key: cord-0999988-6ig1q2as authors: Achadu, Ojodomo J.; Takemura, Kenshin; Khoris, Indra Memdi; Park, Enoch Y. title: Plasmonic/magnetic molybdenum trioxide and graphitic carbon nitride quantum dots-based fluoroimmunosensing system for influenza virus date: 2020-06-22 journal: Sens Actuators B Chem DOI: 10.1016/j.snb.2020.128494 sha: b530b17fd94100059734e23aed41cfd3b7d32aa1 doc_id: 999988 cord_uid: 6ig1q2as A novel magnetic/plasmonic-assisted fluoro-immunoassay system is developed for the detection of influenza virus using magnetic-derivatized plasmonic molybdenum trioxide quantum dots (MP-MoO(3) QDs) as the plasmonic/magnetic agent and fluorescent graphitic carbon nitride quantum dots (gCNQDs) as the monitoring probe. Specific antibody against influenza A virus was conjugated onto the surface of MP-MoO(3) QDs and gCNQDs, respectively. In the presence of influenza A virus (as the test virus), a core-satellite immunocomplex is formed between the antibody-conjugated nanomaterials (Ab-MP-MoO(3) QDs and Ab-gCNQDs) and their interaction resulted in the modulation and gradual enhancement of the fluorescence intensity of the detection probe with the influenza virus concentration-dependent increase. In addition, PL change without influenza A virus was not observed. Limits of detection of 0.25 and 0.9 pg/mL were achieved for Influenza virus A/New Caledonia (20/99/IVR/116) (H1N1) detection in deionized water and human serum, respectively. Clinically isolated influenza virus A/Yokohama (110/2009) (H3N2) was detected in the range of 45 – 25,000 PFU/mL, with a limit of detection ca 45 PFU/mL (as opposed to a minimum of 5000 PFU/mL for a commercial test kit). This developed biosensor provides a robust, sensitive as well as a selective platform for influenza virus detection. The recent outbreak of a novel coronavirus disease (COVID-19) and its potential for adverse effect on the global economy is an example of how quickly new infectious diseases can arise and spread [1] . Outbreaks such as this can unexpectedly cause demands for clinical knowledge, rapid diagnostic strategies, and epidemiological studies before a pandemic occurs. The rapid and ultrasensitive detection of infectious diseases is critical for the prevention and/or control of outbreaks. Therefore, it is crucial to continually deploy innovative materials to further develop the practical applications of biosensing systems capable of detecting biomolecules (DNAs, RNAs, proteins and virus particles) and of diagnozing other potentially harmful infectious diseases. The deployment of carbon-based quantum dots as optical probes has received a tremendous boost due to their excellent optical properties, biocompatibility and low cost of preparation [2] [3] [4] [5] [6] [7] . As carbon-based nanomaterials, graphitic carbon nitrides QDs (gCNQDs) possess excellent optical properties comparable to traditional heavy metal-based QDs, and are not as toxic. So, they are becoming competitive materials in nanosensors development [8] [9] [10] [11] . The presence of graphitic nitride-N-atoms introduces a different kind of "surface state" which impacts some semiconductor-like properties [12] [13] [14] . This feature has attractively bestowed an edge on gCNQDs and their derivatives for utilization in optical and electrochemical-based sensing [15, 16] . For instance, a robust photo-J o u r n a l P r e -p r o o f electrochemical immunosensor was designed by Sun et al. for the sensitive detection of avian viruses using hybrid of gold nanoparticles (AuNPs) and gCNQDs coupled to CdTe QDs [10] . Wang et al. reported a photo-electrochemical platform for methylated RNA detection using gCNQDs/CdS hybrid [17] . Pang et al. reported the fabrication of pcDNA3-HBV nanobiosensor using gCNQDs-sensitized TiO 2 nanopillars [9] . Interestingly, the tailored surface modification of gCNQDs has been achieved with moieties that acted as receptors to capture and detect target analytes. In