Microchimica Acta

Diamond-Conjugated SARS-CoV-2 Spike Protein: Nanoenabled Electrochemical

Impedance Immunosensing on a Gold Microelectrode
--Manuscript Draft--

Manuscript Number:

Full Title: Diamond-Conjugated SARS-CoV-2 Spike Protein: Nanoenabled Electrochemical

Impedance Immunosensing on a Gold Microelectrode

Article Type: Original Paper

Keywords: COVID-19; Spike protein; Biosensor; Respiratory virus; Pandemic

Corresponding Author: Subash C.B. Gopinath

University Malaysia Perlis
MALAYSIA

Corresponding Author Secondary

Information:

Corresponding Author's Institution: University Malaysia Perlis

Corresponding Author's Secondary

Institution:

First Author: Santheraleka Ramanathan

First Author Secondary Information:

Order of Authors: Santheraleka Ramanathan

Subash C.B. Gopinath

Zool Hilmi Ismail

Order of Authors Secondary Information:

Funding Information: ASEAN University Network Southeast Dr. Zool Hilmi Ismail
Asia Engineering Education Development
Network
(R.K130000.7343.4B617)
Universiti Malaysia Perlis Dr. Subash C.B. Gopinath
(9001‐00596)

Abstract: This research proposes a promising immunosensing strategy in diagnosing SARS-

CoV-2 using a 10 µm gap-sized gold interdigitated electrode (AuIDE) to target the
surface spike protein (SP). The microelectrode surface was modified by (3-
glycidyloxypropyl) trimethoxysilane to enforce the epoxy matrix, which facilitates the
immobilization of the anti-SP antibody. The immunosensing performance was
evaluated by integrating a nanosized (~10 nm) diamond-complexed SP as a target.
The proposed immunoassay was quantitatively evaluated through electrochemical
impedance spectroscopy (EIS) with the swept frequency from 0.1 to 1 MHz using a 100
mVRMS AC voltage supply. The immunoassay performed without diamond integration
showed low sensitivity, with the lowest SP concentration measured at 1 pM at a
determination coefficient of R2 = 0.9681. In contrast, the nanodiamond-conjugated SP
on the immunosensor showed excellent sensitivity with a determination coefficient of
R2 = 0.986. SP detection with a nanodiamond-conjugated target on AuIDE reached the
low limit of detection at 189 fM in a linear detection range from 250–8000 fM. The
specificity of the developed immunosensor was verified by interacting influenza-
hemagglutinin and SARS-CoV-2-nucleocapsid protein with anti-SP, showing specific
EIS measurements only with the SP. In addition, the authentic interaction of SP and
anti-SP was validated by enzyme-linked immunosorbent assay.

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1 Diamond-Conjugated SARS-CoV-2 Spike Protein: Nanoenabled Electrochemical

2 Impedance Immunosensing on a Gold Microelectrode
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11 Santheraleka Ramanathan1, Subash C.B. Gopinath1,2*, Zool Hilmi Ismail3,*
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21 Institute of Nano Electronic Engineering, Universiti Malaysia Perlis,
22 01000 Kangar, Perlis, Malaysia
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23 Faculty of Chemical Engineering Technology, Universiti Malaysia Perlis,
24 02600 Arau, Perlis, Malaysia
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25 Centre for Artificial Intelligence and Robotics, Universiti Teknologi Malaysia,
26 Jalan Sultan Yahya Petra, 54100, Kuala Lumpur, Malaysia
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39 *Correspondence: subash@unimap.edu.my (or) zool@utm.my

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40 Abstract
41 This research proposes a promising immunosensing strategy in diagnosing SARS-CoV-2 using
42 a 10 µm gap-sized gold interdigitated electrode (AuIDE) to target the surface spike protein
43 (SP). The microelectrode surface was modified by (3-glycidyloxypropyl) trimethoxysilane to
44 enforce the epoxy matrix, which facilitates the immobilization of the anti-SP antibody. The
45 immunosensing performance was evaluated by integrating a nanosized (~10 nm) diamond-
46 complexed SP as a target. The proposed immunoassay was quantitatively evaluated through
47 electrochemical impedance spectroscopy (EIS) with the swept frequency from 0.1 to 1 MHz
48 using a 100 mVRMS AC voltage supply. The immunoassay performed without diamond
49 integration showed low sensitivity, with the lowest SP concentration measured at 1 pM at a
50 determination coefficient of R2 = 0.9681. In contrast, the nanodiamond-conjugated SP on the
51 immunosensor showed excellent sensitivity with a determination coefficient of R2 = 0.986. SP
52 detection with a nanodiamond-conjugated target on AuIDE reached the low limit of detection
53 at 189 fM in a linear detection range from 250–8000 fM. The specificity of the developed
54 immunosensor was verified by interacting influenza-hemagglutinin and SARS-CoV-2-
55 nucleocapsid protein with anti-SP, showing specific EIS measurements only with the SP. In
56 addition, the authentic interaction of SP and anti-SP was validated by enzyme-linked
57 immunosorbent assay.
58
59 Keywords: COVID-19; Spike protein; Biosensor; Respiratory virus; Pandemic
60

