Cobalt Oxide Porous Nanocubes Based Electrochemical

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Cobalt Oxide Porous Nanocubes Based Electrochemical Immunobiosensing of Hepatitis B Virus DNA in Blood Serum and Urine Samples Palanisamy Kannan, Palaniappan Subramanian, Thandavarayan Maiyalagan, and Zhongqing Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00153 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Analytical Chemistry

Cobalt

Oxide

Porous

Nanocubes

Based

Electrochemical

Immunobiosensing of Hepatitis B Virus DNA in Blood Serum and Urine Samples

Palanisamy Kannan,a* Palaniappan Subramanian,b Thandavarayan Maiyalagan,c* and Zhongqing Jiang d

a

College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, 314001, P. R. China. b

Department of Metallurgy and Materials Engineering, The Katholieke Universiteit Leuven (KU Leuven), Kasteelpark Arenberg 44 - Box 2450, 3001 Leuven, Belgium. c

Electrochemical Energy Laboratory, Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur 603203, India. d

Department of Physics, Key Laboratory of ATMMT Ministry of Education, Zhejiang Sci-Tech University, Hangzhou 310018, P. R. China.

* Corresponding Authors: [email protected] (Kannan); Tel: +86-19857386580; Fax: +86-573 83643264. [email protected] (Maiyalagan); Tel: +91-8220322594 1

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ABSTRACT In this work, we report a new biosensing platform for hepatitis B virus (HBV) DNA genosensing using cobalt oxide (Co3O4) nanostructures. The tunable morphologies of Co3O4 nanostructures such as porous nanocubes (PNCs), nanooctahedra (NOHs), and nanosticks (NSKs) are synthesized, and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction patterns (XRD), nitrogen adsorption/desorption isotherms (BET), and electrochemical impedance spectral (EIS) methods. The HBV probe DNA (ssDNA) is immobilized on the Co3O4 nanostructures through coordinate bond formation between nucleic acid of ssDNA and Co metal, which results in highly stable nanostructured biosensing platform. To the best of our knowledge, first time the target cDNA of HBV is detected using ssDNA/Co3O4 PNCs/GCE electrode by EIS method with a limit of detection (LOD) of 0.38 pM (S/N=3). Moreover, the ssDNA/Co3O4 PNCs/GCE has shown excellent specificity to HBV target cDNA as compared with non-complementary DNA, and 1- and 3-mismatch DNAs. Finally, we explore ssDNA/Co3O4 PNCs/GCE as potential electrode to test HBV DNA in blood serum and urine samples for practical applications.

KEYWORDS:

Co3O4

nanostructures,

hepatitis

B

virus,

spectroscopy, real samples, point-care-of-diagnosis.

2


electrochemical

impedance

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INTRODUCTION The accurate detection of specific nucleic acid sequences of viruses, bacteria and humans are of tremendous importance in the diagnosis of pathogenic and genetic diseases 1-5. Hepatitis B virus (HBV), potentially considered as an important pathogen is very harmful and cause liver diseases in humans including liver cirrhosis, hepatic inflammation, hepatocellular carcinoma and primary liver cancer

6,7.

Recently, vaccination has been effective, and considerably reduced its

prevalence; still 800 million people over the world are infected by HBV 6,8-10. DNA from HBV is identified as a primary biomarker for the detection of HBV infection, and detected in blood serum or dried blood spots in the range about ≥109 copies/ml, by polymerase chain reaction (PCR) analysis

11-13.

The ELISA method has been generally used for clinical identification of

HBV detection, though the use of antigens-antibodies, lack of serological responses, and its stability limits their practical application

14,15.

Other studies were based on optical detection of

HBV by isothermal amplification of virus DNA, which was performed in a microreactor

16.

Recently, nanomaterials based biosensing strategies have enabled substantial developments in the detection of HBV

9,17-24;

however, still some vital disadvantages were identified including

usage of complexed nanomaterials and its pretreatments, consumption of large sample volumes, instability, less sensitivity, time-consuming (at least more than 1-2 days) and requirement of specialized instrumentation. Thus, enhancing its sensitivity, stability and achieving rapid sensing responses, for the detection of HBV DNA is still highly challenging. Transition metal oxide nanoparticles (TMO NPs) have received extensive interest in recent advanced investigations; since the chemical and physical properties, crystalline phases, and catalytic activities are different from its size, morphology, and compositions

25-29.

Among

TMO NPs, cobalt oxide (Co3O4) belongs to the spinel crystal structure family because of its 3



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close-packing cubic array of oxide ions, and received tremendous attention in the comprehensive applications such as magnetic data storage systems, intercalation compounds for energy storage devices, and dynamic catalysts

30-32.