our previous work, the surface of gCNQDs was functionalized with 2, 2, 6, 6-tetramethyl (piperidin-1-yl) oxyl (TEMPO) to detect ascorbic acid in the presence of zinc phthalocyanine [2] . Furthermore, biomolecules including thymine and tannic acid have been grafted onto the surface gCNQDs for the purpose of deriving Hg 2+ , Cu 2+ and ascorbic acid responsive nanosensors, respectively [18, 19] . On the other hand, novel materials which can serve as viable alternatives to Au and Ag are desired and are a hotspot for research [20, 21] . This is due in large part to the high cost of preparation and/or procurement of noble metals nanoparticles (NPs). As a result, a new low-cost, easy-to-prepare and non-toxic molybdenum trioxide QDs (MoO 3 QDs) with excellent plasmonic properties are gaining traction [21] [22] [23] [24] [25] [26] [27] [28] . MoO 3 QDs are few-crystalline-structured nanoparticles with single-layered morphology and oxygen vacancies. Hence, they exhibit a semiconductor-based tunable localized surface plasmonic resonance (LSPR), comparable to noble metals NPs, both in the visible and near-infrared (NIR) regions [27, 28] . To demonstrate that the inherent tunable LSPR of MoO 3 QDs J o u r n a l P r e -p r o o f can be harnessed for opto-electrical sensing, MoO 3 QDs was adopted as a substrate for the surface enhanced Raman spectroscopy (SERS) detection of bovine serum albumin (BSA) and methylene blue (MB), respectively. The Raman signals of the probe molecule (Rhodamine 6G) and MB were strongly amplified due to the interfacial charge transfer effect between the substrates and probe molecule. The intense LSPR absorption of MoO 3 QDs in the near infrared (NIR) region was deployed in the photothermal ablation of cancer, glucose detection in the fluorescence (FL)-based detection of 2, 4, 6-trinitrotoluene (TNT) [23, 25] . Plasmonic nanostructures are known to influence the fluorescence (FL) properties of QDs via plasmon-induced energy transfer [29, 30] . Optical biosensing platforms have been designed based on this kind of interaction [31, 35] . QDs. This step, as expected, resulted in a signal enhancement and sensitivity of the established immunoassay. The target influenza virus was separated easily by an external magnet field allowing an ultrasensitive detection of influenza virus A (H1N1) and clinically isolated influenza virus A (H3N2) RNA, respectively. The biosensing system has been developed to provide a robust performance, as well as high selectivity and ultrasensitivity for influenza virus detection when compared to a commercially available rapid influenza diagnostic test (RIDT) kits. Commercial RIDT kit -QuikNavi Flu 2 was purchased from Denka -Seiken Co. Ltd. (Tokyo, Japan). Zika virus was kindly provided by Prof. K. Morita of Institute of Tropical Medicine, Nagasaki University. Noro virus-like particles (NoV-LPs) were prepared in our lab according to previously reported protocol [36] . HEV-LPs were prepared according to Li et al. [37] . All experiments were carried out using high purity deionized (DI) water (>18 MΩ·cm). Ground state electronic absorption (UV/Vis), fluorescence excitation and emission spectra were recorded on a filter-based multimode microplate reader (Infinite Conjugation of the antibody to the respective QDs and nanoparticles was confirmed by enzyme-linked immunosorbent assay (ELISA) using a microplate (Model 680; Bio-Rad, Hercules, USA). Melamine was used to synthesize graphitic carbon nitride nanosheets (gCNNs) according to reported procedures [38] . Graphitic carbon nitride QDs (gCNQDs) were prepared by the solvothermal treatment of the graphitic nanosheets (gCNNs) according to previously reported method with modifications [18] . Briefly, gCNNs (0.1 g) and citric acid (0.2 g) were dissolved in 10 mL of DMF, stirred for 5 min and sonicated for 20 min to obtain homogenous suspension. The suspension was transferred and sealed in a 50 mL Teflon-lined stainless