61 Introduction
62 The SARS-CoV-2 virus originating from Wuhan, China, in December 2019 was reported to
63 have human-to-human transmission from a seafood market in the city. The outbreak of the
64 virus was rapid across the world, infecting ~200 countries and causing millions of deaths [1].
65 The SARS-CoV-2 virus causes respiratory complications, which manifest very common
66 symptoms such as dry cough, fever, body ache, and tiredness. The severity of the virus results
67 in organ damage, mainly to the lungs, heart, and liver, leading to death [2, 3]. Since no
68 immediate antiviral drug is accessible, the early prognostics, diagnosis and treatment to save
69 humankind from the pandemic outbreak were grievous. However, the existing technology
70 coupled with computed tomography (CT) has been desired as a primary diagnostic strategy to
71 recognize SARS-CoV-2 infection. Following that, reverse transcription-polymerase chain
72 reaction (RT-PCR) is still upholding its stand as the highly sensitive technique to detect SARS-

2
 73 CoV-2 [4, 5]. Due to several drawbacks aroused with early and rapid detection, the invention
74 of various techniques was continued by developing loop-mediated isothermal amplification
75 (LAMP), specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) and serological
76 strategies, yet the performances of these techniques are not in parallel with the performance
77 exhibited by real-time RT-PCR [6]. As the pandemic spreads, an ideal detection technique for
78 recognizing dreadful viruses is expected to be highly sensitive, user friendly, and suitable for
79 point-of-care diagnosis, regardless of the external domains.
80
81 SARS-CoV-2 is composed of four main structural proteins: spike glycoprotein (SP),
82 nucleocapsid (NCP), envelope (E), and matrix (M). SP plays a significant role in viral infection,
83 as it is entangled in viral receptor recognition, attachment, and entry into host cells [7, 8]. SP
84 expedites the viral protein from binding to the host cell receptor angiotensin-converting
85 enzyme 2 (ACE2) and consequently mediates viral cell entry. SP is tremendously critical in
86 rapid cell multiplication in the host body and has the indispensable potential for diagnosing
87 and treating SARS-CoV-2 infectious disease [9, 10]. In the state-of-the-art biosensors,
88 electrochemical approaches have emerged as one of the most promising techniques to target
89 infectious disease antigens [11, 12]. The advantages of electrochemical strategies are that they
90 can identify small changes in the biomolecular system on a sensing plate. In this regard,
91 electrochemical immunoassays have become a well-suited strategy to detect antigens on
92 antibody-immobilized biosensors [13, 14]. Electrochemical quantification was determined
93 through the redox indicator applied, followed by biomolecule interactions occurring on the
94 sensing surface. In addition, electrochemical immunoassays are cost-effective and expeditious,
95 using low-cost equipment and materials with less expert handling involved [15, 16].
96 Nevertheless, electrochemical immunoassay strategies have rarely been reported for targeting
97 SARS-CoV-2 antigens.
98
99 A futuristic electrochemical immunosensing strategy targeting SARS-CoV-2 SP using
100 a gold interdigitated electrode (AuIDE) is proposed in this research. The sensitivity of the
101 immunosensor was evaluated by integrating nanodiamonds with SP and compared with the
102 immunoassay without diamond conjugation. Rigid three-dimensional tetrahedral structured
103 diamond nanomaterials were selected because they exhibit excellent strength and durability
104 compared to other carbon-based nanomaterials [17, 18]. Diamond was recognized for
105 improving signal detection on biosensing surfaces due to its absolute noncytotoxic nature,
106 chemical inertness, and high biocompatibility with multiple types of biomolecules [19, 20].

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107 The inert diamond nanomaterial exhibits a low background current and high resistance to
108 fouling, which are extremely favorable for conjugation with protein for rapid and sensitive
109 target detection [21]. The performance of the developed immunosensor in diagnosing SARS-
110 CoV-2 infectious disease was determined by electrochemical impedance spectroscopy (EIS).
111 The interactions between SP and anti-SP antibody with nanomaterial integration were well
112 defined through impedance measurements by EIS. With the relatively simple and advantages
113 of the detection technique, the proposed strategy is recommended to be widely used in
114 diagnosing SARS-CoV-2 infectious disease, especially for rapid and point-of-care detection to
115 save infected people at every edge of the world.
116

