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Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism
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Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism Ahmad Mobed , Mohammad Hasanzadeh , Nasrin Shadjou , Soodabeh Hassanpour , Arezoo Saadati , Mohammad Agazadeh PII: DOI: Reference:
S0026-265X(19)32262-3 https://doi.org/10.1016/j.microc.2019.104286 MICROC 104286
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
23 August 2019 23 September 2019 24 September 2019
Please cite this article as: Ahmad Mobed , Mohammad Hasanzadeh , Nasrin Shadjou , Soodabeh Hassanpour , Arezoo Saadati , Mohammad Agazadeh , Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism, Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104286
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Highlights
Legionella pneumophila is a causative agent of Pontiac fever and Legionaries’ disease. Cys A/AuNPs modified Ag/GQDs nano-ink was used a novel substrate for the immobilization of ssDNA and probing its hybridization with cDNA. Target DNA was quantified at a linear range from 1 µM to 1 ZM and low limit of quantification (LLOQ) was 1 Zepto-molar
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Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism
Ahmad Mobed a, Mohammad Hasanzadeh
b*
, Nasrin Shadjou
b,*
, Soodabeh Hassanpour c,
Arezoo Saadati d, Mohammad Agazadeh a
a
Department of Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences,
Iran. b
Pharmaceutical Analysis Research Center and Faculty of Pharmacy, Tabriz University of
Medical Sciences, Tabriz, Iran. c
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
d
Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences,
Tabriz, Iran.
Corresponding authors. [email protected]; [email protected]
2
Abstract Rapid screening of pathogenic microorganisms are widely demand in the last decade. According to fastidious and hard to grow nature, Legionella penumophila is important pathogen for bioassay and biomedical analysis. In this work a novel paper-based genoassay was developed for monitoring of L. penumophila. For the first time, silver-graphene quantum dots (Ag/GQDs) ink was synthesized and used for construction of new substrate for bioassay of L. penumophila. The prepared interface was modified by gold nanoparticles grafted by Cysteamine A (CysA/AuNPs) which is necessary for ssDNA immobilization and hybridization by cDNA. All of the genoassay fabrication steps were characterized by field emission scanning electron microscope (FE-SEM), Energy-dispersive X-ray spectroscopy (EDS), Atomic force microscopic (AFM) and also TEM (transmission electron microscopy). Using chronoamperometry technique, the measurement of target cDNA was performed successfully. Also, cDNA was determined in the linear rang of 1µM to 1ZM which low limit of quantification was 1ZM. The results show that the designed bioplatform despite a simple structure with high sensitivity and specificity for the DNA based bioassay of L. penumophila genome. Keywords: biosensor, nanotechnology, bioanalysis, advanced nanomaterial, nucleic acid, hybridization.
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1. Introduction In the last decade development of biosensors has been increased considerably (1). DNA based biosensors for the detection of target sequences have enticed ever increasing interests in association with highly challenging research efforts directed to gene analysis, clinical disease diagnosis, or even scientific applications (2, 3). Different biosensing methods such as optical, electrochemistry, surface plasmon resonance (SPR), have been well established for target DNA detection (4). Amongst them, electrochemistry revealed great advantages such as simple, rapid, low-cost and high sensitivity (5). On the other hand, the use of nanomaterials in biosensor construction shows excessive potential for monitoring biomolecules and sensitive detection of various targets. Nanomaterials can be used as carriers to load signal markers, straight as signal reporters for sensitively detection of analytes. On the other hand, nanomaterials can increase speed of electron transfer used as functional materials on electrode surfaces (6, 7). Because of effective way to develop miniature, light , eventually compostable and low cost, today, paper-based biosensors have gained much consideration as a detection tools (8). The first paper-based sensor can be considered the invention of paper chromatography by Martin and Synge, who were awarded with the Nobel Prize in chemistry in 1952 (9, 10). Printed electrodes on paper have already been used as a detection device in several aspect such as electrochemical (11), electrochemiluminescence (12), photoelectrochemical (13-15). Recently, paper-based biosensors extensively developed for detection of pathogenic bacteria. In 2011, Chen-zhong Li and coworker present a novel paper-based biosensor for detection of Pseudomonas aeruginosa and Staphylococcus aureus. They used gold nanoparticles (AuNPs) to increased sensitivity of biosensor (16). During a
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novel work an immunochromatographic strip test using gold nanoparticles was established for the rapid detection of Salmonella typhi (S. typhi) in human serum. Fabricated paper-based biosensor was ideal for rapid, simple and low-cost detection of S. typhi (17). Reported paperbased biosensor by Saravanan Rengaraj and coworker not only were remarkable simplicity but also were cost-effectiveness and biodegradability (8). Also a paper-based diagnostic tool has been developed for detection of E.coli by S. J. Reinholt and coworker. They used of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs). Finding revealed wide range of sensitive and specific bioassays with noteworthy advantages over their traditional complements (18). Detail of some bacterial paper-based biosensors summarized in Table S1 (see supporting information). L. pneumophila is an important human pathogen found ubiquitously in freshwater environments and According to World Health Organization (WHO), mortality rate associated with Legionnaires' disease is up to 40% among average patients and up to 80% among immunosuppressed patients (19). To date, several methods from microscopic observation to molecular based tools applied for detection of L. pneumophila. Additionally different type of biosensors such as DNA based biosensor (20), immunosensor (21) fabricated for rapidly and sensitively detection of L. pneumophila. According to low cost and simple preparation of paper-based biosensor, a novel genosensor was fabricated on the surface of photographic paper modified by silver nanoparticle-graphene quantum dots (Ag/GQDs) nano-ink grafted by CysA-AuNPs toward detection of mip gene from Legionella. Finally, Ag/GQDs nano-ink modified by CysA-AuNPs not only increased the amount of immobilization of probe (ssDNA) but also improved sensitivity of paper-based genosensor for targeting of dsDNA.