steel autoclave and heated at 160°C for 12 h. The autoclave was allowed to cool to room temperature naturally. The obtained product was filtered through a 0.22 m microporous filter membrane and then dialyzed (using a membrane of MWCO 1.5 kDA) for 48 h to obtain pure gCNQDs solution. The solution was further freeze dried to obtained solid product. Pristine MoO 3 QDs was prepared by a room temperature ultraviolent (UV) irradiation method according to reported procedure [28] . The novel magnetic- The respective antibody conjugated gCNQDs and MP-MoO 3 QDs were used as the detection probe (5% BSA was used for blocking to avoid non-specific binding of the antibody-conjugated probes). Next, 50 L of the probe solution was placed in a 96-well plate, followed by the incubation of the respective target influenza virus (20 L) for 5 min before fluorescence measurements were acquired. After the incubation duration, the antibody-antigen complex solutions were excited at 500 nm, and the fluorescence spectra within the range of 530-800 nm were recorded. The detection of influenza virus (H1N1) was carried out in ultrapure water and human serum within the concentration range of 1 pg/mL -100 ng/mL. Clinically In this work, bulk graphitic carbon nitride nanosheets (gCNNs) were treated under solvothermal conditions to prepare highly fluorescent gCNQDs. In order to characterize the prepared carbon-based QDs, various techniques ranging from materials science to spectroscopy were employed. The transmission electron microscopy (TEM) image obtained showed quasi-spherically shaped particles that are monodispersed with size distribution between 5 -9 nm (7.25±0.7 average diameter) (Fig. 1A) . The HRTEM image shows that the graphitic lattice spacing is 0.23 nm (Fig. 1A inset) , which is a feature of the (002) hexagonal plane of graphitic carbon nitrides [4, 8, 11] . The AFM results showed a narrow distribution of the graphitic QDs with few layers of planar graphitic sheets having nanoscopic size of ~5 nm with a height profile of 1.5 nm (Fig. 1B) . The powder X-ray diffraction (XRD) pattern (Fig. 1C ) obtained for the prepared gCNQDs exhibit a broad peak at 27.9° which corresponds to the graphitic interplanar 002 d-spacing which is known to be exhibited by graphitic QDs [8] . Chemical functional groups were also elucidated by carrying out FTIR spectroscopy. In Fig. 1D , the spectra of gCNQDs and/or nitride functionality of the s-triazine heterocycles expected to be associated with graphitic carbon nitride QDs [41] . The result also shows that the gCNQDs may be dispersed in solution to a moderate degree, as zeta potential values >20 mV are known to result in well-dispersed colloidal solutions due to increased interparticle repulsion [40, 41] . The optical spectroscopy characterization of gCNQDs displayed a typical strong ground-level absorption peak at ~542 nm and a broad absorption at < 500 nm (Fig. 2A) . These absorptions are ascribed to the n-π* and π-π* electronic transitions of the electron lone pairs of N atoms of s-triazine units [4, 8, 11, 18] . Further, the fluorescence properties of the as-synthesized gCNQDs were probed. The FL emission of gCNQDs prepared under solvothermal conditions exhibit emission extending into the yellow region with maximum fluorescence intensity at 652 nm when excited at 500 nm wavelength ( Fig. 2A) . Carbon-based QDs with similar fluorescence emission extending well into the red region have been prepared using solvothermal processes [42, 43] . Another important parameter evaluated for the prepared gCNQDs in terms of their suitability to function as an optical probe is their fluorescence quantum yield (Φ F ). The robustness and sensitivity of a given fluorescence-based material as a probe is dictated by efficiently high Φ F values. Interestingly, gCNQDs possess Φ F of ~44%; using Rhodamine 6G as the reference standard (see Supplementary Information for details). It is plausible to infer that