10-3 to 7.72x10-4 cm.s-1, which confirms that biotin loading is large, covering major electrochemically active regions. On the other hand, the RCT is lower after biotin immobilization than after the Fenton reaction, even though the estimated k0 is also lower (green triangles vs. red circles in Figure 5b and Figure 5d). This is due to a notorious lowering of ΔEp and RCT as the CV and EIS measurements are run on hydroxylated electrodes (not shown). This is due to modifications in the surface state and points out to a partial reversal of hydroxylation, which further enforces the need for immediate APTES treatment of freshly hydroxylated surfaces.



Page 18 of 26

3.4 Label-Free Impedimetric Detection of Avidin At this point, it is worthwhile to mention some aspects regarding the EIS detection of avidin. Since one wants to apply a small amplitude AC perturbation upon the equilibrium potential, both reduced and oxidized species are used at the same molarity so that the electrode is held balanced. Firstly, using more concentrated solutions of [Fe(CN)6]4-/3- favors slower kinetics in terms of the semi-quantitative limit of Equation 1 and, implicitly, provides 𝟎

wider range for bioanalyte quantification. However, since 𝒌𝟎𝟏𝟒.𝟓% ≫ 𝒌𝒍𝒊𝒎 (see subsection 3.2), keeping low electrolyte concentration (1 to 2 mM) is preferable. It minimizes the ionic interactions (e.g. bulk complexation reactions) arising from the inherent proximity of the [Fe(CN)6]4- and [Fe(CN)6]3- anions at higher concentrations, as well as the extent of specific adsorption and changes in the electrodes’ surface state.46 Secondly, extreme care was taken when dealing with impedance temporal drifting, a thoroughly discussed topic in the literature.8,46 To our experience with carbon materials such as DGNP, CVD graphene or laser–induced graphene, it takes a significant amount of incubation time (up to 3 hours) in order to stabilize the AC response in stagnant electrolyte. That signal drift is observable regardless of the functionalization stage of the sensor, being consistent with a progressive equilibration of adsorbates at the electrode/solution interface. In the particular case of DGNP electrodes, the RCT tends to increase until eventually a pseudo steady state is reached (not shown). Since RCT in impedimetric sensors usually denote the same behavior upon specific binding (e.g. CT blockage by large avidin molecules at the electrode surface, antibody-antigen complexation reactions, virus or bacteria attachment), this can constitute a serious source of false-positive results. In this sense, stirring the solution enabled one to attain steady measurements within a more practicable time scale, about 20 minutes, monitored by open circuit potential drift rates below 10 V.min-1, after an initial logarithmic transient period resembling an equilibration isotherm. As important, stirring the solution allowed maintaining reproducibility throughout the measurements. This is seemingly related to the renewal of the diffuse layer and the homogenizing effect on the Nernst diffusion layer thickness caused by convective mass transport.47,48. The stirring was performed at 200 rpm, at which there is not a direct impact of convection on the EIS response other than the above-mentioned considerations. In such conditions, no signal drifting occurs and the low-frequency portion of the Nyquist plot is linear with unitary slope, therefore undistinguishable from the Warburg impedance (black squares in Figure 6).49 On the contrary, if convective mass flow is intense enough the Nyquist 18 ACS Paragon Plus Environment

Page 19 of 26

plots deviate from linear behavior towards another x-intercept at low frequencies, as exemplified by the stirring speeds of 400 and 600 rpm (red circles and green triangles in Figure 6, respectively) below c.a. 1 Hz.