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Additionally, engineered genosensor can be used as a simple and low cost biodevice for the detection of legionella ever for environmental or clinical samples. 2. Material and Methods 2.1. Chemicals and Reagents All of oligonucleotide sequences including probe l. pneumophila: 5ʹ SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG 3ʹ. Complementary target sequence (5ʹ ACA AAA GGG GCG GGG AGA GTA 3ʹ. single nucleotide mismatch target sequence (5ʹ ACA AAA GGAGCG GGG AGA GTA 3ʹ. three nucleotide mismatch target sequence (5ʹ GCA AAA GGG GCG GGG AGA GGG 3ʹ. All DNA oligonucleotide sequences were obtained from Takapouzist Co. (Iran). The oligonucleotide stock solutions were diluted with 0.1 M TrisHCl buffer, pH 7.4 solution (Tris). Dttiothereitol (DTT), Toluidine blue (TB), Potassium ferrocyanide K4Fe(CN)6, potassium ferricyanide K3 Fe(CN)6, Sodium acetate, AgNO3, citric acid, CysA, hydrogen tetrachloroaurate(III)hydrate (HAuCl4·3H2O), Mercaptoethanole (MCE), were obtained from Sigma-Aldrich (Ontario, Canada).A ferricyanide/ferrocyanide solution containing 0.5 M KCl and 0.5 M of K4 Fe(CN)6/K3Fe(CN)6 (1:1) were prepared for the electrochemical measurement of the microscopic surface areas of the working electrodes. Piranha solution was prepared by mixing hydrogen peroxide and concentrated sulfuric acid in a 1:2 (v/v) ratio. It was prepared immediately prior to use to maintain its reactivity. A stock solution of 20 µM TB was prepared in Tris-HCl and employed as redox indicator for revealing DNA hybridization. All the above solutions were kept at -4 ◦C before use. DTT solution containing 10 mM sodium acetate and 500 mM of DTT, pH=5.2 was prepared and kept at -4 ◦C. For preparation of the substrate, polyvinyl pyrrolidone (PVP) K-30 (molecule weight ≈ 300 g), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-
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hydroxysuccinimide (NHS), bovine serum albumin (BSA), potassium ferricyanide K3 Fe(CN)6, potassium ferrocyanide K4 Fe(CN)6, sodium hydroxide, disodium hydrogen orthophosphate, potassium dihydrogen orthophosphate potassium chloride, sodium citrate, cysteamine hydrochloride, hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O), graphite, ethylene glycol (EG), and ethanol were purchased from Sigma-Aldrich (Ontario, Canada). 2.2. Instrument Electrochemical processes were carried out with a PalmSens 4c system, driven with PSTrace5 software. The equilibrium time was 2 s and the employed ac voltage amplitude was 10 mV. Pt wire was applied as a counter electrode and Ag/AgCl (Metrohm, The Netherlands) as a reference electrode. Ag/RGO paper electrode which synthesized and prepared by ourselves was applied as a working electrode. FE-SEM (high-resolution field emission scanning electron microscope), Hitachi SU8020, Czech with an operating voltage of 3 kV was applied for characterization of electrode surface morphology, and EDS (energy dispersive spectroscopy) coupled with the FE-SEM apparatus was applied for analysis of electrode chemical compositions. TEM (transmission electron microscopy), Adelaide, Australia with an operating voltage of 200 kV was applied for investigation of synthesis mechanism. Size distribution and zeta potential of Ag/RGO nano-ink was analyzed by DLS (dynamic light scattering) with zeta potential instrument Malvern Instruments Ltd (Zetasizer Ver. 7.11, MAL1032660, England). Ohmmeter, XIOLE, XL830L, China, multimeter was applied for resistivity and conductivity nano-ink estimate. 3. Result and discussion
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Synthesis of graphene quantum dots doped by silver nanoparticle (Ag/GQDs) conductive nano-ink Graphene quantum dots (GQDs) were synthesized according to our pervious works (22, 23). Next, 2 mL of GQDs solution was heated at 60 0C in water bath. Then 0.6 gr graphite and 0.4 g of PVP were added into synthesized GQDs. The mixture was magnetically stirred until complete dissolving of the PVP and graphite powder. Then, 600 µL aqueous solution of AgNO3 (0.2 M) was directly added into above solution and at least 5 min magnetically stirred in order to guarantee efficient blending of GO and Ag +. The mixed solution maintained 12 h at 60◦C in incubator without agitation. In the following the mixture solution magnetically stirred at 80◦C again. Finally, 400 µL of sodium hydroxide (NaOH, 8 M) solution was drop to mix in order to enhance the reduction impact. Subsequently, the prepared solution centrifuged for 30 min at 8000 rpm and 3 times washed with distilled water (DI) for removing extra reactant. In order to produce hybrid conductive ink Ag/GQDs nano-ink were dispersed into DI water, ethanol, ethylene glycol with volume ratio of 9:9:3 and sonicated for 30 min which resulted in production of carbon water-based ink. After completion of the synthesis process, the produced nano-ink was stored for 24 h at room temperature before. Conductivity study of Ag/GQDs nano-ink For this purpose, different conductive patterns designed on the surface of photographic sheets via Ag/GQDs pellet with different thicknesses. As shown in Fig. S1, different types of conducting lines, a dip LED with a rated voltage of 3.0 V and 15.0 V battery was fabricated on paper substrate. One of the legs of LED (cathode) was connected to battery anode and so cathode surface of battery fixed to one side of conductive line. Once the anode leg of LED touched to other side of conductive line, the electronic circuit switched on and the LED was
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lighted up. Afterwards, the resistance of conductive lines was measured by ohmmeter. Also the conductivity measurements obtained 266 μs for Ag/GQDs nano-ink. In order to demonstrate the reliability and flexibility, the conductive patterns bended outwards by 90 ˚C. CysA-Au NPs synthesis In a brief description, 50 mL of 0.5 mM HAuCl4 was magnetically stirred and heated until boiling points under reflux states. Subsequently, 5 mL of 38.8 mM sodium citrate (Na3C6H5O7) solution was added to the above solution. While the sodium citrate solution added, the yellow color of solution changed into wine-red. The mixture was continued magnetically stirring until wine-red color was fixed. Then, in order to functionalizing citrate capped-Au NPs with CysA through mixing of CysA and Au NPs with 100:1 volume ratio and stirring for approximately 12 h with gradually addition of CysA. The latest color of CysA-Au NPs mixture is deep red color and has the stability up to 2 months at 4˚C. Characterizations The TEM, XRD, Raman spectra, FE-SEM, EDS and DLS of Ag/GQD nano-ink, AgGQD/CysA-Au NPs photographic paper electrode modified with pDNA and cDNA To investigate the mechanism of Ag/GQDs composite formation and validate the linking of AgNPs and GQDs onto graphite sheets, TEM images of Ag/GQD nano-ink were recorded and shown in Fig. S2. It was obviously displayed that the numerous dispersed spherical AgNPs and GQDs were decorated the graphite sheets. Abundant crystal nuclei were created promptly when Ag NPs were added into mixture of nano-ink. PVP played as a mild reluctant and also as protectant which was absorbed onto the nuclei instantly at this moment. Therefore, prevent aggregation and merger of nuclei into larger and irregular nanoparticles.
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All of these results was confirmed by XRD and Raman spectra (Fig. S2 (B and C) (see supporting information)). Fig. S3 (see supporting information), demonstrates the FE-SEM images of bulk Ag/GQDs nano-ink, Ag-GQDs/CysA-Au NPs paper electrode modified with pDNA and AgGQDs/CysA-Au NPs-pDNA paper-based genosensor after incubation with cDNA to investigate the morphology of surface. The images reveal excellent Ag NPs dispersion and distribution on the bulk solution sample of synthesized Ag/GQDs nano-ink. The small sized AgNPs and GQDs with spherical uniform structure decorated on the graphite sheets. The following images approve that AgNPs present in synthesized Ag/GQDs nano-ink as electrical signal amplification element. The formation and distribution of Ag NPs shown in different magnification in Fig. S3B. It is interesting to observe that Ag/GQDs nano-ink prepared in water/Et/Eg demonstrates no aggregation of nanoparticles. When Ag/GQDs nano-ink was applied to make patterns on the surface of photographic papers, it stuck to the surface of paper, aggregate randomly. In following of the standard colloids theory, the colloid stability was controlled by the balance between charged particles, columbic repulsion and van der Waals interaction. It is important to point out that PVP reduced Ag/GQDs nanoink has a remarkable impact on conductivity alteration due to anchoring of Ag NPs and GQDs onto graphitic sheets. As the reduction process continued, more and more Ag NPs and GQDs anchored onto graphitic sheets. The FE-SEM images of CysA-Au NPs/Ag-GQDs nano-ink shows spherical particles with uniform structure (Fig. S3 (G-I) (see supporting information)), revealing that the Au NPs were indeed assembled onto the surface of thiolated Ag-GQDs nano-ink. Interestingly, more dispersed and less aggregated Au NPs are seen upon the electrostatic interactions of CysA-