the presence of citric acid made the gCNQDs water soluble (due to the incorporation of carboxylic group), and also created additional defects/trap sites on the QDs surface (new surface states) [44, 45] . This probably may have resulted in an easy exciton mobility translating into such a high Φ F and red-shifted emission. Novel magnetoplasmonic derivative of molybdenum-based QDs was prepared in the presence of FeCl 2 and FeCl 3 as the magnetic component precursors. QDs at ~3.2 nm was increased to ~18 nm upon magnetic NPs functionalization (Fig. 3C ). X-ray diffractogram (XRD) patterns provided an insight into MP- MoO 3 QDs formation when compared with the pattern of the pristine MoO 3 QDs. As shown in Fig, 3D , pristine MoO 3 QDs are endowed with a broad peak within the range of 22 -33°, which shows that they have poor crystallinity [27, 28] . This peak has been attributed to the (040) positions of a-MoO 3 (JCPDs no. 05-0508) [27] . Conversely, the MP-MoO 3 QDs hybrid displays the characteristic diffraction pattern of MoO 3 QDs in 2θ range from 5 -30° and that of magnetic constituent J o u r n a l P r e -p r o o f at 2θ = 35°, 57° and 63° which corresponds to magnetic NPs marked indices of (311), (511) and (440), respectively [47] . Energy dispersive X-ray spectroscopy (EDX) was employed to elucidate the elemental compositions of MP-MoO 3 QDs. The main compositional elements which are Mo and Fe are found as displayed in the obtained spectra (Fig. 3E) Fig. 4A and B) . The core-satellite immunocomplex formed between the Ab-gCNQDs and Ab-MP-MoO 3 QDs can be easily isolated with a magnetic field leading to sample concentration and an interference-free FL signal modulation. The extent of the core-satellite immunocomplexing was directly proportional to influenza virus (H1N1) concentration; this in turn led to stronger plasmonic coupling effect as more MP-MoO 3 QDs are brought closer to the gCNQDs by the antibody-antigen binding affinity. Consequently, corresponding linear calibration curves were plotted to elucidate the linear dynamic detection range as shown in Fig. 4C . The LOD for the detection of influenza virus (H1N1) in ultrapure water is 0.25 pg/mL, while the LOD in human serum was evaluated to be 0.9 pg/mL. A comparison of the obtained LODs with other detection systems indicated that this developed system has a comparable sensitivity (Table 1) [48] [49] [50] [51] [52] [53] . Furthermore, by analyzing the kinetics of the detection cycle of the time-course change in the FL intensity of gCNQDs ( Fig. S6 in Supplementary data), it can be observed that the FL enhancement occurred rapidly when the target virus was introduced to the 96 well-plate reaction chamber that allowed rapid fluorescence detection up to 5 min after which no major FL modifications had been observed up to 10 min and above after this optimum point. The detection protocol was expedited for influenza virus (H1N1) detection in a matter of ~5 min even in clinical samples containing target influenza virus. Hence, this developed detection platform could achieve faster results for urgent diagnostic measures better than or replace commercial rapid diagnostic kits, which requires ~15 -20 min, and/or (qRT-PCR), which requires several hours (4 -6 h) for detection, such that point-of-care testing could be expected if this detection protocol is fully integrated with portable instrumentation system. Moreover, it is plausible to point out that this detection platform can be tuned appropriately to detect other target viruses. To verify the specificity and selective disposition of the developed immunosensor towards influenza virus (H1N1) as the target virus, some other viruses such as Zika virus, NoV-LPs, HEV-LPs, Dengue were employed to study the potential interferences that may be exhibited by monitoring the response of the fluoroimmunosensors in the presence of ~ 10 ng/mL other viruses. As shown in A novel combination of graphitic carbon