Particularly, cobalt oxides exists as three important

polymorphic forms such as cobaltous oxide (CoO), cobaltic oxide (Co2O3), and cobaltosic oxide (Co3O4); compared to other two polymorphs, Co3O4 is exclusively superior, because of its potential applications in supercapacitors, lithium ion batteries, and catalyst 33-36. Some rare effort has been made for the sensitive detection of staphylococcus aureus nuc gene sequence using ssDNA probe immobilized on sandwich nanocomposite (chitosan–Co3O4–Graphene/carbon ionic liquid electrode) substrate

37.

In addition, biomolecules such as hemoglobin and cholesterol

oxidase were immobilized on the Co3O4 nanostructures for sensing applications

38-40.

In this

work, we report morphology-controlled synthesis of Co3O4 nanostructures such as porous nanocubes, nanooctahedra, and nanosticks, and evaluated their performance as nucleic acid sensor for HBV gene by electrochemical impedance spectroscopy (EIS) method. The proposed EIS nucleic acid sensor achieved limit of detection (LOD) of 0.38 pM (S/N=3), and excellent practical applicability i.e., the detection of HBV gene in real samples such as human blood serum, and urine samples were also demonstrated. To the best of our knowledge, this is the first report on ultrasensitive detection of HBV gene using Co3O4 nanostructures based biosensing platform. EXPERIMENTAL SECTION Chemicals and Materials Cobalt(II) nitrate (Co(NO3)2.6H2O), cobalt(II) chloride (CoCl2.6H2O), sodium hydroxide (NaOH), sodium chloride (NaCl), sodium citrate (C6H7NaO7), urea (NH2-CO-NH2), potassium 4


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ferricyanide (K3[Fe(CN)6]), and potassium ferrocyanide (K4[Fe(CN)6]) were received from Merck, India. The Britton-Robinson buffer (BRB; pH=7.31) solution was prepared for electrochemical experiments according to the reported literature.41 All the DNA sequences and blood serum sample were purchased from Sigma-Aldrich, India and stored in the refrigerator at 20 C with extreme care. The DNA base sequences are given here: Probe DNA: 5’-AAT GTG CTC CCC CAA CTC CTC-3’; Target DNA: 5’-GAG GAG TTG GGG GAG CAC ATT-3’; 1base mismatch to Probe DNA: 5’-GAG GAG TTG GGG GAG CTC ATT-3’; 3-base mismatch to Probe DNA: 5’-GAC GAG TTG CGG GAG CTC ATT-3’; Non-complementary to Probe DNA: 5’-AAA AGG TGT AAG CGT TTG CCG-3’. The target DNA used for proposed DNA biosensor was HBV gene sequences: 21-base target DNA sequences (target ssDNA, namely a 21-base fragment of HBV gene sequences). The probe DNA sequences were completely complementary with the target DNA. The mismatch DNA was indicated as 1-mismatched, and 3-mismatched DNA bases (underlined) used in this study. All other chemicals used in this investigation were of analytical grade. Millipore water (18.2 MΩ.cm) was obtained from an Elix Millipore system and used to prepare the solutions in this investigation. Synthesis of Co3O4 Porous Nanocubes, and Nanooctahedra Co(NO3)2 6H2O (40 mmol) was dissolved in 20 mL of aqueous NaOH (10 mmol) solution, constantly stirred for 10min to obtain homogeneous solution, then transferred into 40 mL Teflon lined stainless steel autoclave and heated at 180°C for 4 hr. The resulting product was filtered, washed with Millipore water and ethanol for several times, then dried under vacuum at 60oC for 2 hr, and finally the product was calcined at 350oC for 3 hr to obtain Co3O4 porous

5



nanocubes. For the synthesis of Co3O4 nanooctahedra, Co(NO3)2.6H2O (80 mmol) and NaOH (5 mmol) were used without altering other synthesis procedure of Co3O4 porous nanocubes. Synthesis of Co3O4 Nanosticks CoCl2.6H2O (40 mmol) was dissolved in 10 mmol of 20 mL aqueous urea solution, constantly stirred for 10 min to obtain homogeneous solution, then transferred into 40 mL Teflon lined stainless steel autoclave and heated at 180°C for 4 hr. The resulting product was filtered, washed with Millipore water and ethanol for several times, then dried under vacuum at 60 oC for 2 hr, and finally the product was calcined at 350 oC for 3 hr to obtain Co3O4 nanosticks. Characterizations The morphological progress of Co3O4 nanostructures were characterized by field emission-scanning electron microscopy (FE-SEM JEOL JSM-7100F, Japan), operating at 10 kV accelerating voltage. The high-resolution transmission electron microscopy (HR-TEM) images were performed using a JEOL JEM 3010 instrument (Japan) with a tungsten filament at an accelerating voltage of 200 kV. The Co3O4 samples were prepared by dropping 3 μl of a colloidal solution onto a carbon-coated copper grid surface. The crystallographic nature was obtained by powder X-ray diffraction technique (XRD, Shimadzu XRD-6000, Japan, Ni filtered CuKα (λ=1.54 Å) radiation operating at 30 kV/40 mA). The 2θ range from 10 to 80 was used in steps of 0.02 with a count time of 2 sec. The specific surface area of the Co3O4 nanostructures was determined using the classic BET method (the Brunauer–Emmett–Teller isotherm). The BET isotherm is the basis for determining the extent of nitrogen adsorption on an exposed surface. The zeta potentials of prepared Co3O4 nanomaterials were determined by electrophoresis light scattering analysis with a Zetasizer Nano-ZS (Malvern Instruments Ltd., UK). The Co3O4 6