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Au NPs with the Ag-GQDs nano-ink. Possibly the binding of positively charged Ag-GQDs to the negatively charged CysA-Au NPs reduced the aggregation of the NPs through steric hindrance, although the loss of some AuNPs from the GQDs surface cannot be eliminated. Interestingly, after incubation of pDNA with CysA-Au NPs/Ag-GQDs nano-ink, the morphology of electrode was changed. It’s found the, a binding between CysA-Au NPs/AgGQDs and nucleic acids was occurred (Fig. S3 (A-C) (see supporting information)). Therefore, pDNA was successfully immobilized on the surface of CysA-Au NPs/Ag-GQDs nano-ink modified paper electrode. More importantly, incubation of cDNA on pDNA modified CysA-Au NPs/Ag-GQDs nano-ink lead to new change on the morphology of electrode. As can be seen, the cDNA was linked to pDNA using hybridization strategy (Fig. S3 (D-F) (see supporting information)). These results confirmed successful assembly of the paper-based genosensor. Also, identification of synthesized Ag/GQDs nano-ink elements is also performed by to Energy-dispersive X-ray spectroscopy (EDS). According to Fig. S4 (see supporting information), the quantitative results confirm the presence of carbon elements with high frequency and Ag NPs in bulk sample. As evident in Fig. S4, signals for O, and C elements belong to both CysA and GQD, while S and N is only attributed to CysA. The large and sharp C peaks are the typical signal for carbon elements in the Ag/GQD nano-ink. Also, in the case of Ag-GQD/CysA-Au NP indicates that Au element overlapped with S element. Zeta potential analysis of Ag/GQD nano-ink was studied and the zeta potential value was 3.55 mV. TEM, FE-SEM, AFM, FTIR, Spectrofluorimetric and spectrophotometric characterization of Au NPs and CysA/Au NPs
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The size and shape of the CysA-Au NPs were approved by TEM (transmission electron microscope) and FE-SEM (Fig. S(5-7) see supporting information). The images exhibited that the spiral shaped particles for both AuNPs and CysA/Au NPs. Despite the secondary functionalization of AuNPs, the spiral shape is conserved while a slight increment in the size of AuNPs could be observed. Images of AFM provide three dimensional structural information about CysA-Au NPs (Fig. S8). Zeta potential analysis of AuNPs was investigated and the zeta potential value was -29.1 mV. Using DLS, the hydrodynamic particle size of AuNPs was about 20 nm. FTIR was used to study of functional group of AuNPs and CysA-AuNPs. As demonstrated in Fig. S9, the main peaks of the AuNPs and CysA-AuNPs are located at 3447, 2359 and 1653, which O−H, C−O and C=O functional groups. The N−H and C=O were located at 3500 and 1630−1690 cm -1 which are finger print peak of amide bond formation. UV-Vis and spectrofluorimetric spectra of Au NPs and CysA/Au NPs are shown in Fig. S9 (B and C). As shown in Fig. S9B, Au NPs has an absorption peak at about 320 nm. As can be seen in Fig. S9C, Au NPs emit fluorescence signal at 580 nm but due to the low quantum yields, the Au NPs were not further utilized in sensing applications. Fabrication of Ag/GQDs-CysA/Au NPs paper-based electrode For this purpose, through utilization of Ag/GQDs composite ink make pattern as working electrode and counter electrode on the surface of photographic sheets and let them dried on the stirrer for 15 min. The prepared Ag/GQDs nano-ink fabricated on the surface of photographic paper were treated with vaporous of hydrazine hydrate which is necessary for further electrodeposition of CysA/Au NPs on the surface of paper deposited by Ag/GQDs nano-ink. So, the paper dipped into CysA/Au NPs solution and electrodeposition was
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performed by chronoamperometry technique at E=0.8 V and duration time of 500 s that leads to attachment of amine groups of CysA/Au NPs with COOH groups of Ag/GQDs paperbased electrode (Fig. 1). Then, CysA/Au NPs-Ag/GQDs paper-based electrode was rinsed with distilled water in order to remove unbound moieties and maintained to dry at room temperature. Genosensor assembly To prepare the DNA biosensor, pDNA combine the designed probe with DTT. DTT was used as a reducing or "de-protecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated DNA have an affinity to form dimers in solution, particularly in the existence of oxygen. Dimerization significantly lowers the efficiency of consequent coupling reactions such as DNA immobilization on gold in biosensors. Usually DTT is mixed with a DNA solution and permitted to react, and then is removed by filtration or by chromatography (for the liquid form). The DTT removal procedure is often called "desalting." Generally, DTT is used as a protecting agent that prevents oxidation of thiol groups DTT is an important smallmolecular reducing agent and has wide applications in chemistry, biochemistry, peptideprotein reaction and clinical medicine (19-21). In the present study, a 10 µL of DTT solution was added to a 10 µL solution of p-DNA, mixed by a vortex and placed at ambient temperature for 30 min. Later 6 h incubation in -4
°C
, and rinsed the surface of
electrode. At this step, p-DNA self-assembling on the AuNPs was occurred, and a pDNA monolayer was formed by Thiol-gold covalent bonds. No further layers (rather than a monolayer) were formed, because it needed attachment of further p-DNA. on the pre-formed p-DNA layer on the electrode surface. In the next step and after rinsing with redistilled water, the electrode was immersed into MCH for 30 min. Self-
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assembled monolayer formed upon p-DNA immobilization may contain pinholes, limbered p-DNA, defects etc. 6-mercapto-1-hexanol immobilization caused to filling the remaining gold sites and caused to form a well-aligned p-DNA monolayer. The obtained electrode is denoted as DNA biosensor throughout the text. Subsequently, electrode were immerged in solution of TB containing Tris-HCl. TB is a basic thiazine metachromatic dye with high affinity for acidic tissue components (22). Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components, thereby staining tissues rich in DNA and RNA. Toluidine blue (TB) has an affinity for nucleic acids, and for that reason binds to nuclear material of tissues with a high DNA and RNA content (23). In this regard, we used the TB to mark the DNA probe. Also, Scheme 1 shows different steps for the fabrication of paper-based genosensor for the detection of L. Penumophila. Optimization of incubation time by TB In the next step, pDNA treated by ditiothreitol (DTT) appropriately for 30 m. DTT, also known as Cleland's reagent, is a small-molecule redox reagent. Its oxidized form is a disulfide-bonded 6-membered ring (24, 25). DTT is widely used in biochemistry works to reduce dissulfide bridges, protect biomolecules, in sample preparation, and to denature proteins before electrophoresis analysis (SDS-PAGE). DTT is commonly used to reduce the disulfide bonds of proteins and peptides (26). DTT cannot reduce buried (solventinaccessible) disulfide bonds, so reduction of disulfide bonds is occasionally approved under denaturing conditions. The terminal sulfur atoms of thiolated DNA have a tendency to form dimers in solution, particularly in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in
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biosensors (27, 28). 10 µL of prepared solution was pipetted onto the working electrode surfaces and incubated in 4℃ for 6 h. After rinsed by DI to remove unbound DNA probe, the electrode immerged in 2-mercaptoethnol (MCE) for 30 min. Several studies revealed that treatment of DNA probe-modified electrode by MCE solution minimized non-specific binding and control the density of DNA probe on the electrode surface by giving a spacer between them to increase hybridization efficiency (29, 30). The hybridization can be exhibited via the redox signal of an electrochemical indicator, which can be an organic molecule (31), metal complexes (32), methylene blue (MB) (33), toluidine blue(TB) (20), enzymes, redox labels or nanoparticles or using technique direct oxidation(34). Label-free indicators are small, electroactive DNA-intercalating or groove-binding substances, which possess a much higher affinity for the resulting hybrid compared to the single-stranded probe. Therefore, the concentration of the indicator at the electrode surface be different when hybridization occurs, resulting in altered electrochemical signals. Besides effective differentiation between ss-DNA and ds-DNA, the indicator should possess a well-defined, low-potential, voltammetric response(35). Therefore, the electrode was immerged in TB in various time (2, 5, 7, 10, and 15 min) for obtain optimum incubation time. To achieve best result ChA technique was applied appropriately. As showed in Fig. 3, the most intensity current recorded in 7 min. Therefore, 7 min was selected as optimum incubation time in this part. Optimization of hybridization time of cDNA DNA hybridization deals notable consideration as result its specificity, selectivity, and sensitivity which is appropriate for miniaturization as it has compatibility with microfabrication technology.