nitride and molybdenum-based QDs was deployed for the fluoroimmunoassay of influenza virus. The sensitivity achieved herein for influenza virus detection was 0.25 pg/mL in DI water and 0.9 pg/mL in human serum. In a clinical sample, influenza virus A (H3N2) was detected using this assay with LOD of 45 PFU/mL within a linear dynamic detection range of 45 -25,000 PFU/mL. The assay showed a good sensitivity for the detection of influenza virus in samples with complex matrices owing to the magnetic J o u r n a l P r e -p r o o f separation/purification protocol of the assay. This work shows that the nanoparticles combination can be adopted as potential materials for constructing efficient platforms for virus detection. In addition, they are highly competitive and low cost alternatives deployable for the plasmonic-induced and optical detection of infectious viral biomolecules by using the desired antigen-antibody pair to devise a vast pool of biosensors to meet the demand for speedy and responsive assessment assays. Fluorescence fiber-optic biosensor H1N1 13.9 pg/mL [48] Magnetofluoro-immunoassay H1N1 6.07 pg/mL [49] Ag-S covalent labelling H1N1 0.1 pg/mL [50] Electrochemical immunosensor H5N1 2.1 pg/mL [51] Metal-enhanced fluoroimmunoassay H1N1 1 ng/mL [52] Peroxidase mimic H1N1 10 pg/mL [53] Magnetoplasmonic fluoroimmunoassay Coronavirus disease 2019 (COVID-19) Situation Report-24 In situ one-pot synthesis of graphitic carbon nitride quantum dots and its 2, 2, 6, 6-tetramethyl (piperidin-1-yl) oxyl derivatives as fluorescent nanoprobes for ascorbic acid Graphene quantum dots in analytical science Graphitic carbon nitride materials: sensing, imaging and therapy Eco-friendly and rapid microwave synthesis of green fluorescent graphitic carbon nitride quantum dots for vitro bioimaging Highly biocompatible phenylboronic acid-functionalized graphitic carbon nitride quantum dots for the selective glucose sensor The photoluminescence mechanism in carbon dots (graphene quantum dots, carbon nanodots, and polymer dots): current state and future perspective Facile bulk production of highly blue fluorescent graphitic carbon nitride quantum dots and their application as highly selective and sensitive sensors for the detection of mercuric and iodide ions in aqueous media A bio-chemical application of N-GQDs and g-C3N4 QDs sensitized TiO2 nanopillars for the quantitative detection of pcDNA3-HBV A dual signal-on photoelectrochemical immunosensor for sensitively detecting target avian viruses based on AuNPs/g-C 3 N 4 coupling with CdTe quantum dots and in situ enzymatic generation of electron donor Two-dimensional graphitic carbon nitride nanosheets for biosensing applications Graphitic carbon nitride as immobilization platform for ssDNA in a genosensor Graphitic Nitrogen Triggers Red Fluorescence in Carbon Dots Synthesis and characterization of nitrogen-rich graphitic carbon nitride Label-free ochratoxin A electrochemical aptasensor based on target-induced non-covalent assembly of peroxidase-like graphitic carbon nitride nanosheets Gold nanoparticles enhanced electrochemiluminescence of graphite-like carbon nitride for the detection of Nuclear Matrix Protein 22 Photoelectrochemical immunosensor for methylated RNA detection based on g-C 3 N 4 /CdS quantum dots heterojunction and Phos-tag-biotin Microwave-assisted synthesis of thyminefunctionalized graphitic carbon nitride quantum dots as fluorescent nanoprobe for mercury (II) Tannic acid-derivatized graphitic carbon nitride quantum dots as an "on-off-on" fluorescent nanoprobe for ascorbic acid via copper(II) mediation A comparative study of different reagentless plasmon sensors based on Ag-Au alloy nanoparticles for detection of Hg Plasmonic ZnO nanorods/Au substrates for protein microarrays with high sensitivity and broad dynamic range Tunable plasmon resonance of molybdenum oxide nanoparticles synthesized in non-aqueous media Preparation of MoO 3 QDs through