117 Experimental Section

118
119 Materials and Reagents
120 A 10 µm gap size gold interdigitated electrode (AuIDE) with 125 electrode fingers connected
121 by pads was purchased from Metrohm DropSens (Malaysia). His-tagged recombinant SARS-
122 CoV-2 SP (native sequence: YP_009724390) produced in HEK293 cells was purchased from
123 InvivoGen (China). Anti-spike antibody produced in mice was purchased from GeneTex, Inc.
124 (North America). The recombinant SARS-CoV-2 nucleocapsid protein (NCP) and
125 hemagglutinin (HA) of human influenza A for specificity determination of the immunosensor
126 were procured from GeneTex, Inc. (North America) and Sino Biological Inc. (China),
127 respectively. Diamond nanopowder was purchased from Sigma Aldrich (USA). Phosphate-
128 buffered saline (PBS, 1 M, pH 7.4), potassium hydroxide (KOH), 99% ethanol, 1,1'-
129 carbonyldiimidazole (CDI), (3-glycidyloxypropyl)trimethoxysilane (GOPTS), potassium
130 hexacyanoferrate (III), K3[Fe(CN)6] potassium hexacyanoferrate (II), and K4[Fe(CN)6] were
131 procured from Sigma Aldrich (USA). Ethanolamine (ETA) obtained from Fisher Scientific
132 (UK) was used as a blocking agent. Enzyme-linked immunosorbent assay (ELISA) plates and
133 anti-mouse IgG horseradish peroxidase (HRP) were procured from Thermo Scientific (USA).
134 ELISA coating buffer (5×) was purchased from Biolegend (Japan). 3,3′,5,5′-
135 Tetramethylbenzidine (TMB) and bovine serum albumin (BSA) were purchased from Promega
136 (USA).
137
138
139

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140 Diamond Conjugation with Spike Protein
141 Approximately 50 mg diamond nanopowder was modified using 0.5 M of CDI solution. The
142 mixture was incubated for 1 hour and washed with distilled water through centrifugation
143 (10,000 x g, 5 min). The diamond pellet was collected and characterized. The diamond
144 morphology was determined through field emission scanning electron microscopy (FESEM,
145 Hitachi, S-4300 SE, Japan) and field emission transmission electron microscopy (FETEM,
146 F20-Tecnai, Columbus). The diamond pellet was then suspended in 500 µL of 1 nM SP and
147 incubated for 1 hour. Then, the unreactive sites on the diamond were blocked using 1 M ETA
148 for 1 hour. The mixture was centrifuged, and the collected pellet was washed several times
149 using 10 mM PBS solution (pH 7.4). Finally, 500 µL of PBS solution was added to the pellet,
150 resulting in 1 nM diamond-conjugated SP. The diamond-conjugated target protein was stored
151 at 4 °C until further use in the immunoassay.
152
153 Immunosensor Development - AuIDE
154 The immunosensor was chemically modified to ensure the specific detection of SP on AuIDE.
155 The microelectrode surface was observed under optical microscopy using a 3D nanoprofiler
156 (Hawk 3D Optical-Surface Profiler, South Korea) and a high-power microscope (HPM, Nikon
157 Corporation, Japan). The AuIDE dimension and precision were determined using scanning
158 electron microscopy (SEM, Jeol Ltd., Japan). The activation of the AuIDE sensing surface was
159 initiated by dropping 20 µL of 1% KOH and incubating for 10 minutes. Then, 2% GOPTS
160 solution was drop-cast on AuIDE to create an epoxy matrix, which is highly reactive with
161 amine and carboxyl bases. The sensing surface was washed with 10 mM PBS solution after a
162 1-hour incubation with the linking agent. As the bioprobe for SP detection, 20 µL of 1 µM anti-
163 spike antibody was immobilized on AuIDE and incubated for 1 hour. The unbound antibodies
164 were removed by washing with PBS solution. Moreover, the unreactive active regions of
165 AuIDE were blocked with 1 M ETA solution. SP, as the target of the assay, was prepared in
166 the femtomolar to nanomolar range of concentrations (1 fM to 1 nM). A 20 µL aliquot of 1 fM
167 SP was immobilized on the sensing surface and incubated for 5 minutes. Then, the surface was
168 washed with PBS solution to remove the unbound target. The detection of SP on AuIDE was
169 evaluated through electrochemical impedance spectroscopy (EIS) using a Novocontrol alpha
170 high-frequency analyzer (Novocontrol Technologies GmbH, Germany). Redox solutions
171 consisting of 10 mM PBS and 5 mM [Fe(CN)6]3- and [Fe(CN)6]4- were used to characterize
172 the Faradaic measurement with EIS analysis. The EIS measurements were recorded with the
173 swept frequency from 0.1 to 1 MHz using a 100 mVRMS AC voltage supply. The efficiency of