Additionally, nanomaterials enhanced the performance of
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electrochemical application through advancing bio-compatibility, accelerating electron transfer rate due to this enhanced signal can be achieved (36). In this work, 10 µl of cDNA (1µM) pipetted on the surface of biosensor and incubated in different time (10, 15, 20, 30, 40, and 60 m) in 37 ℃. For monitoring hybridization, ChA technique was applied properly. As showed in Fig. 4, 30 min was selected as optimum incubation time for appropriate hybridization. After hybridization of pDNA with c-DNA, the peak currents was changed obviously. This shift further confirms the binding of TB with the cDNA structure via hydrophobic attractions at the concentration range employed in this study. Higher positive shift in the formal potential of TB as a result of binding with cDNA can be an indicator for stronger attraction between TB and cDNA. Meanwhile, it has been reported that TB intercalates into the DNA base pairs at low concentrations, whereas it attaches to the negatively charged phosphate groups at higher concentrations (27). It can be concluded thus that TB binds with cDNA in a higher extent compared to cDNA. The peak current can therefore be employed to discriminate between cDNA and pDNA structures at the electrode surface. The decrement in the peak current of TB interacted with cDNA can be also related to both the effect of stronger binding of TB with cDNA (compared to pDNA),
and/or lower concentration of the bonded TB to DNA. Based on presented
hypothesis, it can be concluded that TB more strongly bind to cDNA. This binding suppresses the electron exchange between TB and underlying surface. Therefore, TB was employed as the redox hybridization marker for fabrication of the DNA biosensor. In the following, however, DPVs were measured for the reduction of bound TB into the biosensor to evaluate
its performance because differential pulse voltammetry is more
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sensitive than cyclic voltammetry, and the reduction peak current of TB is greater than the oxidation one. According to this phenomena, electrochemical behavior of the paper-based was evaluated by ChA technique. As displayed in the Fig. S10A (see supporting information), after modification of electrode by Ag/GQDs-Cys A-AuNPs, recorded curve was altered which established the fabrication of nanomaterial on the surface of paper electrode. According to obtained result, after modification of (Ag/GQDs) nanoink-paper electrode, significant change on the currents were observed which is related to the hindrance effect of AuNPs. Interestingly, after immobilization of ssDNA, the current (-I/µA) was increased. This confirmed successful preparation of genosensor. Finally, after hybridization by dsDNA, the current (-I/µA) was further increased which proved interaction of appropriate ssDNAdsDNA. All of those results confirmed appropriate construction of biodevice. Analytical study The interaction between DNA probes and complementary sequence evaluates significantly Ag/GQDs nano-ink modified by CysA-AuNPs which effects the performance of electrochemical DNA genosensor. In this regard, various concentration of cDNA (10 -6, 10-9, 10-17, and 10-21) was immobilized on the surface of genosensor. For this purpose, 10 µl of cDNA pipetted on the surface of created genosensor and incubated for 30 min in 37 ℃. After incubation and washing by DW, ChA technique applied for monitoring of hybridization in different concentration and calibration curve were obtained which the linear rang was 1µM to 1 ZM. Also, low limit of quantification (LLOQ) was 1 ZM (Fig. 4). According to Fig. 4, after hybridization of 5ʹ SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG 3ʹ with complementary target sequence (5ʹ ACA AAA GGG GCG GGG
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AGA GTA 3ʹ., the maximum peak current was appeared at 50 µA for 1 µM of cDNA. Interestingly, there is a linear relationship between peak current and concentration of cDNA. As can be seen in Fig S4B, by reduction of cDNA concentration to 1ZM, the peak current was decreased to 20 µA which regression equation was obtained as I (µA)=-11.38 CcDNA+64.46. These results confirmed that, proposed genosensor is sensitive to detection of cDNA sequence in low concentrations (1 ZM). Also, a wide dynamic range (1 ZM to 1 µM) was obtained by this paper passed biosensor. The appropriate limit of quantification can be considered as a result of several factors involved in the preparation of the desired genosensor, including excellent electrical conductivity, structural properties and high stability of the Ag/GQDs nano-ink, appropriate surface area which providing the feasibility of biological activity was used to identify hybridization of pDNA with cDNA. In addition, Cys A/AuNPs has double role in this biosensor. The terminal sulfur atoms of thiolated DNA has so affinity to the AuNPs which lead to stable interaction of S-Au. Also, Cys A/AuNPs led to signal amplification which is necessary for increment of the genosensor sensitivity. Therefore, using the engineered genosensor excellent hybridization was occurred and appropriated peak current and LLOQ were obtained. Stability assessment Stability of the paper-based genosensor was investigated by ChA technique from 0-4 h. For this purpose, after preparation of biosensor, ChA was recorded immediately, 2 h and 4 h according previous steps. As showed in the Fig.S11A (see supporting information), in earlier time recorded curve indicated good stability but after 4 h stability decreased, significantly. Finding revealed that stability of the fabricated genosensor is acceptable up to 2h.
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According to Fig. S11, immediately after preparation of genosensor, the peak current was appeared at -4µA. But after 2h and 4h incubation, these peaks were observed in -6.1 and -7.3 µA. According to this result, we should be use this genosensor immediately after preparation. The appropriate stability of the genosensor can be considered as a result excellent stability of Ag/GQDs nano-ink on the surface of photographic paper. Selectivity of study Selectivity of electrochemical biodevice are paramount in emerging electrochemical DNA biosensors. Planned genosensors, despite having a very simple and cost effective structure, it has a high selectivity. For monitoring the selectivity, negative control of experiment were DNA sequence with mismatch in 1 and 3 nucleotides. After hybridization with cDNA, mismatch 1 and mismatch 3, ChA were recorded. As displayed in Fig. S12 (see supporting information), engineered DNA biosensor shows selectively response to the target sequence (cDNA) dissimilar from mismatch sequences. Engineered paper-based nanobiosensors revealed to be exceptional biodevice for detection of specific sequence of mip gene from L. penumophila. According to Fig. S12, after hybridization of 5ʹ SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG 3ʹ with complementary target sequence (5ʹ ACA AAA GGG GCG GGG AGA GTA 3ʹ., the peak current was appeared at 12 µA. But by using single nucleotide mismatch target sequence (5ʹ ACA AAA GGA GCG GGG AGA GTA 3ʹ) and three nucleotide mismatch target sequence (5ʹ GCA AAA GGG GCG GGG AGA GGG 3ʹ) peak currents were appeared at 8 µA and 6 µA, respectively. These results confirmed that, engineered biosensor is selective to detection of target sequence of L. pneumophila. 4. Conclusion
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In summary, a novel nanomaterial improved paper-based genosensor for facile and inexpensive detection of L. penumophila was developed. Engineered paper-based nanobiosensors revealed to be exceptional biodevice for detection of specific sequence of mip gene from L. penumophila. Actually the simple nature of the genosensor makes it available for appropriate applications, meanwhile is portable and user friendly. Also, the rapid detection is critical in many situations such as illness diagnostics which lead to a fast treatment. This bio device is fast to response and can be used as an alternative tool for labor and time consuming methods. Therefore, in the near prospect the paper-based diagnostic biodevices are estimated to bring progresses in terms of sensitivity improvement as well as the multiple molecules detection ability. The expected bacterial detection limit was as 1 ZM and linear range were 1 µM to1 ZM which is lower than other comparable sensors previously described. Acknowledgments This research work was supported by Tabriz University of Medical Sciences. Conflict of interest There is no conflict of interest.
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Scheme 1: Schematic representation of paper based genosensor for the detection of L. Penumophila
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Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism Ahmad Mobed , Mohammad Hasanzadeh , Nasrin Shadjou , Soodabeh Hassanpour , Arezoo Saadati , Mohammad Agazadeh PII: DOI: Reference:
S0026-265X(19)32262-3 https://doi.org/10.1016/j.microc.2019.104286 MICROC 104286
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
23 August 2019 23 September 2019 24 September 2019
Please cite this article as: Ahmad Mobed , Mohammad Hasanzadeh , Nasrin Shadjou , Soodabeh Hassanpour , Arezoo Saadati , Mohammad Agazadeh , Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism, Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104286
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Highlights
Legionella pneumophila is a causative agent of Pontiac fever and Legionaries’ disease. Cys A/AuNPs modified Ag/GQDs nano-ink was used a novel substrate for the immobilization of ssDNA and probing its hybridization with cDNA. Target DNA was quantified at a linear range from 1 µM to 1 ZM and low limit of quantification (LLOQ) was 1 Zepto-molar
1
Immobilization of ssDNA on the surface of silver nanoparticles-graphene quantum dots modified by gold nanoparticles towards biosensing of microorganism
Ahmad Mobed a, Mohammad Hasanzadeh
b*
, Nasrin Shadjou
b,*
, Soodabeh Hassanpour c,
Arezoo Saadati d, Mohammad Agazadeh a
a
Department of Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences,
Iran. b
Pharmaceutical Analysis Research Center and Faculty of Pharmacy, Tabriz University of
Medical Sciences, Tabriz, Iran. c
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
d
Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences,
Tabriz, Iran.