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nanostructures were dispersed in Britton-Robinson buffer (pH=7.31), and the obtained values given here. +7.61, +6.42, and +7.73 for Co3O4 porous nanocubes, nanooctahedrons, and nanosticks structures. All the electrochemical measurements were conducted by using computer controlled Autolab PGSTAT30 (Metrohm Autolab, Netherlands) electrochemical workstation with a conventional three-electrode system, where GCE (3 mm diameter), Pt wire, and Ag/AgCl (KCl sat) were used as working, counter, reference electrodes, respectively. The electrochemical impedance spectroscopy (EIS) studies were performed in BR buffer solution containing 1mM Fe(CN)63-/4- (pH=7.31). All electrochemical experiments were performed under nitrogen atmosphere. Preparation of ssDNA Probe Modified Electrode The Co3O4 (porous nanocubes, nanooctahedra, and nanostickes) nanostructures modified GC electrode was obtained as follows: Firstly, glassy carbon (GC) electrode with mirrorpolished surface was obtained using alumina powder (1 µm and 0.3 µm size) followed by ultrasonication in ethanol and Millipore water for 10 min. Secondly, 90 µL of Co3O4 nanostructures sample was mixed with 10 µL of 5 wt% of Nafion solution by ultrasonication for 10 min. Then, 5 µL of the as-prepared Co3O4 nanostructures were drop-casted on the GC electrode surface and then dried under nitrogen atmosphere for further modification steps (Scheme S1). Using BR buffer solution, 5 µM probe DNA (ssDNA) was prepared and then 5 µL probe DNA (ssDNA) solution was dropped onto the GCE/Co3O4 PNCs surface. The probe ssDNA modified GCE/Co3O4 PNCs was dried under nitrogen atmosphere at room temperature for 2 hr and then gently washed with BR buffer solution to remove un-immobilized ssDNA. The ssDNA probe modified electrode was denoted as GCE/Co3O4 PNCs/ssDNA. After that, the 7



GCE/Co3O4 PNCs/ssDNA electrode was washed with Millipore water and rinsed with BR buffer solution before hybridization. The hybridization assay reaction was carried out by dropping 5 µL of hybridized buffer solution (0.5 mM sodium chloride and 0.05 mM sodium citrate) containing different concentrations of the target cDNA on the GCE/Co3O4 PNCs/ssDNA electrode at 37 °C and kept undisturbed for 30 min. Then, the GCE/Co3O4 PNCs/ssDNA/cDNA electrode was washed with BR buffer solution to remove the un-hybridized cDNA and used as working electrode for HBV DNA sensing (Scheme S1). The simialar hybridization procedure was adopted for preparing GCE/Co3O4 PNCs/ssDNA/ncDNA electrode (non-complementary DNA; ncDNA) for control, and GCE/Co3O4 NS/ssDNA/smDNA (single-base mismatched DNA; smDNA), GCE/Co3O4 NS/ssDNA/tmDNA (three-base mismatched DNA; tmDNA) electrodes used for selectivity analysis of this proposed biosensor. Further, the other Co3O4 nanostructures such as nanooctahedra and nanosticks modified electrodes are represented as GCE/ Co3O4 NOHs and GCE/Co3O4 NSs, and above similar DNA modifications were adopted for comparison studies.

RESULTS AND DISCUSSION Morphological and Crystal Structure Characterizations The morphological evaluations of the as-prepared Co3O4 nanostructures were assessed by both scanning and transmission microscopic analysis. We have shown the time-dependent formation of Co3O4 porous nanocubes morphology in Figure 1. As can be seen in Figure 1A, at the initial stage of reaction, the nuclei and growth continued slowly to yield numerous quasispherical or ill-defined cubic nanostructures and irregular array of Co(OH)3 nanoseed particles with a size of 30 ± 8 nm, because of the huge structural anisotropy of Co(OH)3 nuclei, and thus 8


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many anisotropic Co(OH)3 nanoparticles were readily formed during the hydrothermal treatment. The small nanoparticles formed in the reaction solution were highly active and continued to selfaggregate to reduce their surface energies. Under this circumstance, quasi-spherical or ill-defined cubic nanostructures/irregular array of nanoseed particles formed larger assembly by minimizing their surface energy via oriented attachment mechanism (Figure 1B). Then Co(OH)3 was transformed into Co3O4 nanoparticles via rapid dehydration process, and followed by rapid and controlled fusion of neighboring particles, resulting in cubic morphology with characteristics of surface defects and porous outer surface (Figure 1C). Further, Ostwald ripening and surface reconstruction eventually results in the formation of larger well-defined Co3O4 porous nanocubes with the length(s) of 90 ± 5 nm (Figure 1D)

42-44.