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174 diamond-conjugated SP in detecting SARS-CoV-2 infectious disease was evaluated on SP
175 antibody-modified AuIDE. The impedance spectra for the analyses were plotted for the real,
176 Z’ and imaginary parts, Z’’ of impedance.
177
178 Enzyme Linked Immunosorbent Assay (ELISA)
179 The specific binding of recombinant soluble SP with anti-SP antibody was validated through
180 ELISA. SP at concentrations ranging from 1 pM to 100 nM was prepared using 1X coating
181 buffer. Then, 50 µL of target was coated into the ‘ELISA plate’ wells and incubated at 4 °C
182 overnight. Consecutive washings at each step were performed using 300 μL of PBS-Tween 20
183 solution. Then, the wells were blocked using 2% BSA solution. The anti-SP antibody was
184 diluted 1:1000 from the stock. Approximately 50 µL of diluted primary mouse antibody was
185 coated in the wells and allowed to interact with the coated target for 1 hour. Next, anti-mouse
186 antibody-conjugated HRP at 1:1000 was coated and incubated for 1 hour to allow the
187 interaction between primary and secondary antibodies. Finally, TMB substrate respective to
188 HRP enzyme was added to the wells. The color change in the wells was observed closely, and
189 the interaction of SP and anti-SP antibody was determined by recording the absorbance reading
190 using a UV-Nanodrop (DS-11/DS-11, Denovix, USA).
191

192 Results and Discussion

193 An ideal biosensor for rapid detection of SARS-CoV-2 disease is expected to have excellent
194 portability, sensitivity, and precision. Excellence in detecting and discriminating various
195 sample detection limits is preferred to diagnose fatal disease at the early stage of virus infection.
196 In this approach, an interactive analysis was performed on AuIDE to evaluate the detection of
197 SARS-CoV-2 using soluble recombinant SP and its anti-SP antibody. IDE sensors have been
198 reported for their efficiency in recognizing specific interactions with varying biomarkers. To
199 improve the detection sensitivity, nanodiamond-conjugated SP was integrated into the
200 developed immunosensor. EIS measurements provide detailed information on the diamond and
201 biomolecule interactions that take place on the immunosensor and aid in determining SP. Fig.
202 1 shows a schematic illustration of the proposed strategy in detecting SARS-CoV-2 SP by an
203 anti-SP antibody on AuIDE integrated with nanodiamond-conjugated SP.
204
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207 AuIDE Morphology – High Resolution Surface Imaging
208 Morphological examination revealed the precision of the AuIDE sensing surface. Fig. 2a shows
209 the digital image of AuIDE used. Optical images of AuIDE electrodes and junctions were
210 observed under HPM (Fig. 2b-2c). The dimensions of the sensing surface were verified through
211 SEM imaging (Fig. 2d), which revealed that the gap and electrode dimensions differed by ~0.4
212 µm from the designed dimensions. The electron microscopic image of the electrode junction
213 indicates the active surface of AuIDE without any foreign substances (Fig. 2e). The smoothness
214 of AuIDE between its gold electrode and silicon substrate was visualized through a 3D profiler
215 (Fig. 2f–2g). Moreover, the roughness profile indicates that the height of the electrode and gap
216 on AuIDE is exact (Fig. 2h), which further affirmed the indistinguishable color of the electrode
217 and gaps indicated through 3D images. The average height of AuIDE used is ~ 48.9 μm.
218
219 Nanodiamond Morphology – Size and Shape Observations
220 The commercially purchased nanodiamond was modified with CDI linker to activate its
221 functional groups for covalent protein conjugation. The size and shape of the nanodiamonds
222 were determined through FETEM and FESEM analyses. Fig. 2i-2k shows the electron
223 microscopic image of diamond under FETEM at various scales, revealing the uniform size of
224 modified diamonds. The distribution was uniform and compact, as no space between the
225 diamonds was observed in the electron microscopic images. Fig. 2l shows an enlarged electron
226 microscopic image of nanodiamond with the size measured using the FETEM microscopic
227 tool. Fig. 2m-2n shows the uniform spherical shape of nanodiamonds, observed through
228 FESEM imaging. Based on the electron microscopic morphology, the average size of diamond
229 nanoparticles is ~10 nm, observed at their spherical shape. The elemental composition of
230 diamond was identified through energy dispersive spectroscopy (EDS). The EDS spectra
231 shown in Fig. 2o emphasized that the diamond consisted of 94.58% carbon and 2.32% and
232 2.75% oxygen and nitrogen, respectively. The platinum (Pt) that appeared in the EDS spectra
233 was due to Pt used for FESEM coating for electron microscopic analysis.
234
235 Spike Protein-Antibody Interaction on AuIDE
236 The impedance spectra obtained when the anti-SP antibody immobilized AuIDE were
237 evaluated with SP from 1 fM to 1 nM (Fig. 3a). The Nyquist plot obtained from EIS
238 measurements of AuIDE represents the Randles circuit, which expresses the redox solution
239 resistance (Rs), charge transfer resistance (Rct) and double layer capacitance (Cdl). The Rs value
240 is expected to be similar for every EIS reading, as the volume of redox solution (20 µL) used

