Femtomolar Detection of Dengue Virus DNA with Serotype

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Femtomolar detection of Dengue Virus DNA with Serotype Identification Ability Ankan Dutta Chowdhury, Akhilesh Babu Ganganboina, Fahmida Nasrin, Kenshin Takemura, Rueyan Doong, Doddy Irawan Setyo Utomo, Jaewook Lee, Indra Memdi Khoris, and Enoch Y. Park Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01802 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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Analytical Chemistry

Femtomolar Detection of Dengue Virus DNA with Serotype Identification Ability Ankan Dutta Chowdhury1, Akhilesh Babu Ganganboina2, Fahmida Nasrin3, Kenshin Takemura3, Ruey-an Doong2,4, Doddy Irawan Setyo Utomo3, Jaewook Lee1, Indra Memdi Khoris5, Enoch Y. Park1,3,5,* 1Laboratory

of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan

2Department

of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101 Section 2, Kuang-Fu Road, Hsinchu, 30013, Taiwan

3Laboratory

of Biotechnology, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan

4Institute

of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu 30010, Taiwan

5College

of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan

E-mail: [email protected] (ADC) [email protected] (ABG) [email protected] (FN) [email protected] (KT) [email protected], [email protected] (RAD) [email protected] (DISU) [email protected] (JL) [email protected] (IMK) [email protected] (EYP) * Corresponding

author: [email protected] (Tel (Fax): +81-54-238-4887)

1 ACS Paragon Plus Environment

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

ABSTRACT: Dengue surveillance trusts only on reverse transcription-polymerase chain reaction (RT-PCR) type methodologies for confirmation of dengue virus serotypes; however its real time application is restricted due to expensive, complicated and time consuming process. In search of a new sensing system, here, we have reported a two-way-detection method for Dengue virus (DENV) serotype identification along with DNA quantification by using a new class of nanocomposite of gold nanoparticles (AuNP) and nitrogen, sulphur codoped graphene quantum dots (N,S-GQDs). The N,S-GQDs@AuNP has been used for serotype detection via simple fluorescence technique using four dye-combined probe DNAs which is further validated by confocal microscopy. The quantification of the DNA has been measured by Differential Pulse Voltammetric (DPV) technique using methyelene blue as redox indicator. Results obtained in this study, clearly demonstrate that the N,SGQDs@AuNP can efficiently detect the four serotypes of DENV individually in the concentration range of 10-14 to 10-6 M with the LOD of 9.4 fM. In addition, to confirm its applicability in long chained complex DNA system, the sensor was also applied to the clinically isolated DENV DNA and showed satisfactory performances for serotype identification as well as quantification. We hope this simple and reliable method can pave an avenue for the development of sensitive and robust sensing probes in biomedical applications.

Keywords: gold nanoparticle; sulphur co-doped graphene quantum dots; dengue virus detection; differential pulse voltammetric biosensor; fluorometric biosensor; cyclic voltammetry.


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INTRODUCTION The specific and sensitive DNA detection is of great demand in recent days for its various biomedical applications including gene profiling, drug diffusion, and clinical diagnostics.1-4 Proper advancement of this method has a great potential to accumulate maximum information efficiently from a small volume of analyst at a low cost. In this regard, a costeffective, simple and reliable DNA detection method is in demand for the bio-monitoring aspect. For the detection of viral diseases, successful DNA biosensors also can provide considerable advantages over existing diagnostic technologies like antigen antibody detection or protein identification due to their poor reliability and false positive responses. In the last decade, several attempts have been made to find a suitable method for DNA sensing such as fluorescence-based assay, surface-enhanced Raman scattering (SERS), electrochemical sensing and mass changes.5-9 Though few reports have claimed to meet the desired sensitivity however, achieving optimum reproducibility and reliability for detecting target analytes without any further amplification still remain as challenging tasks. Dengue fever, which affects a significant and ever-increasing portion of the world's population, is an important example of one such viral disease. Dengue virus (DENV) belongs to the Flaviviridae family comprising of 4 unique serotypes.10 As the induced antibody concentration in the early stage of infection is quite low, the proper diagnosis is becoming extremely difficult by IgM capture enzyme-linked immunosorbent

assay

(MAC-ELISA),

hemagglutination

inhibition

(HAE),

immunofluorescence assay and the plaque reduction neutralization test (PRNT).11-13 The fatality is increased severely when a secondary infection is occurred by a virus serotype different from that of the initial infection. The specific virus after infection induces life-long immunity towards the same serotype, but renders significantly less protection against infection from other serotypes. To achieve reliable and sensitive detectability of the serotype 3 ACS Paragon Plus Environment