Interestingly, after calcination at 350 C for 3

hr, surface of the Co3O4 nanocubes became rough and rich porosity was developed (inset of Figure 1D). At the same time, the original cubic morphology was significantly retained and highly stable, which is the key foundation for highly sensitive electrochemical sensing platform 45.

On the other hand, we have increased the concentration of Co(NO3)2 6H2O (80 mmol) and

decreased the concentration of NaOH (5 mmol) in the synthesis procedure, which resulted in Co3O4 nanooctahedra morphology with a size of 130 ± 16 nm from the center point and 105 ± 7 nm from the sidewalls (Figure 2A and 2B). At the beginning stage, numerous polyhedral and spherical nanoparticles were formed with high index crystallography planes; then the reaction continues to grow polyhedral nanoparticles into different directions due to insufficient amount of OH- ions in the reaction mixture. Thus, the conversion to Co(OH)3 was low and slow, which resulted in the formation of pre-octahedral morphology. Then definite formation of preoctahedral morphology of the nanoparticles appeared, and it became prevailing to grow Co3O4 nanooctahedra via self-assembly and subsequent Ostwald ripening mechanisms 46-48. 9



Figure 1. Time-dependent SEM images (1 hr (A), 2 hr (B), 3 hr (C), and 4 hr (D)) of Co3O4 nanocubes structure obtained by hydrothermal process. The rich porous structures were obtained by 3 hr calcination treatment of Co3O4 nanocubes structure at 350 C.

Next, we have used the precursors such as CoCl2 6H2O (40 mmol), and urea (10 mmol) instead of Co(NO3)2 6H2O and NaOH in the synthesis procedure without changing the experimental conditions, and the corresponding SEM images of the sample are shown in Figure 2C and 2D. The SEM images exhibited well-defined stick-like Co3O4 nanostructures with a length of 600-800 nm and the width of 100 ± 8 nm, which is consistent with the recently reported nanosticks morphology 49,50. In the early stage, the urea molecule adsorbs on the hydrated cobalt salt and initiated the growth of nanoparticle nuclei, then simultaneously aggregated via randomaggregation mechanism, and resulted in networked sheet-like morphology while the reaction continues. Finally, the CO2 decomposition from urea molecule acted as the soft-template to grow 10


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extended stick-like morphology. It is important to point out here that our procedure establishes a widespread protocol for synthesizing variety of Co3O4 nanostructures without adopting change in the reaction conditions, injection of foreign reagents, no external surfactant and templates.

Figure 2. Low (A, C), and corresponding high (C, D) resolution SEM images of Co3O4 nanooctahedra and nanostickes structures obtained by hydrothermal and calcination treatments. The TEM morphological characterization of Co3O4 nanostructures is displayed in Figure 3. The low magnification TEM image of Co3O4 nanostructures showed well-defined cubic nanoparticles (Figure 3A). High-resolution magnification TEM image (Figure 3B) shows all Co3O4 nanocubes made by the porous nanoparticle assembly (surface and interior portions) and possess perfect sharp edges (indicated in the dotted marks), corners and well-defined faces with the uniform size of 90 nm. Further, the close view from HR-TEM image shows that the observed d-spacing fringes correspond to the (400) plane (d = 0.207 nm) of spinel Co3O4 (Figure 3C). Fast Fourier 11



Transform (FFT) pattern of Co3O4 nanocubes exposed the (112) high-index facet and showed a single crystal feature.51,52 All the FFT spots can be indexed along [100] zone axis of spinel Co3O4 nanocubes (Figure 3C; inset). The lattice fringe can be observed clearly, and the interplanar distances of adjacent lattice fringes was about 0.26 nm (2.6 Å), which is in good agreement with the d value of (040) plane of cubic Co3O4 nanocrystal. Besides, the sharp diffraction spots of the corresponding FFT pattern reveal the pure crystalline nature of Co3O4 nanocubes. As a result, the high-index faceted porous nanocubes would further facilitate the catalysts to expose more edges and coordinated surface atoms, serving as the active sites to the efficient catalytic process.51,52 Thus, the as-synthesized nanostructures obtained via simple hydrothermal method were of Co3O4 nanocubes morphology with pure crystalline nature. Figure 3D displayed the TEM image of Co3O4 nanostructures surrounded by smooth surfaces and has a perfect octahedral morphology with sharp corners and edges with fine porous structures. The average particle size measured was about 130 nm and 100 nm from its center portion and sidewalls, respectively, which is in good agreement with SEM images. Further, the TEM image of an individual Co3O4 octahedral nanoparticle taken along the (111) direction in the TEM illustrates an octahedral shape that fits well with the hexagon (bounded by the dashed-yellow lines). Figure 3E showed the TEM image of Co3O4 stick-like nanoparticles with a length of 800 nm and the width of 100 nm and were composed of small nanospheres with numerous porous on its surface (dashed-green lines). From the side view, top of the nanosticks with solid stick-like surface were observed, different from hollow nanotubes and nanorods morphology.