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241 is the same for every measurement. The increment in the Rct value represents the weight of
242 charge transfer upon interaction between the developed immunosensor and different
243 concentrations of SP. The impedance spectra were discussed based on the changes observed in
244 the real part impedance, Z’, which indicates the charge transfer resistance from the redox
245 solution to the base of the immunosensor. The Nyquist spectra generated with 1 fM and 10 fM
246 SP were lower than those generated with ETA. The intensity of charge transfer was
247 insignificant with the concentration, indicating insignificance in detecting the interaction
248 between the SP antibody and protein. From 100 fM to 1 nM, a notable increase was observed
249 in the Nyquist plot, revealing that SP and anti-SP antibody interactions occurred. A huge gap
250 was noticed between Z’ of 1 pM to 10 pM SP and then to 100 pM SP. The gap between the
251 Nyquist plots indicates that the detection range of SP on the developed immunosensor falls in
252 the picomolar range of concentration. Thus, the detection of SP on anti-SP immobilized AuIDE
253 was performed in the narrowed range of target concentrations. Fig. 3b shows the impedance
254 spectra plotted by analyzing SP from 1 pM to 64 pM. A linear accretion in Z’ was observed
255 from 1 to 8 pM. Z’ increases by ~1.5-fold from 1 to 8 pM at each interval target. The Z’
256 difference between 8 and 16 pM was small. In addition, the Nyquist spectra of 32 and 64 pM
257 SP went downward against the linear direction. This indicates that the saturation point for SP
258 interaction with 1 µM anti-SP immobilized AuIDE was at 8 pM SP. The linear detection of SP
259 at the lower picomolar range emphasizes the success of immunosensors developed for
260 detecting SP for diagnosing SARS-CoV-2 infectious disease.
261
262 Fig. 3c shows the Bode plot generated with the total ‘Z’ values against the frequency
263 supplied (0.1 to 1 MHz) for SP and anti-SP interaction on the developed immunosensor. The
264 Bode plot shows that the total ‘Z’ values increase from 1 pM to 16 pM SP, resembling the
265 similar trend observed in impedance spectra (Fig. 3b). A significant variation in the total ‘Z’
266 was observed at 23 Hz before it reached a stagnant state at 102 Hz up to 106 Hz. Fig. 3d shows
267 the phase angle revealed by the immunosensor with immobilized SP. In good agreement with
268 the total ‘Z’ (Fig. 3c), the phase angle spectra show a significant change at ~23 Hz. The active
269 frequency and its real part impedances for different concentrations of SP were evaluated to
270 determine the sensitivity performances.
271
272 Diamond Conjugated Spike Protein-Antibody Interaction on AuIDE
273 The electrochemical measurements of anti-spike antibody immobilized AuIDE were recorded
274 by analysis with diamond-conjugated SP from 0.25 to 8 pM. The concentration range selected

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275 for evaluation was reduced from the previous immunoassay (1 – 64 pM), as the conjugation of
276 nanodiamonds is expected to improve the detection strength of SP on the sensing plate. Fig. 4a
277 shows the impedance spectra obtained from the diamond-conjugated SP interaction on the
278 developed immunosensor. It was proven that the performance of the immunosensor was
279 excellent when diamond-conjugated SP was used as the target. Linear detection of SP was
280 apparent from the 0.5 to 8 pM diamond-conjugated protein. The Z’ impedance value was
281 immensely reduced from 25 kΩ (Fig. 3b) to 4 kΩ (Fig. 4a) when a 1 pM target concentration
282 interacted with the immunosensor. The reduction in Z’ denotes the highly moving electron with
283 the target interaction and conjugation of diamond on the sensing plate. This further enhances
284 the electrical conductivity of the sensor, ~6250-fold better than the interaction exhibited
285 without diamond. ETA exhibited 3 kΩ of Z’, and the value was increased to 4 kΩ at 1 pM. The
286 small increase in the Z’ value indicates that diamond-conjugated SP is detected on chemically
287 modified AuIDE. As the diamond-conjugated protein concentration was increased from 1 to 2
288 pM, the increase in Z’ was ~1.6-fold higher, which indicates the significant interaction of SP-
289 antibody on AuIDE. Following that, a linear detection trend was observed from 2 pM (7.8 kΩ)
290 to 8 pM (9.2 kΩ), emphasizing the significance of diamond in enhancing the sensitivity and
291 linearity in detecting the target protein on the developed immunosensor. The linear accretion
292 in Z’ explains the increase in the rate of charge transfer aroused with countless SP and anti-SP
293 on AuIDE. However, the immense reduction in Z’ with linear detection of SP is aided by the
294 conjugation of diamond with biomolecules. Diamond enables high-density SP immobilization
295 on the sensing plate. This enhances SP detection and improves electron mobility, which directly
296 boosts the sensitivity of the immunosensor. Fig. 4b and 4c show the total Z and phase angle
297 against the swept frequency, respectively. The plots indicated that a high rate of charge transfer
298 occurred in the low frequency range (0.1–100 Hz). The active frequency in detecting SP using
299 the developed immunosensor was determined at 30 Hz based on the active frequency range in
300 the Bode plot and the maximum phase angle of the system.
301
302 Immunosensor Sensitivity: Comparative Analysis
303 The sensitivity of the developed immunosensor was evaluated with SP and diamond-
304 conjugated SP as targets for detecting SARS-CoV-2. Fig. 4d shows a linear calibration curve
305 generated by the linear detection measurements using SP and diamond-conjugated SP on the
306 immunosensor. Fig. reveals that anti-SP immobilized AuIDE has a lower Z’ in the low
307 frequency range using diamond-conjugated SP as the target compared to the immunoassay
308 performed without diamond. Moreover, the linear regression lines plotted by comparing