identification process, instead of indirect methods of IgM or IgG detection, a direct virus particle or DNA detection from infected blood samples should be the most promising way. Thus, in addition to quantifying the presence of the DENV in human serum, a biosensor that can accurately identify virus serotype is in an utmost need. Many reports can be found and classified as either direct or indirect method of DENV detection.14-19 The direct diagnostic method constitutes live virus isolation or detection of RNA in serum, plasma, whole blood and infected tissues within 3 – 5 days of manifestation of symptoms. Though the direct methods are the most reliable for the quantification of disease, however, selectivity becomes a problem due to the cross-reactivity with other infectious diseases like zika, malaria and leptospirosis. To circumvent the problems of specificity, molecular assays based on nucleic acid amplification by reverse transcriptase polymerase chain reaction (RT-PCR) are assumed to rapidly detect and differentiate DENV serotypes.20 In spite of its high cost, serotype identification is also difficult by this technique due to its complexity and requirement of highly skilled professionals. Among the techniques used, fluorescence and electrochemical detection methods have drawn great attention, recently. Conventional fluorometric methods have limited applications in ultrasensitive detection due to its inability to measure extremely small changes in intensity. The increased interest in electrochemical methods for hybridization detection arises from the high sensitivity, low cost, and their compatibility with microfabrication technology. One of the most often used approaches for the electrochemical detection is based on using enzyme labels in order to generate an amplified signal through different schemes, such as via bioelectrocatalytic redox transformations or biocatalyzed precipitation of an insoluble product at the electrode as a result of the formation of the double-stranded DNA complexes. Although large signal amplification from enzyme labels can be achieved, labeling of an oligonucleotide by an enzyme is not a straightforward route, and once prepared, the activity 4 ACS Paragon Plus Environment

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of the conjugate must be periodically controlled owing to the inherent poor stability of enzymes. Here we have presented a two-way-DNA-detection method based on nitrogen, sulphur doped graphene quantum dots with gold nanoparticle nanoassembly (N,SGQDs@AuNP) that can be used for monitoring the serotype of DENV qualitatively as well as quantitatively, while requiring the use of a low volume of the analyte. The fluorometric signal from the sensor defines the nature of the serotype whereas the DPV signal further allows the quantitative determination of identified serotype. Multiple serotypes of DNA could be successfully detected by employing this method with a linear range of 10-14 to 10-6 M, demonstrating the applicability of developed convenient system for clinical diagnosis and biomolecular interaction studies.

Scheme1. Schematic representation for the detection mechanism of DENV DNA detection by N,S-GQDs@AuNP nanocomposite in two-way-detection method; fluorometric sensing for serotype identification and electrochemical DPV sensing for DNA quantification.

EXPERIMENTAL SECCTION 5 ACS Paragon Plus Environment


Chemicals. Citric acid, urea, thiourea, HAuCl₄, N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and methyelene blue were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Potassium hydrogen phosphate and dihydrogen phosphate were purchased from Merck, Germany. All other reagents were of analytical grade and were used as received without further purification. Solutions used in this study were prepared using bi-distilled deionized water purified through a UV treated Rephile water system (18.2 MΩ cm) unless otherwise mentioned. Synthesis of N,S-GQDs. The nitrogen, sulphur doped graphene quantum dots (N,SGQDs) were prepared by standard route of hydrothermal synthesis.21 In brief, 0.23 g citric acid and 0.23 g thiourea were dissolved into 5 mL of deionized water and the solution was then transferred into a 20-mL of Teflon lined stainless steel autoclave tube to heat up to 160 °C for 4 h. The obtained brown suspension was added into ethanol solution and centrifuged at 3500 × g for 5 min. The separated N,S-GQDs then re-dispersed in water and dialyzed through a 1 kD dialysis bag for 12 h. Synthesis of AuNPs. Seed particle solutions were prepared by the standard citrate reduction method.22 Briefly, 2.5 mL of a HAuCl4,·3H2O solution (0.2 % w/v) in 50 mL of water were heated to boiling and then 2 mL of sodium-citrate solution (1 % w/v, containing 0.05 % w/v citric acid) were added quickly under vigorous stirring. The solution was kept boiling for 5 min and was then allowed to cool down. A certain amount of seed solution was diluted to 20 mL and placed into a three-necked flask. A 10 mL aliquot of HAuCl4 and 10 mL of trisodium citrate were added separately at room temperature under vigorous stirring over a time of about 45 min. After the addition was complete, the mixture was brought to boiling and maintained at this temperature for about 30 min. Finally the solution was allowed to cool down and UV-Vis and TEM investigations were carried out.