600

200 rpm 400 rpm 600 rpm

-Zim = (1.0015  0.0020) Zre - (133.03  0.80) () R2=0.9999

0.1 Hz

500 400

-Zim, 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300

1 Hz

200 100 0 100

200

300

400

500

600

700

Zre, 

Figure 6. Consecutive Nyquist plots of unmodified DGNP14.5% electrodes within the 1kHz to 0.1 Hz frequency range obtained at different stirring conditions and linear fit of the Warburg impedance at the low-frequency portion of the spectrum (solid red line). The electrolyte is a mixture of [Fe(CN)6]4- (1 mM) with [Fe(CN)6]3- (1 mM) in PBS (10 mM). The biotin-avidin biorecognition pair is used in many contexts. It is commonly employed in protein purification and to improve the immobilization process of bioreceptors in sandwich-type biosensors, e.g. enzymes and antibodies or aptamers in enzymo and immuno sensors, respectively 50–52. Given its large affinity constant, it can also be used to evaluate the suitability of an electrode material for label-free impedimetric sensors. The avidin EIS detection measurements and the corresponding DGNP sensor calibration curve are shown in Figure 7. Figure 7a, Figure 7b and Figure 7c show the Nyquist plots of the biotin-modified electrodes and corresponding controls (all in duplicate) upon incubation in blank and avidincontaining solutions at concentrations up to 1000 μg.mL-1, whilst Figure 7d shows the corresponding normalized response. Control A consists of APTES/biotin (no EDC and NHS) electrodes, allowing studying the influence of EDC and NHS in biotin immobilization. Control B consists of APTES/EDC/NHS/SRE electrodes (i.e., a specific recognition element other than biotin). In particular, the SREs were anti-hCG and anti-E.coli at the concentration of 500 μg.mL-1. These controls validate the avidin sensors’ specific recognition events and provide an estimation of the extent of non-specific binding, and thus should be included as relevant factors in the calculation of the effective limit of detection (LOD) and of quantitation (LOQ). 19 ACS Paragon Plus Environment


a)

Blank signal (Sensor #1) 10 µg.mL -1 100 µg.mL -1 1000 µg.mL -1

140 120

60

60 40

20

20 0

160 180 200 220 240 260 280

140 160 180 200 220 240 260

A ve ra g e d re s pons e

1 .5

0

160

Zre, Ω

R C T /R C T bla nk =

2 .0

40

20

Zre, Ω

2 .5

d)

S e ns or C ontrol A C ontrol B

1

1.5

S e ns or S e ns or C ontrol C ontrol C ontrol C ontrol

2.0

2.5

3.0

log 10 ([A v idin])

bla blank nk g /m L )-1) (0 µg.mL

220

(0 .8 6 2  0 .0 8 1 ) log 1 0 [A vidin] - (0 .5 9 8  0 .1 6 3 )

R = 0 .9 7 4

1.0

0 .5

200

Zre, Ω

0

1 .0

180

2

2

c)

60

80

40

Blank signal (Control A#1) 10 µg.mL -1 100 µg.mL -1 1000 µg.mL -1

80

-Zim, Ω

-Zim, Ω

-Zim, Ω

b)

100

80

0

Blank signal (Control B#1) 10 µg.mL -1 100 µg.mL -1 1000 µg.mL -1

140 120

100

bla nk blank ,, arb. RΔRC TCT//RR CT a rb.units units CT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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#1 #2 A #1 A #2 B #1 B #2

0 .0 -1-1 .0-0 .0 5 .9 0 5 0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

3 .0

log ([A vidin]),log log101(µg.mL ( g /m-1L) ) log1 10 ([Avidin]), 0 0 Figure 7. Nyquist plots of the a) NHS/EDC/Biotin (sensor), b) NHS/EDC/anti-hCG (control B) and c) Biotin-only (control A) samples after incubation in blank, 10, 100 and 1000 μg.mL-1 avidin solutions. d) Normalized response of NHS/EDC/Biotin sensors and control samples upon exposition to 0 (blank), 1, 10, 100, 500 and 1000 μg.mL-1 avidin in 10 mM PBS solution. Inset: Calibration curve of NHS/EDC/biotin sensors’ averaged response (red circles) and averaged responses of control B (black diamonds) and control A (green triangles). Error bars are propagated equivalent circuit fitting errors. The electrolyte is a mixture of [Fe(CN)6]4- (1 mM) with [Fe(CN)6]3- (1 mM) in PBS (10 mM). The sensors’ normalized response, 𝑹𝑪𝑻 ― 𝑹𝒃𝒍𝒂𝒏𝒌 𝑪𝑻 𝑹𝒃𝒍𝒂𝒏𝒌 𝑪𝑻

=

∆𝑹𝑪𝑻 𝑹𝒃𝒍𝒂𝒏𝒌 𝑪𝑻

,

(6)

starts to differentiate from controls at c.a. 10 μg.mL-1, scaling logarithmically up to the maximum concentration studied, 1000 μg.mL-1. All controls’ RCT increase slightly until c.a. 20 ACS Paragon Plus Environment

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100 μg.mL-1 is attained and tend to stabilize afterwards. There is not a distinct response from control A and B (inset of Figure 7d), supporting the importance of covalently binding the biotin to the electrodes using EDC and NHS. The sensors’ 3σ/m LOD and 10σ/m LOQ are 1.3 and 6.4 μg.mL-1 (19 and 93 nM), respectively. However, more realistic calculations must encompass the estimation of non-specific binding contributions. Therefore, the Control B#1 and B#2 averaged response was included in the final sensor’s figure of merit. The LOD and LOQ corrected by the non-specific response are 2.3 and 13.8 μg.mL-1 (33 and 200 nM), respectively. In parallel with the exposition to avidin solutions, biotin-modified sensors were also consecutively exposed to passivating bovine serum albumin (BSA) solutions having concentrations up to 500 μg.mL-1 in PBS buffer. The corresponding impedance spectra are representatively shown in Figure 8.