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309 immunoassay with and without diamond incorporation indicate the performance of diamond-
310 based immunoassay in detecting the target at lower concentration range and hence emphasize
311 the highly sensitive immunoassay. Fig. 4e shows the calibration curve plotted with the mean
312 Z’ values obtained at 23 Hz versus the logarithm of SP concentrations. The linear calibration
313 curve (n=3) represents the linearity in detecting SP targets from 1 to 32 pM. A linear equation
314 obtained from the calibration curve was y = 736.37 log10 x + 22014. The coefficient
315 determination attained with the linear calibration was R2 = 0.9681. In comparison, Fig. 4f
316 shows the calibration curve attained by EIS measurements of the immunosensor using
317 diamond-conjugated SP. The linear equation attained from the analysis was y = 464.2 log10 x
318 + 8116. The determination coefficient obtained was R2 = 0.986. This emphasizes that the
319 diamond-conjugated SP interaction on anti-SP immobilized AuIDE has shown good sensitivity
320 and linearity in detecting SP in the low picomolar range. Nonetheless, SP without diamond
321 conjugation exhibited lower sensitivity, revealed by the lower R2 value attained. Diamond-
322 conjugated SP showed remarkable linear detection with a higher R2, indicating the high
323 sensitivity and performance of the developed immunosensor. A linear equation was evaluated
324 for computing the limit of blank (LOB) and the limit of detection (LOD) based on the
325 quantification reported [22], shown as:
326 LoB = mean blank + 1.645 (SD blank)
327 LoD = LoB + 1.645 (SD low concentration sample)
328 Based on the computational evaluation, the LOD of the developed immunosensor with
329 diamond-conjugated SP is 189 fM for approximately 116 fM LOB. The values obtained
330 through the linear regression plot and LOD calculations justified the high-performance
331 immunoassay incorporating nanodiamonds for targeting the SARS-CoV-2 SP on a gold
332 microelectrode. The immunoassay denoted a rapid immunoassay strategy with 10 minutes of
333 reaction time for probe-target interaction. Immediate EIS readings are an added advantage for
334 the prompt evaluation of immunosensor analytical performance. The propitiousness of
335 nanodiamonds is perceptible to addressing the high sensitivity of immunoassays, where the
336 LOD reaches the femto level of SP detection, without additional modification of the
337 nanodiamond chemistry and microelectrode sensing functionalization. The LODs attained in
338 the present research were compared with those in recent work on detecting SARS-CoV-2 SP
339 (Table 1).
340
341
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343 Immunosensor Selectivity and Specificity Analysis – Spiking in Human Serum
344 The prospect of the developed immunosensor for clinical SP detection was determined using
345 diamond-conjugated SP (1 fM to 100 pM) spiked in human serum. Fig. 5a shows the Nyquist
346 plot generated with different serum concentrations to determine the optimized serum dilution
347 for SP spiking. Based on the plot, a 1:100 dilution of serum was chosen for spiking with targets,
348 as it shows a negative trend (high value) in Z’ with the stock serum, which exhibits a low Z’
349 value. The impedance spectra shown in Fig. 5b indicate the detection of diamond-conjugated
350 SP spiked in human serum on AuIDE. A prominent detection range was observed at 0.25 – 1
351 pM of diamond-conjugated protein. The Z’ value attained by 250 fM human serum-diluted
352 diamond-conjugated SP (12 kΩ) was 3-fold higher than the target diluted in PBS (4 kΩ). The
353 significant elevation in the Z’ value indicates the large noise in the electrolyte system due to
354 the presence of multiple types of biomacromolecules in human serum. However, the linear
355 detection Nyquist plot observed in Fig. 5b represents the excellent performance of the
356 developed immunosensor using AuIDE in detecting diamond-conjugated SP in human serum.
357 The detection of SP spiked in human serum consists of large and intensive macromolecules
358 such as globulin (~66 kDa) and albumin (~150 kDa) [23]. This emphasizes the potential of the
359 developed immunosensor with diamond integration for clinical applications of infectious
360 disease diagnosis. The specificity of the immunosensor developed for detecting SP was
361 examined using HA and NCP as negative proteins. Fig. 5c shows the mean Z’ values revealed
362 by the immunosensor when tested with negative proteins, followed by positive diamond-
363 conjugated SP. The results denote that HA and NCP protein give nonspecific binding, as shown
364 in the schematic diagram insert in Fig. 5c, whereas diamond-conjugated SP gives specific
365 detection. The results justify the ideal condition of the developed immunosensor in recognizing
366 single and complementary target antigens. This condition is attained by the ideal
367 functionalization of the anti-SP antibody on a gold microelectrode, which is able to capture SP
368 in the presence of multiple macro- and micromolecules found in serum. In addition, the carbon
369 nanodiamond integrated with SP enhances the high-speed and high-density antibody-antigen
370 specific binding with its advantageous large surface area and its resistance to biofouling,
371 especially in the presence of other molecules. Hence, the results emphasized the significant
372 selection and sensitive detection of SARS-CoV-2 SP in serum and denote a promising strategy
373 for implementation in clinical trials.
374
375
376