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Preparation of Oligomeric solutions and clinically isolated DENV DNA. A calculative amount of Diethyl pyrocarbonate (DEPC) treated water was added to DNA as well as dye labelled DNA container, purchased from FASMAC (Atsugi, Japan) to get the concentration of 1 mM. The DNA concentration was further diluted to 2 µM to serve as the stock solution for the experiment. Exact concentration of the DNA solutions were further verified by the absorption value at 260 nm in UV-Visible spectra. Clinically isolated DENV 3 was used for this test. Prior to use for sensing analysis, the RNAs were extracted from the samples and amplified in PCR (Takara, Shiga, Japan) with poly T addition. The designed primers for PCR are given in Figure S6 of supporting information. According to Scheme 1, following DNAs are used in this present work: dT-Target DNAs: 1. (a) 5’-TTTTTTTTTT-GGG AAG CTG TAT CCT GGT GGT AAG G -3’ (DENV-1) (b) 5’- TTTTTTTTTT-ATG AAG CTG TAG TCT CAC TGG AAG G -3’ (DENV-2) (c) 5’- TTTTTTTTTT-AGG GAA GCT GTA CCT CCT TGC AAA G -3’ (DENV-3) (d) 5’- TTTTTTTTTT-GAG GAA GCT GTA CTC CTG GTG GAA G -3’ (DENV-4) (e) 5’- TTTTTTTTTT-AGG GAA GCT GTT CCT CCT TGC AAA G -3’ (single base mismatch in DENV-3) (e) 5’- TTTTTTTTTT-AGG GAA GCT GTT GCT CCT TGC AAA G -3’ (double base mismatch in DENV-3) 2. 5’- TTTTTTTTTT-GAA TTA GCT TGT ATG ATG TCG CTG A-3’ (Norovirus) 3. 5’- TTTTTTTTTT-GCT AAA ACG CGG AGT AGC CCG T-3’ (Zika virus)



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4. 5’- TTTTTTTTTT-ATT TCA GTT ATT ATG CCG TTG TAT T-3’ (Influenza virus) 5. 5’- TTTTTTTTTT-AAAGGGCCC TTTAAAGGGTTTCCCA -3’ (noncomplementary) Probe 1 (dA): SH-AAAAAAAAAAAA Probe 2: (a) CCC TTC GAC ATA GGA CCA CCA TTC C-Cy3 (complementary of DENV-1) (b) TAC TTC GAC ATC AGA GTG ACC TTC C-FITC (complementary of DENV-2) (c) TCC CTT CGA CAT GGA GGA ACG TTT C-Rho-X (complementary of DENV-3) (d) CTC CTT CGA CAT GAG GAC CAC CTT C-Tex red (complementary of DENV-4) Synthesis of N,S-GQDs@AuNP-dA Nanocomposites.

The nanocomposite of N,S-

GQDs@AuNP was formed by simple mixing of these two solutions in 1:1 ratio, overnight. The dialyzed N,S-GQDs was mixed with as prepared AuNP under slow but continuous stirring to make the nanoassembly between sulphur atom of N,S-GQDs and AuNP via soft acid soft base interaction. Then, the nanocomposite was centrifuged again to remove excess N,S-GQDs and re-dispersed with 10 µL of 2 µM thiolated dA (probe 1) for further 2 h stirring. Finally the N,S-GQDs@AuNP-dA nanocomposite was again centrifuged at 11500 × g to remove the excess dA and collected for its further applications. Characterizations. The morphology as well as size distribution of AuNP and N,SGQDs were characterized using a transmission electron microscope (JEM-2100F; JEOL, Ltd., Tokyo, Japan) and a high-resolution transmission electron microscopy (JEOL JEM-2010, Taipei, Taiwan HRTEM) at 300 kV. X-ray diffraction (XRD) patterns were recorded using RINT ULTIMA XRD (Rigaku Co., Tokyo, Japan) with a Ni filter and a Cu-Kα source. UV−visible and fluorescence spectra of nanocomposites were recorded using a filter-based 8 ACS Paragon Plus Environment

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multimode microplate reader (Infinite F500; TECAN, Ltd, Männedorf, Switzerland). Dynamic light scattering (DLS) measurement was performed using a Zetasizer Nano series (Malvern Inst. Ltd., Malvern, UK). The thermal properties were examined by thermogravimetric analysis using Mettler Toledo DSC/TGA 3+ Stare system (Taipei, Taiwan) in the presence of N2. Optical Measurements for Determining the Specific Serotype of DENV. To prepare the standard identification curve for different serotypes of DENV, the N,S-GQDs@AuNP-dA nanocomposite was dispersed in PBS buffer and dT attached target DNAs of different serotype with known concentration of 10-9 M were incubated separately at 37 °C for 10 minutes. Then the complementary DNA sequences of serotypes, tagged with different dyes (Probe 2, (a) DENV-1 Cy3, (b) DENV-2 FITC, (c) DENV-3 RhX and (d) DENV-4 Texas red) were added to the above mixture and further incubated at 37 °C for 10 mins. The mixture was then centrifuged at 11500 × g to settle down the nanocomposites along with specific probe 2 conjugated to it. Discarding the supernatant, the separated nanocomposites were further re-dispersed in 1 ml of fresh buffer and fluorescence measurements at 4 different excitations were performed. For confocal microscopic imaging (LSM-700, Carl Zeiss, Tokyo, Japan), the final dispersed solution was placed on 1 × 1 cm rubber box and were taken for the images. Electrochemical Detection for Quantifying the Detected Serotypes. To determine the electrochemical

properties,

cyclic

voltammetry

(CV)