Corresponding authors. [email protected]; [email protected]
2
Abstract Rapid screening of pathogenic microorganisms are widely demand in the last decade. According to fastidious and hard to grow nature, Legionella penumophila is important pathogen for bioassay and biomedical analysis. In this work a novel paper-based genoassay was developed for monitoring of L. penumophila. For the first time, silver-graphene quantum dots (Ag/GQDs) ink was synthesized and used for construction of new substrate for bioassay of L. penumophila. The prepared interface was modified by gold nanoparticles grafted by Cysteamine A (CysA/AuNPs) which is necessary for ssDNA immobilization and hybridization by cDNA. All of the genoassay fabrication steps were characterized by field emission scanning electron microscope (FE-SEM), Energy-dispersive X-ray spectroscopy (EDS), Atomic force microscopic (AFM) and also TEM (transmission electron microscopy). Using chronoamperometry technique, the measurement of target cDNA was performed successfully. Also, cDNA was determined in the linear rang of 1µM to 1ZM which low limit of quantification was 1ZM. The results show that the designed bioplatform despite a simple structure with high sensitivity and specificity for the DNA based bioassay of L. penumophila genome. Keywords: biosensor, nanotechnology, bioanalysis, advanced nanomaterial, nucleic acid, hybridization.
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1. Introduction In the last decade development of biosensors has been increased considerably (1). DNA based biosensors for the detection of target sequences have enticed ever increasing interests in association with highly challenging research efforts directed to gene analysis, clinical disease diagnosis, or even scientific applications (2, 3). Different biosensing methods such as optical, electrochemistry, surface plasmon resonance (SPR), have been well established for target DNA detection (4). Amongst them, electrochemistry revealed great advantages such as simple, rapid, low-cost and high sensitivity (5). On the other hand, the use of nanomaterials in biosensor construction shows excessive potential for monitoring biomolecules and sensitive detection of various targets. Nanomaterials can be used as carriers to load signal markers, straight as signal reporters for sensitively detection of analytes. On the other hand, nanomaterials can increase speed of electron transfer used as functional materials on electrode surfaces (6, 7). Because of effective way to develop miniature, light , eventually compostable and low cost, today, paper-based biosensors have gained much consideration as a detection tools (8). The first paper-based sensor can be considered the invention of paper chromatography by Martin and Synge, who were awarded with the Nobel Prize in chemistry in 1952 (9, 10). Printed electrodes on paper have already been used as a detection device in several aspect such as electrochemical (11), electrochemiluminescence (12), photoelectrochemical (13-15). Recently, paper-based biosensors extensively developed for detection of pathogenic bacteria. In 2011, Chen-zhong Li and coworker present a novel paper-based biosensor for detection of Pseudomonas aeruginosa and Staphylococcus aureus. They used gold nanoparticles (AuNPs) to increased sensitivity of biosensor (16). During a
4
novel work an immunochromatographic strip test using gold nanoparticles was established for the rapid detection of Salmonella typhi (S. typhi) in human serum. Fabricated paper-based biosensor was ideal for rapid, simple and low-cost detection of S. typhi (17). Reported paperbased biosensor by Saravanan Rengaraj and coworker not only were remarkable simplicity but also were cost-effectiveness and biodegradability (8). Also a paper-based diagnostic tool has been developed for detection of E.coli by S. J. Reinholt and coworker. They used of electrospun nanofibers as functional material in paper-based lateral flow assays (LFAs). Finding revealed wide range of sensitive and specific bioassays with noteworthy advantages over their traditional complements (18). Detail of some bacterial paper-based biosensors summarized in Table S1 (see supporting information). L. pneumophila is an important human pathogen found ubiquitously in freshwater environments and According to World Health Organization (WHO), mortality rate associated with Legionnaires' disease is up to 40% among average patients and up to 80% among immunosuppressed patients (19). To date, several methods from microscopic observation to molecular based tools applied for detection of L. pneumophila. Additionally different type of biosensors such as DNA based biosensor (20), immunosensor (21) fabricated for rapidly and sensitively detection of L. pneumophila. According to low cost and simple preparation of paper-based biosensor, a novel genosensor was fabricated on the surface of photographic paper modified by silver nanoparticle-graphene quantum dots (Ag/GQDs) nano-ink grafted by CysA-AuNPs toward detection of mip gene from Legionella. Finally, Ag/GQDs nano-ink modified by CysA-AuNPs not only increased the amount of immobilization of probe (ssDNA) but also improved sensitivity of paper-based genosensor for targeting of dsDNA.
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Additionally, engineered genosensor can be used as a simple and low cost biodevice for the detection of legionella ever for environmental or clinical samples. 2. Material and Methods 2.1. Chemicals and Reagents All of oligonucleotide sequences including probe l. pneumophila: 5ʹ SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG 3ʹ. Complementary target sequence (5ʹ ACA AAA GGG GCG GGG AGA GTA 3ʹ. single nucleotide mismatch target sequence (5ʹ ACA AAA GGAGCG GGG AGA GTA 3ʹ. three nucleotide mismatch target sequence (5ʹ GCA AAA GGG GCG GGG AGA GGG 3ʹ. All DNA oligonucleotide sequences were obtained from Takapouzist Co. (Iran). The oligonucleotide stock solutions were diluted with 0.1 M TrisHCl buffer, pH 7.4 solution (Tris). Dttiothereitol (DTT), Toluidine blue (TB), Potassium ferrocyanide K4Fe(CN)6, potassium ferricyanide K3 Fe(CN)6, Sodium acetate, AgNO3, citric acid, CysA, hydrogen tetrachloroaurate(III)hydrate (HAuCl4·3H2O), Mercaptoethanole (MCE), were obtained from Sigma-Aldrich (Ontario, Canada).A ferricyanide/ferrocyanide solution containing 0.5 M KCl and 0.5 M of K4 Fe(CN)6/K3Fe(CN)6 (1:1) were prepared for the electrochemical measurement of the microscopic surface areas of the working electrodes. Piranha solution was prepared by mixing hydrogen peroxide and concentrated sulfuric acid in a 1:2 (v/v) ratio. It was prepared immediately prior to use to maintain its reactivity. A stock solution of 20 µM TB was prepared in Tris-HCl and employed as redox indicator for revealing DNA hybridization. All the above solutions were kept at -4 ◦C before use. DTT solution containing 10 mM sodium acetate and 500 mM of DTT, pH=5.2 was prepared and kept at -4 ◦C. For preparation of the substrate, polyvinyl pyrrolidone (PVP) K-30 (molecule weight ≈ 300 g), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-