combining intercalation and thermal exfoliation Room-Temperature and Aqueous-Phase Synthesis of Plasmonic Molybdenum Oxide Nanoparticles for Visible-Light-Enhanced Hydrogen Generation Highly Photoluminescent Molybdenum Oxide Quantum Dots: One-Pot Synthesis and Application in 2,4,6-Trinitrotoluene Determination Localized Surface Plasmon Resonances in Plasmonic Molybdenum Tungsten Oxide Hybrid for Visible-Light-Enhanced Catalytic Reaction Plasmonic molybdenum trioxide quantum dots with noble metal-comparable surface enhanced Raman scattering Rapid roomtemperature preparation of MoO 3−x quantum dots by ultraviolet irradiation for photothermal treatment and glucose detection Thiol-ligand-Catalyzed Quenching and Etching in Mixtures of Colloidal Quantum Dots and Silver Nanoparticles Long-Range Resonance Coupling-Induced Surface Energy Transfer from CdTe Quantum Dot to Plasmonic Nanoparticle Localized surface plasmon resonance-mediated fluorescence signals in plasmonic nanoparticlequantum dot hybrids for ultrasensitive Zika virus RNA detection via hairpin hybridization assays A plasmon-assisted fluoro-immunoassay using gold nanoparticledecorated carbon nanotubes for monitoring the influenza virus Versatility of a localized surface plasmon resonance-based gold nanoparticle-alloyed quantum dot nanobiosensor for immunofluorescence detection of viruses Ultrasensitive detection of norovirus using a magnetofluoroimmunoassay based on synergic properties of gold/magnetic nanoparticle hybrid nanocomposites and quantum dots Single-step detection of norovirus tuning localized J o u r n a l P r e -p r o o f surface plasmon resonance-induced optical signal between gold nanoparticles and quantum dots Size-controlled preparation of peroxidase-like graphene-gold nanoparticle hybrids for the visible detection of norovirus-like particles Expression and self-assembly of empty virus-like particles of hepatitis E virus Graphitic carbon nitride embedded hydrogels for enhanced gel electrophoresis Improving the detection limits of near infrared spectroscopy in the determination of aromatic hydrocarbons in water employing a silicone sensing phase Carboxylic Carbon Quantum Dots as a Fluorescent Sensing Platform for DNA Detection Carbon Dots: Diverse Preparation, Application, and Perspective in Surface Chemistry Full-Color Light-Emitting Carbon Dots with a Surface-State-Controlled Luminescence Mechanism Bright Multicolor Bandgap Fluorescent Carbon Quantum Dots for Electroluminescent Light-Emitting Diodes Microwave Growth and Tunable Photoluminescence of Nitrogen-doped Graphene and Carbon Nitride Quantum Dot Theoretical Investigations of Optical Origins of Fluorescent Graphene Quantum Dots PVP-capped nickel nanoparticles: synthesis, characterization and utilization as a glycerol electrosensor Electrochemical genoassays on gold-coated magnetic nanoparticles to quantify genetically modified organisms (GMOs) in food and feed as GMO percentage Detection of swine-origin influenza A (H1N1) viruses using a localized surface plasmon coupled fluorescence fiberoptic biosensor Plasmonic/magnetic graphene-based magnetofluoro-immunosensing platform for virus detection Highly sensitive fluorescent immunosensor for detection of influenza virus based on Ag autocatalysis Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum Metal enhanced fluorescence on nanoporous gold leaf-based assay platform for virus detection Detection of influenza virus using peroxidase-mimic of gold nanoparticles Dynamics of Glycoprotein Charge in the Evolutionary History of Human Influenza The authors sincerely thank Professor K. Morita of the Institute of Tropical [55] Y. Kobayashi He is currently interested in preparation and engineering of nanobiomaterials such as virus-like particles and detection of infectious viruses. Also, he is interested in the expression of eukaryotic proteins in silkworm larvae and their applicability on biological detection of molecules.