was

carried

out

on

a

potentiostat/galvanostat (SP-150, Bio-Logic, Tokyo, Japan) workstation. The gold (Au) millielectrode, used for electrochemical studies, was first cleaned by standard method and then the N,S-GQDs@AuNP nanocomposite was drop-casted on the surface of Au milli-electrode and CV was taken in PBS buffer. To determine the sensitivity of the Au||N,S-GQDs@AuNP-dA as the sensor electrode, Differential Pulse Voltammetry (DPV) was employed in 10 mM PBS 9 ACS Paragon Plus Environment


buffer at pH 6.7 containing 100 mM NaCl. Initially the N,S-GQDs@AuNP-dA nanoprobe was dispersed in the PBS buffer and the known serotype of DENV DNA having poly T tail was added in separate mixtures in of different concentration range (10-14 to 10-6 M). Then the corresponding dye combined DNA probe 2 was added to the mixtures and incubated for further 10 minutes. After that, the mixture was centrifuged at 11500 × g to separate the nanocomposites from unbounded DNA. The precipitate was then drop-casted on clean Au electrode and incubated in 3 mL of 20 µM of methylene blue (MB) solution to intercalate in the grooves of single stranded DNA (ssDNA) of nanocomposite. In absence of any target DNA, the Au||N,S-GQDs@AuNP-dA nanoprobe consumed maximum amount of MB for the existence of free ssDNA on nanoprobe, resulting highest peak in DPV which was gradually decrease with increasing concentration of target DNA. The target DNA with its complementary strand got attach on the free ssDNA to form duplexes hence lowering the amount of MB loading on nanoprobe, decreasing the signal intensity of DPV curve. The ratio of signal intensity of without target DNA loaded nanoprobe with after duplex formation by different concentrations of DNA, the calibration plot was established.

RESULTS AND DISCUSSIONS In this study, a two way DNA detection technique has been proposed to detect the DENV along with quantitative determination of serotype found. To obtain serotype determining ability, the N,S-GQDs@AuNP-dA nanoprobe is employed to known concentration of DENV serotype which is qualified successfully by fluorescent dye-combined probes. Thereafter the quantification calibration of each known serotype has been established by subsequent electrochemical DPV technique using methylene blue as a redox indicator where the different intercalating ability of MB with ssDNA and dsDNA has been employed. The central theme 10 ACS Paragon Plus Environment

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of the work is to build a reliable technique for the detection of an unknown concentration of an unknown serotype of DENV DNA which can serve as a superior platform as a reliable kit for Dengue diagnosis. Characterizations of N,S-GQDs@AuNP Nanocomposites. Large sized (60 – 70 nm) AuNP has been synthesized via citrate reduction method to anchor maximum amount of N,SGQDs and ssDNA on the surface. In Figure 1A, the TEM image of homogeneously distributed AuNP is presented where the particle size distribution is given in the inset. The size of the nanoparticle is around 78.2 ± 2.1 nm (n = 30) which is also observed in the high magnified image in inset. On the other hand, the N,S-GQDs, prepared by hydrothermal route (Figure 1B) show homogeneously distributed dots with uniform lateral sizes. The particle size distribution shown in Figure 1C is narrow (2 – 9 nm) and the average lateral size, determined by histogram, is 4.5 ± 0.6 nm (n = 80). It is expected that such narrow-distributed particle size with superior homogeneous dispersion can significantly enhance the conjugation property with synthesized AuNP. In addition, the fringes of graphitic lattice can be viewed clearly from HRTEM image (Figure 1D) where the surface roughness indicates that the N,SGQDs are graphitic single crystal with a spacing of 0.21 nm, which corresponds to the (100) plane of graphene (Figure 1E).23 The sulphur present in N,S-GQDs, allows the conjugation of N,S-GQDs with the AuNP to produce N,S-GQDs@AuNP nanocomposites by only physical mixing, employing the soft acid - soft base interaction. The fluorescence intensity of the bare N,S-GQDs, presented in Figure S1, has significantly quenched after N,S-GQDs@AuNPs nanocomposite formation due to the conjugation of the Au metal with the sulphur doped graphene matrix which perturbs the π-π delocalization of the N,S-GQDs electronic structure. The purified N,SGQDs@AuNP nanocomposites is further analyzed by TEM, presented in Figure 1F. The close agglomeration of small dots of N,S-GQDs is mainly oriented on the surface of the big 11 ACS Paragon Plus Environment


black dots of AuNP, clearly supports the hypothesis of N,S-GQDs attachment on AuNP. The HRTEM images of an isolated N,S-GQDs@AuNP nanocomposite (inset of Figure 1F), clearly shows two types of their characteristic fringes of 0.21 nm for N,S-GQDs (100 planes)21 and 0.24 nm for Au (111 planes).24 The synthesis of N,S-GQDs@AuNP nanocomposites has been further corroborated by the DLS study (Figure 1G). The 5 – 6 nm N,S-GQDs and 60 – 70 nm AuNPs become 110 – 120 nm (the hydrodynamic diameters from DLS) for N,S-GQDs@AuNP nanocomposites, which are closely supported by the TEM image. Though the agglomeration of N,S-GQDs@AuNP nanocomposites is little disadvantageous for the application of sensing which may reduce the active surface area of nanoprobe as well as the target DNA capture, hence lowering sensitivity, however, it shows distinct advantages for the analysis in the confocal microscopy where the larger sized N,SGQDs@AuNP nanocomposites helps to show clear images for serotype identification.