12


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Figure 3. Low to high-resolution TEM images of Co3O4 porous nanocubes (A-C), and the TEM image of other Co3O4 nanostructures such as nanooctahedra (D), and nanostickes (E). We further examined the crystalline nature of as-synthesized Co3O4 nanostructures by XRD method, and patterns of the three Co3O4 nanostructures were shown in Figure 4. All the diffraction peaks could be indexed on the standard PDF card (JCPDS No. 09-0418). This means that Co3O4 nanocube (Figure 4A), octahedron (Figure 4B), and nanosticks (Figure 4C) morphologies belong to the face-centered-cubic (fcc) space group with a lattice constant of 8.084 Å and no other peaks were related to CoO which is consistent with the previously reported literatures

53,54.

The patterns such as 18.9, 30.8, 36.7, 44.7, 55.6, 59.3, and 64.9 were

corresponding to the planes of 111, 220, 311,400, 422, 511, and 440, respectively. Notably, ratio of the relative peak intensities in the rich high index planes/facets (511) and (440) is higher for Co3O4 nanocubes structures (Figure 4A; indicated as dashed circle) than the nanooctahedrons, and nanosticks like morphologies, which reveals that the Co3O4 nanocubes structures were 13



possesses high-index facets, and useful for electrochemical sensing and catalysis applications.28,51,52 Nitrogen adsorption-desorption isotherms of Co3O4 nanostructures are displayed in Figure 4D, and the inset illustrate the respective Barrett-Joyner-Halenda (BJH) pore size distribution plots. The isotherms were indicated as type IV model with H3 hysteresis loop, which is characteristic of porous nanostructures. The BJH pore size distribution indicates that all the Co3O4 nanostructures contains mesoscale pores i.e., 7.4 nm for nanocubes, 7.1 nm for nanooctahedra, and 10.8 nm for nanosticks morphologies. The Brunauer-Emmett-Teller (BET) surface areas and pore volumes of the Co3O4 nanostructures were 96 m2/g and 63 mm3/g, and 54 m2/g, which are corresponding to the Co3O4 nanocubes, nanooctahedra and nanosticks-like morphologies, respectively. Such high surface area of Co3O4 nanocubes was mainly due to the existence of numerous mesopores in nanostructures as compared with nanooctahedra, and nanosticks. We have characterized DNA functionalization (coordinate bond interaction) on the nanoporous surface of Co3O4 nanocubes by FT-IR spectroscopy method. As can be seen, the FTIR spectra of Co3O4 nanocube sample shows two fingerprint characteristics peaks at 577, 668 cm-1, corresponding to the spinel structure of Co3O4 (Figure 4E, curve “i”) 55. The other peaks at 1548 and 1636 cm-1 were assigned to the vibration bands of H2O and OH- ions, which interacted with Co metal in the nanostructures. After functionalization of DNA on the surface of Co3O4 nanocubes, FT-IR spectra showed additional sharp intense peak at 1398 and a broad peak at 1102 cm-1 corresponding to asymmetric stretching vibration of PO4 backbone (P=O) and C-O-H inplane bending vibration of carbohydrate molecules in the DNA molecules, respectively.56 In addition to the above peaks, a new peak at 941 cm-1 was characteristically ascribed to the vibrational bond of Co-O-P functionalities (red color dotted circle in Figure 4E, curve “ii”). The oxygen species in the phosphoric group of ssDNA was reacted with the Co element in the Co3O4 14


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nanostructures to form a stable coordinate bond during the nucleic acid immobilization process. Thus, the FT-IR spectral analysis clearly evidenced that probe DNA was successfully attached to the porous Co3O4 cubic nanostructures.

Figure 4. XRD of Co3O4 porous nanocubes (A), nanooctahedra (B), and nanostickes (C) morphologies, and corresponding BET analysis (D). The FT-IR spectra of Co3O4 porous nanocubes (E-i), and ssDNA functionalized on Co3O4 porous nanocubes (E-ii). 15