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377 Surface Chemistry Analysis - Functionalization
378 The Z’ value against the analyte added in the step of surface chemistry on immunosensor
379 development is shown in Fig. 5d. Modification of AuIDE with GOPTS created a moderate
380 resistive layer with ~48 kΩ real part impedance. Following that, the immobilization of anti-SP
381 generated a high Z’ at ~88 kΩ, indicating the highly insulating characteristics of the antibody
382 and its high electron mobility in the presence of its active sites. The blocking of AuIDE with
383 ETA reduces the charge resistance, as it has blocked the nonreactive sites and reduced the
384 electron mobility on the sensing plate. The addition of a target on antibody-immobilized
385 AuIDE gives a higher Z’ (44 kΩ) than ETA (24 kΩ), indicating the specific binding of SP and
386 anti-SP and thus justifying the precision of surface chemistry in detecting SP.
387
388 Immunosensor and Spike Protein Binding: Validation
389 The stability of AuIDE used in the development of the immunosensor was validated through
390 reproducible EIS measurements (Fig. 6a). The Nyquist plot attained by testing three bare
391 AuIDEs indicates the Rs and Rct of the microelectrodes. The kick-off Rs value for the three bare
392 AuIDEs is the same [Fig. 6a(i)], which demonstrates the accuracy in reproducibility of the
393 devices. The variation observed with the Z’ of the three bare devices may arouse due to the
394 contaminants existing on the sensing plate. Moreover, the binding between recombinant SP
395 and anti-SP was validated through ELISA as a preliminary SP detection assay before biosensor
396 applications. Fig. 6b shows the absorbance readings recorded at 620 nm with tested SP targets
397 in the range of 1 pM to 100 nM concentrations. A gradual increase in the absorbance
398 measurement was notable from 1 pM to 1 nM SP, validating the specific binding of
399 recombinant SP and its antibody procured for this research. Maximum absorbance numbers
400 were recorded at 10 nM and 100 nM SP, which were ~3-fold higher than 1 nM SP, indicating
401 innumerable protein-antibody binding of the SARS-CoV-2 target. This result is in good
402 agreement with the dark blue color that appeared in the ELISA well (Fig. 6b; inset),
403 corresponding to the high amount of SP immobilized. In addition, the specificity of SP protein-
404 antibody binding was evaluated by immobilizing 1 nM NCP target in the ELISA well. The
405 negligible absorbance and its corresponding clear ELISA well evidenced the specific detection
406 of SP using procured protein and antibody for recognizing SARS-CoV-2 infectious disease.
407
408
409

12
410 Conclusion
411 The global threat caused by the SARS-CoV-2 infectious disease urges the development of
412 rapid, sensitive, and selective diagnostics in controlling and alleviating the pandemic
413 consequences. The presented research proposed a prognosticative electrochemical
414 immunoassay strategy using AuIDE in detecting SP by anti-SP antibody as a promising
415 strategy for diagnosing SARS-CoV-2 virus. The electrochemical immunoassays were
416 evaluated with and without nanodiamond conjugation to SP. It is apparent that the diamond-
417 conjugated SP showed a superior linear detection trend in the lower picomolar range of
418 concentration in comparison to the immunoassay performed without integration of diamond
419 nanomaterial. Linear detection ranging from 0.25 to 8 pM of diamond-conjugated SP
420 evidenced its outstanding sensitivity with a lower detection limit, which is equal to 189 fM and
421 a determination coefficient of R2 = 0.986. Although SP-antibody has revealed a linear detection
422 trend, the absence of diamond causes failure in recognizing the target at a very low picomolar
423 range of concentrations. The immunoassay without nanodiamond integration revealed lower
424 performance justified by the low R2 value. The developed immunoassay worked well with
425 diamond-conjugated SP spiked in human serum, which justifies the satisfactory performance
426 of the proposed strategy in terms of selective and stable detection. The presented work
427 emphasizes the significance of nanodiamond-conjugated SP in boosting the performance of
428 immunosensing strategies for rapid identification and quantification of SARS-CoV-2 virus
429 infection.
430
431 Acknowledgments
432 The authors acknowledge a grant (R.K130000.7343.4B617) under ASEAN University
433 Network Southeast Asia Engineering Education Development Network (AUN/Seed-Net)
434 Special Program for Research against COVID-19 (SPRAC) and a grant (9001‐ 00596) from
435 Universiti Malaysia Perlis.
436
437 Declaration of Competing Interest
438 All authors have no conflicts of interest to disclose.
439
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530