Figure 1. Characterisations: (A) TEM image of as synthesized AuNP (inset high magnification of single AuNP and particle distribution of AuNP from 60 - 100 nm), (B) TEM


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image of N,S-GQDs (inset shows the image of N,S-GQDs and buffer solution under 360 nm UV lamp), (C) Particle size distribution, (D) HRTEM of N,S-GQDs, (E) lattice spacing of asprepared N,S-GQDs along the line shown in figure 1D, (F) TEM image of N,SGQDs@AuNP (inset: high resolution image of an isolated particle), (G) DLS of AuNP, N,SGQDs and N,S-GQDs@AuNP, (H) TGA of AuNP and N,S-GQDs@AuNP and (I) XRD of AuNP, N,S-GQDs and N,S-GQDs@AuNP.

The successful formation of N,S-GQDs@AuNP nanocomposites is further verified by TGA and XRD analysis. In Figure 1H, the TGA of as synthesized AuNP shows excellent stability up to 700 °C in N2 atmosphere. The 3.7 % decay may be due to the presence of water molecules and oxidized layer of Au3+.25 However, stability of N,S-GQDs@AuNP nanocomposites, is significantly decreased due to the presence of large amount of carbon matrix of N,S-GQDs. Moreover, the sudden drop at 350 – 400 °C for carbon oxidation clearly shows the signature mass loss of graphene based structures.26 From the TGA weight loss percentage calculation, we have estimated the approximate GQDs loading of ~42 % compared to AuNPs which is very close to their added precursor ratio of 1:1. The XRD of AuNP shows two characteristic peaks at 2θ = 37.9 and 44.2 for (111) and (200) crystal planes of gold (Figure 1I).27 The XRD patterns of N,S-GQDs exhibit a broad peak centered at 2θ = 24.2°, corresponding to the (002) plane of graphene which is also visible prominently in N,SGQDs@AuNP nanocomposites.23 In addition, the characteristic peaks of AuNP are also appeared here proving the successful formation of N,S-GQDs@AuNP nanocomposites. Similar observation was also noted in the XPS analysis of the AuNPs before and after N,SGQDs addition, as shown in Figure S2. The deconvoluted peak of bare AuNPs shows exclusively Au0. However a significant contribution of deconvoluted Au-S peak has been introduced after formation of N,S-GQDs@AuNP nanocomposites, clearly corroborating the 13 ACS Paragon Plus Environment


TEM, XRD and TGA analysis for successful formation of N,S-GQDs@AuNP nanocomposites. Serotype Detection by Optical Method. The detection of the specific type of DNA (serotype) among four DENV serotypes is the main aim for the optical analysis which can be further validated by confocal images. As shown in scheme presented in Figure 2A, the N,SGQDs@AuNP-dA nanocomposite was mixed with 1 nM concentration of 4 serotypes of DENV DNA separately (DENV-1, 2, 3 and 4). Then all four types of dye-combined DNA (probe 2) were mixed with four solutions followed by 20 min incubation at 37 °C to get maximum binding with the target strand. According to our hypothesis, in four vials containing different serotype DNA, the nanocomposites capture only their dye-combined complementary one, keeping other three dye-combined DNAs free. Therefore, from four different vials, we can get four different dye-combined N,S-GQDs@AuNP-dA/targetDNA nanocomposites, representing four different targets. The fluorescence intensity was recorded at the wavelength range of 500 – 700 nm under four different excitation wavelengths of 485 (for FITC), 540 (for Cy3), 570 (for Rhodamine X) and 590 nm (Texas red) for each sample. To optimize the nanomolar concentration for fluorometric analysis, we have standardized the concentration dependent fluorescence response in the range of 1 fM − 1 µM where the femtomolar detection is also detectable in fluorescence shown in Figure S3, supplementary information. However to get best results in confocal microscopy, a minimum concentration of nanomolar has been maintained. Before the fluorometric analysis for DENV serotype detection, bare dye conjugated DNAs are examined for any possible quenching with the N,SGQDs as control experiments, presented in Figure S4. The fluorescence of all dye labeled DNAs remained almost same as the excitation wavelengths of dues used are highly different from the emission of N,S-GQDs. In addition, the quantum yield of these fluorophores are relatively high compared with the N,S-GQDs which is also a strong reason for the ignorable 14 ACS Paragon Plus Environment

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effect. It is clear from the Figure 2B, C, D and E, the nanocomposites can distinctly capture their complementary dye-combined probe DNA and show their respective fluorescence signals. Each vial is excited in four different wavelengths to verify the cross excitation of dyes and purity of target. The separated solutions were further analyzed in confocal microscope for confirmation, presented in Figure 2Bi, Ci, Di and Ei. Each sample shows clear image of fluorescence when excited at their corresponding wavelengths. Therefore, from these above results of fluorescence spectroscopy and confocal microscopy, it can be concluded that the N,S-GQDs@AuNP-dA nanocomposites can successfully identify the DENV DNA serotype where 570 nm emitted Cy3 represents DENV-1, 530 nm emitted FITC represents DENV-2, 610 nm emitted RhX represents DENV-3 and 615 nm emitted Texas red represents DENV-4.