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hydroxysuccinimide (NHS), bovine serum albumin (BSA), potassium ferricyanide K3 Fe(CN)6, potassium ferrocyanide K4 Fe(CN)6, sodium hydroxide, disodium hydrogen orthophosphate, potassium dihydrogen orthophosphate potassium chloride, sodium citrate, cysteamine hydrochloride, hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O), graphite, ethylene glycol (EG), and ethanol were purchased from Sigma-Aldrich (Ontario, Canada). 2.2. Instrument Electrochemical processes were carried out with a PalmSens 4c system, driven with PSTrace5 software. The equilibrium time was 2 s and the employed ac voltage amplitude was 10 mV. Pt wire was applied as a counter electrode and Ag/AgCl (Metrohm, The Netherlands) as a reference electrode. Ag/RGO paper electrode which synthesized and prepared by ourselves was applied as a working electrode. FE-SEM (high-resolution field emission scanning electron microscope), Hitachi SU8020, Czech with an operating voltage of 3 kV was applied for characterization of electrode surface morphology, and EDS (energy dispersive spectroscopy) coupled with the FE-SEM apparatus was applied for analysis of electrode chemical compositions. TEM (transmission electron microscopy), Adelaide, Australia with an operating voltage of 200 kV was applied for investigation of synthesis mechanism. Size distribution and zeta potential of Ag/RGO nano-ink was analyzed by DLS (dynamic light scattering) with zeta potential instrument Malvern Instruments Ltd (Zetasizer Ver. 7.11, MAL1032660, England). Ohmmeter, XIOLE, XL830L, China, multimeter was applied for resistivity and conductivity nano-ink estimate. 3. Result and discussion
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Synthesis of graphene quantum dots doped by silver nanoparticle (Ag/GQDs) conductive nano-ink Graphene quantum dots (GQDs) were synthesized according to our pervious works (22, 23). Next, 2 mL of GQDs solution was heated at 60 0C in water bath. Then 0.6 gr graphite and 0.4 g of PVP were added into synthesized GQDs. The mixture was magnetically stirred until complete dissolving of the PVP and graphite powder. Then, 600 µL aqueous solution of AgNO3 (0.2 M) was directly added into above solution and at least 5 min magnetically stirred in order to guarantee efficient blending of GO and Ag +. The mixed solution maintained 12 h at 60◦C in incubator without agitation. In the following the mixture solution magnetically stirred at 80◦C again. Finally, 400 µL of sodium hydroxide (NaOH, 8 M) solution was drop to mix in order to enhance the reduction impact. Subsequently, the prepared solution centrifuged for 30 min at 8000 rpm and 3 times washed with distilled water (DI) for removing extra reactant. In order to produce hybrid conductive ink Ag/GQDs nano-ink were dispersed into DI water, ethanol, ethylene glycol with volume ratio of 9:9:3 and sonicated for 30 min which resulted in production of carbon water-based ink. After completion of the synthesis process, the produced nano-ink was stored for 24 h at room temperature before. Conductivity study of Ag/GQDs nano-ink For this purpose, different conductive patterns designed on the surface of photographic sheets via Ag/GQDs pellet with different thicknesses. As shown in Fig. S1, different types of conducting lines, a dip LED with a rated voltage of 3.0 V and 15.0 V battery was fabricated on paper substrate. One of the legs of LED (cathode) was connected to battery anode and so cathode surface of battery fixed to one side of conductive line. Once the anode leg of LED touched to other side of conductive line, the electronic circuit switched on and the LED was
8
lighted up. Afterwards, the resistance of conductive lines was measured by ohmmeter. Also the conductivity measurements obtained 266 μs for Ag/GQDs nano-ink. In order to demonstrate the reliability and flexibility, the conductive patterns bended outwards by 90 ˚C. CysA-Au NPs synthesis In a brief description, 50 mL of 0.5 mM HAuCl4 was magnetically stirred and heated until boiling points under reflux states. Subsequently, 5 mL of 38.8 mM sodium citrate (Na3C6H5O7) solution was added to the above solution. While the sodium citrate solution added, the yellow color of solution changed into wine-red. The mixture was continued magnetically stirring until wine-red color was fixed. Then, in order to functionalizing citrate capped-Au NPs with CysA through mixing of CysA and Au NPs with 100:1 volume ratio and stirring for approximately 12 h with gradually addition of CysA. The latest color of CysA-Au NPs mixture is deep red color and has the stability up to 2 months at 4˚C. Characterizations The TEM, XRD, Raman spectra, FE-SEM, EDS and DLS of Ag/GQD nano-ink, AgGQD/CysA-Au NPs photographic paper electrode modified with pDNA and cDNA To investigate the mechanism of Ag/GQDs composite formation and validate the linking of AgNPs and GQDs onto graphite sheets, TEM images of Ag/GQD nano-ink were recorded and shown in Fig. S2. It was obviously displayed that the numerous dispersed spherical AgNPs and GQDs were decorated the graphite sheets. Abundant crystal nuclei were created promptly when Ag NPs were added into mixture of nano-ink. PVP played as a mild reluctant and also as protectant which was absorbed onto the nuclei instantly at this moment. Therefore, prevent aggregation and merger of nuclei into larger and irregular nanoparticles.
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All of these results was confirmed by XRD and Raman spectra (Fig. S2 (B and C) (see supporting information)). Fig. S3 (see supporting information), demonstrates the FE-SEM images of bulk Ag/GQDs nano-ink, Ag-GQDs/CysA-Au NPs paper electrode modified with pDNA and AgGQDs/CysA-Au NPs-pDNA paper-based genosensor after incubation with cDNA to investigate the morphology of surface. The images reveal excellent Ag NPs dispersion and distribution on the bulk solution sample of synthesized Ag/GQDs nano-ink. The small sized AgNPs and GQDs with spherical uniform structure decorated on the graphite sheets. The following images approve that AgNPs present in synthesized Ag/GQDs nano-ink as electrical signal amplification element. The formation and distribution of Ag NPs shown in different magnification in Fig. S3B. It is interesting to observe that Ag/GQDs nano-ink prepared in water/Et/Eg demonstrates no aggregation of nanoparticles. When Ag/GQDs nano-ink was applied to make patterns on the surface of photographic papers, it stuck to the surface of paper, aggregate randomly. In following of the standard colloids theory, the colloid stability was controlled by the balance between charged particles, columbic repulsion and van der Waals interaction. It is important to point out that PVP reduced Ag/GQDs nanoink has a remarkable impact on conductivity alteration due to anchoring of Ag NPs and GQDs onto graphitic sheets. As the reduction process continued, more and more Ag NPs and GQDs anchored onto graphitic sheets. The FE-SEM images of CysA-Au NPs/Ag-GQDs nano-ink shows spherical particles with uniform structure (Fig. S3 (G-I) (see supporting information)), revealing that the Au NPs were indeed assembled onto the surface of thiolated Ag-GQDs nano-ink. Interestingly, more dispersed and less aggregated Au NPs are seen upon the electrostatic interactions of CysA-
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Au NPs with the Ag-GQDs nano-ink. Possibly the binding of positively charged Ag-GQDs to the negatively charged CysA-Au NPs reduced the aggregation of the NPs through steric hindrance, although the loss of some AuNPs from the GQDs surface cannot be eliminated. Interestingly, after incubation of pDNA with CysA-Au NPs/Ag-GQDs nano-ink, the morphology of electrode was changed. It’s found the, a binding between CysA-Au NPs/AgGQDs and nucleic acids was occurred (Fig. S3 (A-C) (see supporting information)). Therefore, pDNA was successfully immobilized on the surface of CysA-Au NPs/Ag-GQDs nano-ink modified paper electrode. More importantly, incubation of cDNA on pDNA modified CysA-Au NPs/Ag-GQDs nano-ink lead to new change on the morphology of electrode. As can be seen, the cDNA was linked to pDNA using hybridization strategy (Fig. S3 (D-F) (see supporting information)). These results confirmed successful assembly of the paper-based genosensor. Also, identification of synthesized Ag/GQDs nano-ink elements is also performed by to Energy-dispersive X-ray spectroscopy (EDS). According to Fig. S4 (see supporting information), the quantitative results confirm the presence of carbon elements with high frequency and Ag NPs in bulk sample. As evident in Fig. S4, signals for O, and C elements belong to both CysA and GQD, while S and N is only attributed to CysA. The large and sharp C peaks are the typical signal for carbon elements in the Ag/GQD nano-ink. Also, in the case of Ag-GQD/CysA-Au NP indicates that Au element overlapped with S element. Zeta potential analysis of Ag/GQD nano-ink was studied and the zeta potential value was 3.55 mV. TEM, FE-SEM, AFM, FTIR, Spectrofluorimetric and spectrophotometric characterization of Au NPs and CysA/Au NPs