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531 Figure Legends
532
533 Fig. 1: Schematic illustration of SP detection. Anti-SP was used on a gold microelectrode as
534 a prognostic approach in diagnosing life-threatening SARS-CoV-2. Impedance
535 measurements of AuIDE in the presence of SP integration with and without
536 nanodiamonds were evaluated by electrochemical spectroscopy.
537
538 Fig. 2: Morphological analysis of AuIDE and nanodiamond. (a) Digital image of AuIDE.
539 HPM images of AuIDE (b) electrode fingers and (c) electrode junctions (d) Electron
540 microscopic image under SEM. Shows the dimensions of electrode and gaps
541 measured. (e) Sharp edges of electrode junction with AuIDE. (f)-(g) 3D optical
542 images of AuIDE. The height difference between the electrode and gap on the sensing
543 plate was visualized. (h) Roughness profile of AuIDE analyzed under a 3D profiler.
544 (i)-(l) FETEM images of nanodiamonds. Uniform nanosized particles were revealed
545 at various scales. (m)-(n) Spherical shape of diamond observed from the FESEM
546 images. (o) EDS analysis of diamond from FESEM. Diamond structure is inserted in
547 the plot.
548
549 Fig. 3: Electrochemical characterization of SP and anti-SP on AuIDE. (a) Impedance spectra
550 of AuIDE immobilized with anti-SP interaction. SP concentrations from 1 fM to 1
551 nM were used. (b) Impedance spectra with narrowed SP concentration. The target
552 concentration constricted at a detection range of 1 pM to 64 pM indicates the lowest
553 detection of SP on bioprobe-attached AuIDE at 1 pM. (c) Response of total
554 impedance against the frequency. The represented Bode plot reveals the equilibrium
555 response of SP detection observed at 23 Hz. The Fig. inset shows the overall
556 impedance response against swept frequency from 0.1 – 1 MHz. (d) Phase angle
557 versus the frequency plot of SP detection on AuIDE.
558
559 Fig. 4: Analytical performances of nanodiamond-incorporated AuIDE for detecting NCP. (a)
560 Nyquist plot shows the detection of diamond-conjugated SP with anti-SP
561 immobilized on AuIDE. (b) Bode plot constructed with the total impedance against
562 frequency. The active range of SP detection occurs at 30 Hz. (c) Phase angle versus
563 frequency of the immunosensing system. (d) Linear calibration lines of immunoassay
564 performed with and without nanodiamond integration. (e) Calibration plot for SP and

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565 anti-SP interaction. Detection ranging from 1 pM to 32 pM shows a determination
566 coefficient of R2 = 0.9681. (f) Calibration plot of AuIDE immunosensing with
567 diamond-conjugated SP. The linear detection range from 0.25 pM to 8 pM with R2 =
568 0.986 shows a limit of detection equal to 0.189 pM.
569
570 Fig. 5: Selectivity, and specificity assessments. (a) Impedance response of human serum and
571 its dilution on anti-SP immobilized AuIDE. (b) Detection of diamond-conjugated SP
572 diluted in serum on the developed immunosensor. (c) Cross-specificity analyses of
573 anti-SP immobilized AuIDE. The mean Z’ value of HA and NCP indicates the
574 nonspecific detection of negative proteins. (d) Mean Z’ against the steps of surface
575 chemistry performed on the AuIDE.
576
577 Fig. 6: (a) Reproducibility assessment conducted with three bare AuIDEs. (b) Validation of
578 SP and anti-SP interaction. The absorbance readings and inserted digital images of
579 the ELISA justified the SP interactions.

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580 Table 1: Recent reports on SARS-CoV-2 diagnosis using SP as the targeted antigen.

Detecting Electrode Nanomaterial Detection LOD Reference

strategy material range
Microfluidic Glass substrate Gold nanospikes 0.1 – 10 ng/mL 0.08 ng/mL [5]
assay
Lateral flow Screen printed Conducting 0 – 200 ng/mL 1.86x105 [7]
immunoassay electrodes polymer copies/mL
Electrochemical Screen printed Graphene sheets 100 fg/ml to 100 1.6 × 101 [24]
immunoassay electrodes pg/mL pfu/mL
Electrochemical Screen printed Nil 1 pg/mL to 10 1 pg/mL [25]
immunoassay electrode ng/mL
Electrochemical Indium tin oxide Gold 0.002 – 100 0.577 fg/mL [26]
immunoassay electrode nanoparticles pg/mL
Electrochemical Gold Carbon 1 pM – 8 pM 0.189 fM Current work
immunoassay nanodiamond
581

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Figure 1 Click here to access/download;Figure;Figure 1.TIF
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Figure 6 Click here to access/download;Figure;Figure 6.tif