Figure 2. Optical detection of DENV serotypes: (A) methodologies for optical analysis, (BE) fluorescence spectra for four DENV DNAs (DENV-1, 2, 3 and 4) with N,S15 ACS Paragon Plus Environment


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GQDs@AuNP-dA and all probes 2, excited each at four different excitations of 485, 540, 570 and 590 nm, (Bi-Ei) Confocal images from their corresponding samples. Each of these four reactions, all parameters are constant, varying only DENV serotype DNA.

Quantification of DNA by Electrochemical Method. After successful identification of serotype by optical analysis, the quantity of the target DNA is examined by electrochemical DPV metric method. The mechanism of DNA detection by DPV is a well-known process in electrochemical sensing where MB is used as a redox indicator as well as a standard DNA intercalator due to its different binding ability towards ssDNA and dsDNA.28 According to the schematic diagram of Figure 3A, the process of analysis is quite similar for first few steps with the previous method in addition of varying concentration. However, after centrifugal separation, the precipitated dye-combined target DNA with N,S-GQDs@AuNP-dA nanocomposites were drop-casted on a finely polyaniline coated Au electrode to make the sensor. The polyaniline film was coated on Au electrode by electrodeposition of aniline in H2SO4 medium.29 This thin layer of polyaniline is used as a well porous matrix to absorb the drop-casted N,S-GQDs@AuNP-dA nanocomposites. The synergistic effect between these two nanomaterials enhanced their electrochemical property largely. The cyclic voltammetry of the bare Au electrode and the N,S-GQDs@AuNPs nanocomposites deposited on the same Au electrode shown in Figure S5 of supporting information indicates the superior electroactivity

of

the

N,S-GQDs@AuNP-dA

nanocomposites.

In

addition,

the

nanocomposites also get enhanced electrochemical property than the bare N,S-GQDs due to the metal-GQDs synergistic effect. Therefore, using the N,S-GQDs@AuNPs nanocomposites, the DPV responses are enhanced largely which is mostly needed for the quantification of small amount of target DNA. The sensor electrode was initially tested for cyclic voltammetry and thereafter DPV metric analysis in a range of DNA concentration of femto to micromolar. 16 ACS Paragon Plus Environment

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CV was carried out to monitor the electrochemical behavior of the sensor electrode before and after of target DNA attachment. As shown in Figure 3B, the Au||N,S-GQDs@AuNP-dA electrode has an excellent electroactivity due to the presence of AuNP and graphitic matrix with a prominent redox peak of MB at -0.42 V.30 Moreover, the sensor electrode can maintain its activity even after loading of micromolar concentration of target DNA which confirms the reliability of the given electrical signal of the sensor. Four concentration dependent DPV metric signal curves are given in Figures 3C, D, E and F which are representing four serotypes of DENV DNA. The calibration lines from the graphs are also plotted in Figures 3Ci, Di, Ei and Fi. Theoretically, the slope of these lines should be equal as the calibration of all duplexes should be same. However, due to small difference in guanidine bases content of four serotypes duplexes, the DNA concentration vs. DPV signal calibration is slightly different from each other.

Figure 3. Electrochemical quantification of DENV DNA: (A) methodologies for DPV analysis,

(B)

CV

of

Au||AuNP@N,S-GQDs-dA

nanocomposites


with

increasing


concentration of DNA loading, (C – F) DPV of four DENV DNAs (DENV-1, 2, 3 and 4) with AuNP@N,S-GQDs-dA, all four probes 2 and 20 µM MB incubation with pulse height of 50 mV and pulse width of 70 ms, at + 0.15 V, (Ci – Fi) Calibration curves from their corresponding DPV in the concentration range of 10-14 to 10-6 M DNA. Each of these four reactions, all parameters are constant, varying only DENV serotype DNA with its concentration.