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The size and shape of the CysA-Au NPs were approved by TEM (transmission electron microscope) and FE-SEM (Fig. S(5-7) see supporting information). The images exhibited that the spiral shaped particles for both AuNPs and CysA/Au NPs. Despite the secondary functionalization of AuNPs, the spiral shape is conserved while a slight increment in the size of AuNPs could be observed. Images of AFM provide three dimensional structural information about CysA-Au NPs (Fig. S8). Zeta potential analysis of AuNPs was investigated and the zeta potential value was -29.1 mV. Using DLS, the hydrodynamic particle size of AuNPs was about 20 nm. FTIR was used to study of functional group of AuNPs and CysA-AuNPs. As demonstrated in Fig. S9, the main peaks of the AuNPs and CysA-AuNPs are located at 3447, 2359 and 1653, which O−H, C−O and C=O functional groups. The N−H and C=O were located at 3500 and 1630−1690 cm -1 which are finger print peak of amide bond formation. UV-Vis and spectrofluorimetric spectra of Au NPs and CysA/Au NPs are shown in Fig. S9 (B and C). As shown in Fig. S9B, Au NPs has an absorption peak at about 320 nm. As can be seen in Fig. S9C, Au NPs emit fluorescence signal at 580 nm but due to the low quantum yields, the Au NPs were not further utilized in sensing applications. Fabrication of Ag/GQDs-CysA/Au NPs paper-based electrode For this purpose, through utilization of Ag/GQDs composite ink make pattern as working electrode and counter electrode on the surface of photographic sheets and let them dried on the stirrer for 15 min. The prepared Ag/GQDs nano-ink fabricated on the surface of photographic paper were treated with vaporous of hydrazine hydrate which is necessary for further electrodeposition of CysA/Au NPs on the surface of paper deposited by Ag/GQDs nano-ink. So, the paper dipped into CysA/Au NPs solution and electrodeposition was
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performed by chronoamperometry technique at E=0.8 V and duration time of 500 s that leads to attachment of amine groups of CysA/Au NPs with COOH groups of Ag/GQDs paperbased electrode (Fig. 1). Then, CysA/Au NPs-Ag/GQDs paper-based electrode was rinsed with distilled water in order to remove unbound moieties and maintained to dry at room temperature. Genosensor assembly To prepare the DNA biosensor, pDNA combine the designed probe with DTT. DTT was used as a reducing or "de-protecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated DNA have an affinity to form dimers in solution, particularly in the existence of oxygen. Dimerization significantly lowers the efficiency of consequent coupling reactions such as DNA immobilization on gold in biosensors. Usually DTT is mixed with a DNA solution and permitted to react, and then is removed by filtration or by chromatography (for the liquid form). The DTT removal procedure is often called "desalting." Generally, DTT is used as a protecting agent that prevents oxidation of thiol groups DTT is an important smallmolecular reducing agent and has wide applications in chemistry, biochemistry, peptideprotein reaction and clinical medicine (19-21). In the present study, a 10 µL of DTT solution was added to a 10 µL solution of p-DNA, mixed by a vortex and placed at ambient temperature for 30 min. Later 6 h incubation in -4
°C
, and rinsed the surface of
electrode. At this step, p-DNA self-assembling on the AuNPs was occurred, and a pDNA monolayer was formed by Thiol-gold covalent bonds. No further layers (rather than a monolayer) were formed, because it needed attachment of further p-DNA. on the pre-formed p-DNA layer on the electrode surface. In the next step and after rinsing with redistilled water, the electrode was immersed into MCH for 30 min. Self-
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assembled monolayer formed upon p-DNA immobilization may contain pinholes, limbered p-DNA, defects etc. 6-mercapto-1-hexanol immobilization caused to filling the remaining gold sites and caused to form a well-aligned p-DNA monolayer. The obtained electrode is denoted as DNA biosensor throughout the text. Subsequently, electrode were immerged in solution of TB containing Tris-HCl. TB is a basic thiazine metachromatic dye with high affinity for acidic tissue components (22). Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components, thereby staining tissues rich in DNA and RNA. Toluidine blue (TB) has an affinity for nucleic acids, and for that reason binds to nuclear material of tissues with a high DNA and RNA content (23). In this regard, we used the TB to mark the DNA probe. Also, Scheme 1 shows different steps for the fabrication of paper-based genosensor for the detection of L. Penumophila. Optimization of incubation time by TB In the next step, pDNA treated by ditiothreitol (DTT) appropriately for 30 m. DTT, also known as Cleland's reagent, is a small-molecule redox reagent. Its oxidized form is a disulfide-bonded 6-membered ring (24, 25). DTT is widely used in biochemistry works to reduce dissulfide bridges, protect biomolecules, in sample preparation, and to denature proteins before electrophoresis analysis (SDS-PAGE). DTT is commonly used to reduce the disulfide bonds of proteins and peptides (26). DTT cannot reduce buried (solventinaccessible) disulfide bonds, so reduction of disulfide bonds is occasionally approved under denaturing conditions. The terminal sulfur atoms of thiolated DNA have a tendency to form dimers in solution, particularly in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in
14
biosensors (27, 28). 10 µL of prepared solution was pipetted onto the working electrode surfaces and incubated in 4℃ for 6 h. After rinsed by DI to remove unbound DNA probe, the electrode immerged in 2-mercaptoethnol (MCE) for 30 min. Several studies revealed that treatment of DNA probe-modified electrode by MCE solution minimized non-specific binding and control the density of DNA probe on the electrode surface by giving a spacer between them to increase hybridization efficiency (29, 30). The hybridization can be exhibited via the redox signal of an electrochemical indicator, which can be an organic molecule (31), metal complexes (32), methylene blue (MB) (33), toluidine blue(TB) (20), enzymes, redox labels or nanoparticles or using technique direct oxidation(34). Label-free indicators are small, electroactive DNA-intercalating or groove-binding substances, which possess a much higher affinity for the resulting hybrid compared to the single-stranded probe. Therefore, the concentration of the indicator at the electrode surface be different when hybridization occurs, resulting in altered electrochemical signals. Besides effective differentiation between ss-DNA and ds-DNA, the indicator should possess a well-defined, low-potential, voltammetric response(35). Therefore, the electrode was immerged in TB in various time (2, 5, 7, 10, and 15 min) for obtain optimum incubation time. To achieve best result ChA technique was applied appropriately. As showed in Fig. 3, the most intensity current recorded in 7 min. Therefore, 7 min was selected as optimum incubation time in this part. Optimization of hybridization time of cDNA DNA hybridization deals notable consideration as result its specificity, selectivity, and sensitivity which is appropriate for miniaturization as it has compatibility with microfabrication technology.