The X-ray photoelectron spectroscopy (XPS) was carried out for further confirming the attachment of probe DNA (ssDNA) on the Co3O4 PNCs surface. Figure S1A, and B displayed the wide scan survey XPS spectra of Co3O4 PNCs and Co3O4 PNCs-ssDNA samples. As can be seen the survey XPS survey spectra of Co3O4 PNCs-ssDNA sample, noticeable O1s peak intensity changes were observed at O1s peak due to the attachment/interaction of ssDNA with the surface of Co3O4 PNCs.57 Unlike Co3O4 PNCs (Figure S1C), the core-level O 1s XPS spectra of Co3O4 PNCs-ssDNA sample showed two peaks; the main peak at 529.3 eV in the O 1s spectrum corresponds to oxygen species in the nanoparticle crystal lattice (Olatt), whereas the peak located at 530.4 eV can be ascribed to the adsorbed oxygen species on the Co3O4 PNCsssDNA sample (Figure S1D).58,59 In addition, N 1s (398.7 eV) belongs to the amino functionalities in the ssDNA, and O-Auger (971.6 eV) peaks were also observed in Co3O4 PNCsssDNA sample, which clearly confirms the attachment of ssDNA on the Co3O4 PNCs surface. EIS Detection of HBV DNA Using Co3O4 Nanostructured Electrodes Despite numerous common features in the pathogenesis of HBV- related diseases, this virus obviously differs in their virological properties, immune escape and survival strategies. Hence, there is an urgent demand to find a simple, rapid and sensitive method to detect DNA sequence of HBV. Electrochemical methods have outstanding advantages including provision of rapid and effective response, analysis simplicity, quantitative application and miniaturization in developing sensitive sensor platforms. Electrochemical impedance spectroscopy (EIS) is a highly sensitive technique used to monitor the resistive properties and is sensitive to the interfacial electron transfer as referenced for the characterization of biomolecules-functionalized electrodes and biocatalytic transformation at the electrodes 60. We have monitored the DNA immobilization 16


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and hybridization steps by EIS analysis in 1mM K3[Fe(CN)6//K4[Fe(CN)6] in Britton-Robinson (BR) buffer (pH = 7.31). Before DNA modification process, we have conducted the EIS analysis of Co3O4 nanostructured electrodes in 1mM K3[Fe(CN)6//K4[Fe(CN)6] in BR-Buffer (pH = 7.31) under optimized conditions. Figure 5A showed the impedance changes from unmodified GCE to Co3O4 nanostructures modified GCE. The complex impedance responses were derived as the sum of real and imaginary components (Z' and Z'') in the system. The charge transfer resistance (Rct) mainly depends on the dielectric and insulating features at the GCE/electrolyte interface. Thus, to obtain the accurate and more detailed information of the EIS, a simple equivalent circuit model (Inset in Figure 5A) was used to fit the obtained results. The semicircle portion at higher frequency region of Nyquist plot represented the electron transfer limited process and the linear portion at lower frequencies could be accredited to the rapid diffusive limiting step of the electrochemical process

61.

As shown in Figure 5A, curve “a”, the unmodified GCE showed a

semi-circle portion of EIS response with a diameter of 282.6  (Figure 4A; curve a), implying Ret of the redox probe. When Co3O4 nanostructures were immobilized on the GCE surface, the EIS of the resulting nanostructured GCE electrodes showed an obvious decrease in the semicircle portion with a diameter of 183.2 , 126.8  and 84.9  for Co3O4 NPCs, Co3O4 NOHs, and Co3O4 NSs respectively, implying rapid electron transfer in the redox probe (Figure 4A; curves b-d). The obtained EIS responses were attributed to the excellent conductive properties of Co3O4 nanostructures, which would highly enhance the electron transfer of the electrochemical redox probe. After probe DNA (ssDNA) modification step, changes observed in the Rct value is a strong evidence for the successful immobilization of ssDNA onto the Co3O4 nanostructures surface. The larger Rct values i.e., 301.5 , 338.2 , and 381.3  were observed for Co3O4 NPCs, Co3O4 NOHs, and Co3O4 NSs, respectively (Figure 5B; curves b’-d’), 17



demonstrated that ssDNA immobilization plays a key role in the electron transfer-limited process. Considerably, Co3O4 NPCs/GCE showed almost five folds (Figure 5B; curves d’) higher Rct values compared to the other two Co3O4 nanostructured electrodes; i.e., ssDNA prevented redox reaction due to the following reasons (i) the existence of numerous mesopores on the surface of Co3O4 NPCs with higher active surface area (see the BET analysis), accessing large amount of ssDNA for specific dsDNA detection (ssDNA/Co3O4 NPCs/GCE); (ii) charge repulsion between negatively charged phosphate backbone of oligonucleotide and [Fe(CN)6]3-/4leading to a reduced electron transfer property at the electrode surface i.e., Co3O4 NPCs have shown higher repulsion due to the large adsorption of ssDNA its surface, as well as specific detection of dsDNA.