Figure 8. Nyquist plots of biotin-functionalized sensors after consecutive incubations in BSA solutions in 10 mM PBS during 45 minutes. The measuring electrolyte and parameters are as in the avidin detection tests of Figure 7.

There is no appreciable response of the biotin-functionalized sensor after the successive incubations in BSA solutions, in a similar trend to the control samples in Figure 7. In addition to (i) the relevant contribution of biotin to the XPS N1s spectrum (Figure 4b), (ii) the relevant increase (decrease) in RCT (k0) after biotin immobilization (Figure 5), and (iii) the relatively low effect of non-specific binding on the measured signal estimated through the control samples in Figure 7, this further corroborates that a significant DGNP surface coverage by biotin has been achieved. More specifically, since BSA did not significantly altered RCT, one can state that the majority of electrochemically active sites are biotinterminated and BSA proteins are mainly bound to electrochemically inactive sites. Some 21 ACS Paragon Plus Environment


authors have reported similar lack of response after immunosensor surface passivation,53,54 also attributing it to a major covering of the active surface by antibody recognition elements. Altogether, the presented results validate the DGNP thin films as adequate and reliable transducers for label-free impedimetric sensing. Nevertheless, high-selectivity measurements still must be demonstrated for other important biomarkers in real samples to definitely implement the DGNP hybrid materials as a reliable alternative. The lowering of LOD and LOQ down to ng.mL-1 levels is also mandatory for many important biomarkers, e.g. prostrate cancer diagnosis demands prostate-specific antigen to be quantified below c.a. 10 ng.mL-1. Yet, direct comparisons with other materials applied to same type of sensors ought to be done considering the same target analyte. In this sense, regardless the existence of several reports on the detection and quantification of avidin via affinity interaction with biotin,55–59 none are based on impedimetric methods, but rather on voltammetric and transistor approaches, for which the reported LODs and LOQs are generally of the same order of magnitude as the ones found in this work. The extension of the concept to immunosensors for the detection of important biomarkers such as prostate-specific antigen, human chorionic gonadotropin, cortisol, E.coli and Legionella is currently undergoing. The selective detection of such biomarkers must be accomplished in real and complex samples containing much more interfering molecules. In order to guarantee reliable quantification at the required levels, more comprehensive studies regarding DGNP immunosensors’ passivation and non-specific response as well as sensor miniaturization will be undertaken in the future.


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4. Conclusion The full process of producing label-free biosensors based on diamond-graphite nanoplatelet (DGNP) thin hybrid films is discussed in detail, from the CVD synthesis to the preliminary studies as faradaic impedimetric biosensors. The DGNP films’ building unit consists in an inner 2D-like nanocrystalline diamond platelet covered by a covalently-bonded nanographite film with a {111}diamond||{0002}graphite epitaxy. These units present preferential vertical alignment and conspicuous edges, which constitute interesting characteristics for electrochemical impedimetric biosensing. Moreover, the charge transfer rate constants in a ferrocyanide solution in PBS buffer rose from c.a. 2.0x10-3 cm.s-1 up to c.a. 5.6x10-3 cm.s-1 upon N2 addition during DGNP synthesis. The DGNP facile charge transfer is a crucial characteristic envisaging the reliable detection and quantification of bioanalytes via faradaic impedimetric approaches. As a proof of concept, biotin-functionalized DGNP electrodes are shown to quantify avidin within the 10 to 1000 μg.mL-1 range following a logarithmic dependency. The limits of detection and of quantitation after correction for the non-specific response are 2.3 and 13.8 μg.mL-1 (33 and 200 nM), respectively.

Supporting Information. Complementary information on the methods used to assess the electrochemical response of the DGNP electrodes.

Acknowledgements N. F. Santos thanks FCT for the PhD grant (SFRH/BD/90017/2012) and i3N for the grant BI/UI96/5177/2018. S. O. Pereira thanks i3N for the BPD grant (BPD/UI96/5808/2017). The authors acknowledge financial support from FEDER funds through the COMPETE 2020 Programme and National Funds through FCT - Portuguese Foundation for Science and Technology under the projects UID/CTM/50025/2013.



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Graphical Abstract


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Physical Structure and Electrochemical Response of Diamond

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