Additionally, nanomaterials enhanced the performance of
15
electrochemical application through advancing bio-compatibility, accelerating electron transfer rate due to this enhanced signal can be achieved (36). In this work, 10 µl of cDNA (1µM) pipetted on the surface of biosensor and incubated in different time (10, 15, 20, 30, 40, and 60 m) in 37 ℃. For monitoring hybridization, ChA technique was applied properly. As showed in Fig. 4, 30 min was selected as optimum incubation time for appropriate hybridization. After hybridization of pDNA with c-DNA, the peak currents was changed obviously. This shift further confirms the binding of TB with the cDNA structure via hydrophobic attractions at the concentration range employed in this study. Higher positive shift in the formal potential of TB as a result of binding with cDNA can be an indicator for stronger attraction between TB and cDNA. Meanwhile, it has been reported that TB intercalates into the DNA base pairs at low concentrations, whereas it attaches to the negatively charged phosphate groups at higher concentrations (27). It can be concluded thus that TB binds with cDNA in a higher extent compared to cDNA. The peak current can therefore be employed to discriminate between cDNA and pDNA structures at the electrode surface. The decrement in the peak current of TB interacted with cDNA can be also related to both the effect of stronger binding of TB with cDNA (compared to pDNA),
and/or lower concentration of the bonded TB to DNA. Based on presented
hypothesis, it can be concluded that TB more strongly bind to cDNA. This binding suppresses the electron exchange between TB and underlying surface. Therefore, TB was employed as the redox hybridization marker for fabrication of the DNA biosensor. In the following, however, DPVs were measured for the reduction of bound TB into the biosensor to evaluate
its performance because differential pulse voltammetry is more
16
sensitive than cyclic voltammetry, and the reduction peak current of TB is greater than the oxidation one. According to this phenomena, electrochemical behavior of the paper-based was evaluated by ChA technique. As displayed in the Fig. S10A (see supporting information), after modification of electrode by Ag/GQDs-Cys A-AuNPs, recorded curve was altered which established the fabrication of nanomaterial on the surface of paper electrode. According to obtained result, after modification of (Ag/GQDs) nanoink-paper electrode, significant change on the currents were observed which is related to the hindrance effect of AuNPs. Interestingly, after immobilization of ssDNA, the current (-I/µA) was increased. This confirmed successful preparation of genosensor. Finally, after hybridization by dsDNA, the current (-I/µA) was further increased which proved interaction of appropriate ssDNAdsDNA. All of those results confirmed appropriate construction of biodevice. Analytical study The interaction between DNA probes and complementary sequence evaluates significantly Ag/GQDs nano-ink modified by CysA-AuNPs which effects the performance of electrochemical DNA genosensor. In this regard, various concentration of cDNA (10 -6, 10-9, 10-17, and 10-21) was immobilized on the surface of genosensor. For this purpose, 10 µl of cDNA pipetted on the surface of created genosensor and incubated for 30 min in 37 ℃. After incubation and washing by DW, ChA technique applied for monitoring of hybridization in different concentration and calibration curve were obtained which the linear rang was 1µM to 1 ZM. Also, low limit of quantification (LLOQ) was 1 ZM (Fig. 4). According to Fig. 4, after hybridization of 5ʹ SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG 3ʹ with complementary target sequence (5ʹ ACA AAA GGG GCG GGG
17
AGA GTA 3ʹ., the maximum peak current was appeared at 50 µA for 1 µM of cDNA. Interestingly, there is a linear relationship between peak current and concentration of cDNA. As can be seen in Fig S4B, by reduction of cDNA concentration to 1ZM, the peak current was decreased to 20 µA which regression equation was obtained as I (µA)=-11.38 CcDNA+64.46. These results confirmed that, proposed genosensor is sensitive to detection of cDNA sequence in low concentrations (1 ZM). Also, a wide dynamic range (1 ZM to 1 µM) was obtained by this paper passed biosensor. The appropriate limit of quantification can be considered as a result of several factors involved in the preparation of the desired genosensor, including excellent electrical conductivity, structural properties and high stability of the Ag/GQDs nano-ink, appropriate surface area which providing the feasibility of biological activity was used to identify hybridization of pDNA with cDNA. In addition, Cys A/AuNPs has double role in this biosensor. The terminal sulfur atoms of thiolated DNA has so affinity to the AuNPs which lead to stable interaction of S-Au. Also, Cys A/AuNPs led to signal amplification which is necessary for increment of the genosensor sensitivity. Therefore, using the engineered genosensor excellent hybridization was occurred and appropriated peak current and LLOQ were obtained. Stability assessment Stability of the paper-based genosensor was investigated by ChA technique from 0-4 h. For this purpose, after preparation of biosensor, ChA was recorded immediately, 2 h and 4 h according previous steps. As showed in the Fig.S11A (see supporting information), in earlier time recorded curve indicated good stability but after 4 h stability decreased, significantly. Finding revealed that stability of the fabricated genosensor is acceptable up to 2h.
18
According to Fig. S11, immediately after preparation of genosensor, the peak current was appeared at -4µA. But after 2h and 4h incubation, these peaks were observed in -6.1 and -7.3 µA. According to this result, we should be use this genosensor immediately after preparation. The appropriate stability of the genosensor can be considered as a result excellent stability of Ag/GQDs nano-ink on the surface of photographic paper. Selectivity of study Selectivity of electrochemical biodevice are paramount in emerging electrochemical DNA biosensors. Planned genosensors, despite having a very simple and cost effective structure, it has a high selectivity. For monitoring the selectivity, negative control of experiment were DNA sequence with mismatch in 1 and 3 nucleotides. After hybridization with cDNA, mismatch 1 and mismatch 3, ChA were recorded. As displayed in Fig. S12 (see supporting information), engineered DNA biosensor shows selectively response to the target sequence (cDNA) dissimilar from mismatch sequences. Engineered paper-based nanobiosensors revealed to be exceptional biodevice for detection of specific sequence of mip gene from L. penumophila. According to Fig. S12, after hybridization of 5ʹ SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG 3ʹ with complementary target sequence (5ʹ ACA AAA GGG GCG GGG AGA GTA 3ʹ., the peak current was appeared at 12 µA. But by using single nucleotide mismatch target sequence (5ʹ ACA AAA GGA GCG GGG AGA GTA 3ʹ) and three nucleotide mismatch target sequence (5ʹ GCA AAA GGG GCG GGG AGA GGG 3ʹ) peak currents were appeared at 8 µA and 6 µA, respectively. These results confirmed that, engineered biosensor is selective to detection of target sequence of L. pneumophila. 4. Conclusion
19
In summary, a novel nanomaterial improved paper-based genosensor for facile and inexpensive detection of L. penumophila was developed. Engineered paper-based nanobiosensors revealed to be exceptional biodevice for detection of specific sequence of mip gene from L. penumophila. Actually the simple nature of the genosensor makes it available for appropriate applications, meanwhile is portable and user friendly. Also, the rapid detection is critical in many situations such as illness diagnostics which lead to a fast treatment. This bio device is fast to response and can be used as an alternative tool for labor and time consuming methods. Therefore, in the near prospect the paper-based diagnostic biodevices are estimated to bring progresses in terms of sensitivity improvement as well as the multiple molecules detection ability. The expected bacterial detection limit was as 1 ZM and linear range were 1 µM to1 ZM which is lower than other comparable sensors previously described. Acknowledgments This research work was supported by Tabriz University of Medical Sciences. Conflict of interest There is no conflict of interest.
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35
30
25
I/ µA
20
15
10
5
0 0
100
200
300
400
500
600
time/ s
Fig. 1: ChA for the electrodeposition of CysA/Au NPs on the surface of Ag/GQDs paper-based electrode. E=0.8V and duration time was 500s.
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Scheme 1: Schematic representation of paper based genosensor for the detection of L. Penumophila
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0
-5
I/ µA
-10
-15
-20
7MIN
2MIN
5MIN
15MIN
-25 0
20
40
60
80
100
120
time/ s
Fig. 2: ChA of (Ag/GQDs) nano-ink modification by Cys A-AuNPs in different incubation time (2, 5, 7, and 15) of ssDNA by TB.E=-0.24V and the duration time was 100s
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0 -5 -10
I/ µA
-15 20min
-20
30min -25
40min 50min
-30 -35 -40 0
20
40
60
80
100
120
time/ s
Fig. 3: ChA of (Ag/GQDs)-nano-ink-CysA-AuNPs-pDNA in different incubation time (20, 30, 40, and 50 min) with cDNA. E=- 0.24V and the duration time was 100s.
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0 -6
A -10
-9 -17
-20
-21
I/ µA
-30
-40
-50
-60
0
20
40
60
80
100
120
time/ s 60 50
B
Ip/µA
40 30 20 10 0 10^-6
10^-9
10^-17
10^-21
C/ M
Fig. 4: A) ChA of CysA/Au NPs on the surface of Ag/GQDs paper-based electrode after hybridization with different concentration of cDNA (1 µM-1 ZM). E=-0. 24V and duration time was 100s B) Histogram of paper based genosensor in different concentrations of dsDNA.
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