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Figure 5. The EIS responses obtained for bare GCE (A-curve a), Co3O4 NSs/GCE (A-curve b), Co3O4 NOHs/GCE (A-curve c), and Co3O4 PNCs/GCE (A-curve d) in BR buffer solution (pH=7.31) containing 1 mM [Fe(CN)6]3-/4-. The EIS responses obtained for ssDNA immobilized on Co3O4 NSs/GCE (B-curve b’), Co3O4 NOHs/GCE (B-curve c’), and Co3O4 PNCs/GCE (Bcurve d’) and corresponding complementary DNA (cDNA; 100 pM) functionalized electrodes (c’’ to d’’) in BR buffer solution (pH=7.31) containing 1 mM [Fe(CN)6]3-/4-. The EIS responses obtained on ssDNA/Co3O4 PNCs/GCE in BR buffer solution (pH=7.31) containing 1 mM [Fe(CN)6]3-/4- with various concentration of HBV DNA from 0.001 nM to 1000 nM (C), and corresponding calibration sensor curve (D). The LOD of proposed HBV sensor was obtained from inset plot of Figure 5D.

Further, to examine the hybridization process, the ssDNA modified Co3O4 nanostructured electrodes were monitored by EIS measurements. As shown in the Figure 5B, the semicircle portion in the Nyquist plot of ssDNA modified Co3O4 nanostructured electrodes were enhanced significantly while it was hybridized with 100 pM complementary DNA (cDNA) molecule, demonstrating the formation of double-stranded DNA (dsDNA) nanohybrids on the Co3O4 nanostructured electrode surface (Figure 5B; curves b”-d”). The enhancement of Rct was due to further increase of negative charges on Co3O4 nanostructured electrode surfaces (especially Co3O4 NPCs interface; 790.1 ), which significantly hindered the electron transfer process. Next, we have tested the wide range applicability of the proposed sensor using ssDNA/Co3O4 NPCs/GCE vs. the concentrations of HBV cDNA, and obtained results are presented in Figure 5C and D. Thus, the sensitivity of ssDNA/Co3O4 NPCs/GCE was assessed by analyzing the dependence of EIS signals upon the addition of cDNA concentrations from 0.001 nM to 1000 nM and displayed corresponding calibration curve with a correlation coefficient of 0.9891 (Figure 5C and D). Using the inset of Figure 5D, the LOD of proposed EIS based HBV DNA biosensor was about 0.38 pM using 3 (S/N=3), where 3 was the standard deviation value from the 12 blank experiments. The proposed Co3O4 nanostructured electrode displayed superior LOD 19



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as compared with other transition metal/metal oxide or non-precious nanomaterials based HBV sensors (Table 1).

Table 1 Comparison of proposed HBV biosensor with other transition metal/metal oxides or non-precious nanomaterials based HBV sensors. Electrode Materials

Linear Range

Detection Method

LOD

Cu2O Hollow Microspheres

0.1 nM to 1 µM

DPV

0.1 nM

62

ꞵ-Cyclodextrin/Magnetic Nanoparticles

1.505 pM to 0.3010 nM

DPV

0.993 pM

63

4,4’-Diaminoazobenzene (4,4’-DAAB)/

79.4 nM to 1.58 µM

DPV

11 nM

64

25 µg/mL to 200 µg/mL

DPV

0.21 µM

65

10 nM to 500 nM

DPV

1 nM

22

0.001 nM to 1000 nM

EIS

0.38 pM

Multiwalled Carbon Nanotube (MWNT)

Single-Walled Carbon Nanotubes

Graphene Quantum Dot (GQD)

Co3O4 Porous Nanocubes

20


References

This Work

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Selectivity, Reproducibility, and Stability of HBV DNA Biosensors The selectivity of the proposed HBV DNA biosensor was verified by exposing the biosensor electrodes to three types of target sequences, including single base-mismatch DNA (smDNA), three base-mismatch DNA (tmDNA), and non-complementary DNA (ncDNA), and the results are shown in Figure 6A,and B. Compared to the cDNA sequence (Figure 6A; curve c), the EIS changes of smDNA (Figure 6A; curve d), tmDNA (Figure 6A; curve e), and ncDNA (Figure 6A; curve f) sequences were gradually decreased due to low or no efficient hybridization on ssDNA probe on Co3O4 NPCs/GCE. Thus, the electrochemical EIS method based biosensor was highly effective at discriminating between the complementary DNA sequence and mismatched DNA. These results suggest that the proposed HBV DNA biosensor unveiled excellent selectivity. The ssDNA/Co3O4 NPCs/GCE biosensor electrode was highly stable upon continuous cycling analysis of in the presence of cDNA with a concentration of 100 pM (Figure S2A). Next, we have tested mechanical stability of as-prepared ssDNA/Co3O4 NPCs/GCE (Figure S2B) by washing with BR buffer solution and followed by ultrapure water. No changes in the EIS responses were noticed for both set of washings indicating that the high stability of fabricated HBV biosensor electrode. In addition, the repeatability of ssDNA/Co3O4 NPCs/GCE was also evaluated using four independent HBV DNA biosensor electrodes prepared under similar experimental procedure and tested in presence of cDNA with a concentration of 100 pM (Figure S2C). The obtained relative standard deviation (RSD) on the reproducibility of ssDNA/Co3O4 NPCs/GCE electrode was about of 6.34%. Thus, the excellent repeatability and reproducibility significantly confirmed that the ssDNA/Co3O4 NPCs/GCE is potentially sensitive and excellent candidate for HBV DNA biosensor application. Further, long-term stability was another crucial factor of the proposed HBV biosensor in practical application, which can be 21



accessed by EIS measurements. The EIS response was retained as 93.2% from its initial response after 14 days storage at 3-4ºC, suggesting the satisfactory long-term stability of the proposed biosensor.