There are three kinds of binding can take place between MB and DNA; electrostatic, groove and intercalative.31 Our earlier investigations have revealed that MB can interact with ssDNA more compared with dsDNA due to spacious availability in single strand.28, 32 At the same time MB has a strong affinity toward guanine bases of ssDNA; therefore MB signal is very high in presence of ssDNA accumulated on electrode (as in every case of Figure 3C – F). With increase in concentration of complementary strand, extent of duplex (dsDNA) formation increases; bases are wrapped in duplex structure, thus preventing MB-ssDNA interactions. Therefore, the DPV peak height of MB decreases with increase in the complementary target concentration from 1 × 10−14 M to 1 × 10−6 M for all cases with the correlation coefficient (R) of 0.959, 0.969, 0.969 and 0.9981, respectively and LOD of 14.3, 12.6, 9.4 and 9.7 fM, respectively (based on 3 × standard deviation of lowest signal / slope of the calibration line). For the reproducibility of the sensor, we have repeated all the experiments at least thrice and results obtained are satisfactory with small standard deviation values. Two-way-detection. Investigation of known serotype DENV DNA with its detectable range of concentration has been already achieved in previous two sections. The N,SGQDs@AuNP-dA nanocomposites have successfully recognized the serotype of given analytes and simultaneously determined the quantity in the range of 10-14 to 10-6 M 18 ACS Paragon Plus Environment

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concentration for all serotypes. Now, the nanocomposites has been applied for the detection of an unknown concentration of unknown serotype DENV DNA by the followed method presented in Figure 4A, with the help of the preserved optical results and electrochemical calibration lines, established from earlier experiments. To validate our methodology, we prepared a dT labeled DENV-1 DNA solution of 10-8 M concentration and treated it as an unknown sample. Our aim is to find out the serotype as well concentration from our proposed technique. Initially, from fluorometric graph, the separated solution significantly excited at 540 nm, clearly indicating the presence of Cy3 labeled DENV-1 serotype (Figure 4B) whereas at other excitation wavelengths could not emit any noticeable spectra. In addition, the confocal images in Figure Bi also visible only at 540 nm excitation, confirming the presence of DENV-1 serotype. From the electrochemical analysis, we have obtained two curves of Au||N,S-GQDs@AuNP-dA sensor electrode in Figure 4C with and without target DNA. The I/Ii found from the graph has been plotted compared with the calibration lines obtained from previous data of electrochemical analysis (Figure 3Ci – Fi). As the serotype of DNA is already confirmed by fluorescence signal which is DENV-1, the obtained point of I/Ii is validated from the DENV-1 calibration line (Figure 4Ci) and the concentration is found as 10-8 M. The data obtained from our proposed technique is completely corroborated with our spiked sample by qualitatively as well as quantitatively, confirming the detectability of our sensing system. In some recent reports, few attempts have been made on DENV DNA detection as well as serotype identification by different fluorometric or electrochemical techniques, listed in Table 1. Most of studies are mainly focused on the DNA detection using their complementary strand attached on the sensing platform irrespective of any serotype specification. Though some reports are able to detect the DENV DNA by electrochemical process with ultra-low sensitivity, however the processes are either fail to determine the exact serotype or possess high possibilities of false signal. Moreover, very limited literatures



demonstrated about the serotype detection based on fluorophore combined complementary DNA; however their studies highly suffered from the lack of quantification. In this work, our proposed process can successfully determine the serotype of a DENV DNA in presence of strong fluorophore tagged probe. In the simultaneous process, the sensor can quantify the DNA concentration also via electrochemical process. Two different methods confirm two different determining parameters, establishing the proposed sensor ahead from those published reports. However, the pre-labeling process of target analyte DNA with dT is a serious drawback for the applicability of this proposed method. In near future, the poly T generated controlled PCR reaction of unknown DENV DNA could be an option to overcome its limitation. Up to this stage, it can be concluded that the sensor can efficiently detect the DNA serotype and concentration of labeled DENV DNA which can lead a new methodology for the most challenging biomedical application of DENV detection.


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Figure 4. Two-way-detection of DENV DNA by fluorometric for serotype identification and electrochemical for quantification: (A) methodologies for two way analysis, (B) Fluorescence spectrum and (Bi) confocal images for unknown serotype identification at four different excitations, The known serotype of DENV is treated for (C) DPV analysis and (Ci) corresponding point to measure unknown concentration of DNA compared with four different calibration lines, obtained in Figures 3Ci, Di, Ei and Fi. Selectivity test. To check the selectivity of the N,S-GQDs@AuNP-dA sensing probe, both DPV and fluorescence response has been tested in presence of different viral oligomers like, norovirus, zika and influenza along with a single and double mutated target oligomers of DENV-3. The change in fluorescence as well as DPV signal was very less in comparison with for any non-complementary DNAs as shown in Figure 5, confirming the high specificity of developed sensor for serotype identification. The change in DPV signal is quite significant even for the double base mismatched target DENV-3 DNA. However, the change in DPV signal is not satisfactory when the single mismatched target DNA was analysed. The results of experiments performed to prove the selectivity of developed sensor indicate the high specificity other than very close target interference when single base mismatch was used. This disadvantage can be overcome in future by addition of multiple target DNA oligomers from a single whole target DENV DNA and modifying the sensing matrix.