Figure 6. The EIS responses obtained for bare GCE (A-curve a), ssDNA/Co3O4 PNCs/GCE (Acurve b), cDNA/ssDNA/Co3O4 PNCs/GCE (A-curve c), smDNA/ssDNA/Co3O4 PNCs/GCE (Acurve d), tmDNA/ssDNA/Co3O4 PNCs/GCE (A-curve e), and ncDNA/ssDNA/Co3O4 PNCs/GCE (A-curve e) in BR buffer solution (pH=7.31) containing 1 mM [Fe(CN)6]3-/4- solution and corresponding bar plots (B). The EIS responses obtained on Co3O4 PNCs/GCE (C-curve a), ssDNA/Co3O4 PNCs/GCE (C-curve b), and various concentrations of cDNA i.e., 100 pm (Ccurve c), 300 pM (C-curve d), 600 pM (C-curve e), and 1000 pM (C-curve f) in BR buffer solution (pH = 7.31) containing 1 mM [Fe(CN)6]3-/4- and blood serum samples. The EIS responses obtained on ssDNA/Co3O4 PNCs/GCE in BR buffer solution (pH=7.31) containing 1 mM [Fe(CN)6]3-/4- and urine samples with cDNA 100 pM (D-curve a), 300 pM (D-curve b), 600 pM (D-curve c), and 1000 pM (D-curve d).

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Detection of HBV DNA in Real Samples Using Co3O4 Nanostructured Electrodes Accurate detection of HBV DNA in blood serum or urine samples is significant for early diagnosis of many infectious diseases (see above). A recovery experiment was conducted to verify the validity of proposed Co3O4 NPCs GCE biosensor electrode in the presence of HBV DNA. The experiments were carried out by using four different blood serum and urine samples, i.e., 0.50 ml of blood serum or urine samples were mixed with 4.5 mL of BR buffer containing 1mM Fe(CN)63-/4- and respective amount of (100, 300, 600, and 1000pM) HBV DNA were used for the experiments to validate the results in the practical applications. The experimental results are summarized in Table S1 and S2. Thus, our proposed Co3O4 NPCs GCE biosensor electrode showed satisfactory recovery results in the ranges between 93.73% and 96.2% with RSD values from 3.8% to 6.27% (n=4), and 91.47% and 94.3% with RSD values from 5.70 to 8.53% (n=4) while using blood serum and urine samples, respectively indicating the potential use in clinical applications. CONCLUSIONS In summary, we have demonstrated a simple and highly sensitive electrochemical HBV immunobiosensor using Co3O4 nanostructured electrodes for the first time. Highlights of our investigations were summarized as follows: (i) Co3O4 NPCs have numerous nanopores with higher active surface area (see the BET analysis), allowing access to large amount of ssDNA on their surface to initiate efficient hybridization with cDNA; (ii) presence of rich high indexed facets in the Co3O4 NPCs nanostructures displayed highly sensitive detection of HBV DNA when compared to the other morphologies such as Co3O4 NOHs and Co3O4 NSs under identical experimental conditions; (iii) Co3O4 NPCs showed higher sensitivity, wide linear response of 23



HBV DNA concentration ranging from 0.001 nM to 1000 nM, with a lowest LOD of 0.38 pM (S/N=3); and (iv) practical application of Co3O4 NPCs was demonstrated by determining the concentration of HBV DNA in blood serum and urine samples. Our method holds great promise for further applications in early diagnosis of HBV-related infectious diseases. AUTHOR INFORMATION Corresponding Authors: [email protected] (Kannan) [email protected] (Maiyalagan) NOTES The authors declare no competing financial interest. ASSOCIATED CONTENT Supporting Information Available: Supporting Information is available free of charge from the Analytical Chemistry home page (http://pubs.acs.org/journal/ancham). Figure S1. XPS spectra of Co3O4 porous nanocubes before and after the immobilization of probe DNA (ssDNA); Figure S2. EIS cycle stability, washing stability and reproducibility analysis of Co3O4 PNCs modified electrodes; Scheme S1. Schematic representation and EIS detection of the as-prepared HBV DNA biosensor; Table S1. Recovery table for HBV DNA biosensor in blood serum samples; Table S2. Recovery table for HBV DNA biosensor in urine samples.

ACKNOWLEDGEMENTS PK thanks the Jiaxing University for providing Start-up Research Grant. REFERENCES

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Graphical Abstract

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Cobalt Oxide Porous Nanocubes Based Electrochemical

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