Figure 5. Selectivity test of the proposed sensor with single, double base mismatch of DENV-3 oligomers, Zika, Influenza, Norovirus and completely noncomplementary oligomers along with DENV-3 target DNA, (A) fluorometric response for serotype identification, (B) DPV metric electrochemical quantification and (C) comparative bar diagram. Detection of clinically isolated DENV RNA with the proposed sensor. Clinically isolated DENV-3 RNA was extracted and subjected to amplification using PCR. The amplified product contained a poly T chain at their 5’ end. After the 38th cycle in PCR, the dye-combined probes and N,S-GQDs@AuNP-dA was added into the collected PCR products as shown in the scheme of Figure 6A. The melting temperature of 72°C was maintained for 22 ACS Paragon Plus Environment

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the incubation process to get the best annealing possibility between the probes and the target DNAs. The centrifuged product after incubation was tested for optical as well as electrochemical process. As presented in Figure 6B, the fluorescence signal of Rhodamine B (RhB) has been clearly appeared, indicating the presence of DENV-3 serotype. Additionally, the 10-fold diluted sample of the same PCR product also shows a small peak at 615 nm, attributing the presence of RhB which further prove the successful serotype detection ability of the proposed sensor. Similarly the DPV signal of the sensor electrode, presented in Figure 6C shows the quantity of the target oligomers of 2 × 10-12 M. The 10-fold diluted products shows a little lower concentration of 8 × 10-14 M compared to its expected concentration of 2 × 10-13 M which may be due the matrix effect and very low concentration of the analyte solution. To verify the amplification process using PCR for the target oligomers, the agarose gel analysis was carried out and presented in Figure S5.



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Fugure 6. Detection of clinically isolated DENV-3: (A) PCR supported methodologies for the analysis, (B) Fluorescence spectra for serotype identification and (C) DPV analysis for quantification (inset: ratio of intensities of DENV-3 DNA and its 10-fold diluted solution, compared with the calibration line obtained in DI water).

Table 1. Comparison of the detectability of the proposed sensor with recently reported DENV sensors:

Materials/Method of

Detection

detection

range

Serotype LOD

detection

References

ability

Electrochemical methods Silicon nanowire(GATE)

10-8–10-15 M

2.0 fM

No

14

PEG surface (DPV)

10–100 nM

3.09 nM

DENV-3

33

10 nM–1 µM

6.9 nM

No

34

10 nM–5 µM

9 nM

No

35

1–103 pfu mL-1

1 pfu mL-1

DENV-2

15

-

pM

DENV-1, 2, 3

36

-

yes

17

SPR (LSV) duplex-specific nuclease (DPV) Alumina electrode (DPV) AuNP-PANI (EIS) Fluorometric methods stem/loop-forming

No

oligonucleotide

quantification

Ag nanocluster

0–500 nM

100 nM

yes

37

5–700 aM

4 aM

No

38

10 fM–µM

9.4 fM

Yes

This work

(FAM)-encapsulated liposomes N,S-GQDs@AuNP (Fl for serotype detection and DPV for quantification)


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CONCLUSIONS In this study, we have successfully developed a new class methodology using N,SGQDs@AuNP nanocomposite for the detection of DENV serotype along with DNA quantification. The target DNAs are labeled with a small poly T chain and believed to conjugate with N,S-GQDs@AuNP-dA nanocomposites which is further captured by dyecombined complementary probe DNA for optical detection. The nanocomposites can successfully identify the DENV serotype showing peaks at 570 nm Cy3 emission for DENV1, 530 nm FITC emission for DENV-2, 610 nm RhX emission for DENV-3 and 615 nm Texas red emission for DENV-4. In addition, the Au electrode deposited with N,SGQDs@N,S-GQDs-dA nanocomposites can also quantify of the target DNA in the range of 10-14 to 10-6 M with a detection limit of 9.4 fM. To confirm its applicability in long chained complex DNA system, the sensor was also applied to the clinically isolated DENV-3 and showed satisfactory performances for serotype identification as well as quantification. To the best of our knowledge, this is the first attempt on the DENV serotype detection along with DNA quantification by two way techniques for the detection of DENV. In near future, the poly T generated controlled PCR reaction of unknown DENV DNA with multiple region amplifications can make this proposed mechanism as a potential tool for real sample monitoring.

ASSOCIATED CONTENT Supporting Information



Fluorescence spectra of N,S-GQDs, deconvoluted Au XPS spectra, fluorescence detection of all 4 serotypes of DENV DNAs, effect of N,S-GQDs on dyes, CV of N,SGQDs@AuNP electrode, agarose gel of amplified PCR. This information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected]. Tel (Fax): +81-54-238-4887.

ORCID Enoch Y. Park: 0000-0002-7840-1424 Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS We thank Professor Kouichi Morita of Institute of Tropical Medicine Nagasaki University for providing clinically isolated DENV-3 strain. ADC and JL sincerely thank the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship (Nos. P17359 and P16361). This work was supported partly by the Bilateral Joint Research Project of the JSPS, Japan.

REFERENCES


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Graphical abstract:


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Femtomolar Detection of Dengue Virus DNA with Serotype

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