Direct Electrochemical Vibrio DNA Sensing Adopting Highly Stable

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Direct electrochemical Vibrio DNA sensing adopting highly stable graphene-flavin mononucleotide aqueous dispersion modified interface Tao Yang, Huaiyin Chen, Zhiwei Qiu, Renzhong Yu, Shizhong Luo, Weihua Li, and Kui Jiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18212 • Publication Date (Web): 15 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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ACS Applied Materials & Interfaces

Direct electrochemical Vibrio DNA sensing adopting highly stable graphene-flavin mononucleotide aqueous dispersion modified interface

Tao Yang a,1, Huaiyin Chen a,b,1, Zhiwei Qiu a, Renzhong Yu a, Shizhong Luo a, Weihua Li b,c,d,*, and Kui Jiao a a

Key Laboratory of Sensor Analysis of Tumor Marker of Education Ministry, Shandong

Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China b

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China

c

Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, PR China

d

School of Chemical Engineering and Technology, Sun Yat-sen University, Zhuhai 519082, PR

China

* Corresponding author. E-mail: [email protected] Tel: +86-532-82897531; Fax: +86-532-82897531 1

These authors contributed equally to this work.

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ABSTRACT: A bio-functionalized graphene nanohybrid was prepared by simultaneously sonicating graphene and riboflavin 5'-monophosphate sodium salt (FMNS). FMNS, as a bio-dispersant, shows an efficient stabilization for obtaining highly dispersed graphene nanosheets in an aqueous solution. Due to the superior dispersion of graphene and the excellent electrochemical redox activity of FMNS, a direct electrochemical DNA sensor was fabricated adopting the inherent electrochemical redox activity of Gr-FMNS. The comparison between using traditional electrochemical indicator ([Fe(CN)6]3-/4-) and using the self-signal of Gr-FMNS was fully conducted to study the DNA sensing performance. The results indicate that the proposed DNA sensing platform displays fine selectivity, high sensitivity, good stability and reproducibility using either [Fe(CN)6]3-/4- probe or the self-signal of Gr-FMNS. The two methods display the same level of detection limit: 7.4 × 10-17 M (using [Fe(CN)6]3-/4-) and 8.3 × 10-17 M (using self-signal), respectively, and the latter exhibits higher sensitivity. Furthermore, the sensing platform also can be applied for the DNA determination in real samples.

KEYWORDS: graphene, flavin mononucleotide, graphene aqueous dispersion, self-signal, DNA sensing


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1. INTRODUCTION

Electrochemical sensor has been considered as a promising tool for bioanalysis on account of the merits of high sensitivity, inherent bio-selectivity, simpleness and low cost etc.1-3 Hitherto, a variety of electrochemical DNA sensors using different construction principles, such as the adoption of electrochemical redox indicator or redox labels, enzymatic catalytic reactions, functionalized nanomaterials, have been fabricated and yielded great achievement.4-7 However, most of the DNA sensors are based on extraneous indicators or labeled DNA to generate detection signals, which adds the operational complexity.4,8 Therefore, the direct electrochemical DNA sensors that fabricated based on the self-signals of materials or interfaces, are actively being sought owing to their diverse advantages such as label-free, simple and convenient procedure. In recent years, a few of direct electrochemical DNA sensors have been constructed. For instance, Wang et al. constructed an electrochemical DNA sensor utilizing the electrochemical self-signal of MoS2−thionin composite, which could sensitively detect double-stranded DNA (dsDNA) as well as single-stranded DNA (ssDNA).9 Our group has also successfully designed a electrochemical DNA sensor using

the

nanocomposite

of

reduced

graphene

oxide

and

poly(m-aminobenzenesulfonic acid) for the direct DNA detection.10 However, at present, the instability and feebleness of self-signal directly limit the performances and application of this kind of electrochemical sensor to a large extent.11 The stability and strength of the self-signal play an important role in constructing a direct electrochemical sensor, and have great influence on the sensing performance.9



Therefore, developing stable and signal-amplified nanomaterials is very urgent.12,13 Graphene, as a multifunctional 2D nanomaterial, has attracted enormous research interests in electrochemical sensing field, owing to its characteristic physical and chemical properties, such as remarkable conductivity, large specific surface area, high electrocatalytic performance, as well as favorable biocompatibility.14,15 A mass of reports have proved the enhancing efficacy of graphene in the electrochemical performance and signal amplification of DNA sensors.16,17 However, graphene is hydrophobic and difficult to be stably and highly dispersed in aqueous media, especially for a long time, limiting its application in electrochemical DNA sensors to some extent. Tremendous efforts have been putted into the how to gain highly stable graphene aqueous dispersion, in order to bring graphene’s superiority into full play in electrochemical sensing field. Flavin mononucleotide, a biosurfactant, has been successfully used as an efficient dispersant for the stable dispersion of graphene nanosheets and carbon nanotubes in aqueous phase.18-20 Recently, Ayán-Varela et al. reported that low amounts of riboflavin 5'-monophosphate sodium salt (FMNS) could stabilize very high concentrations of graphene sheets in an aqueous medium.21 FMNS molecule can be intensely adsorbed onto the basal plane of graphene through its isoalloxazine moiety, and the hydrophilic phosphate group with negative charge in aqueous phase could supply graphene with colloidal stability via the electrostatic repulsion. Besides the high efficiency for aqueous dispersion of graphene, FMNS also possesses high electrochemical activity. However, the utilization of the electrochemical self-signal of


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graphene-FMNS (Gr-FMNS) nanocomposite has not been reported at present. In this paper, FMNS was used as a bio-dispersant for stabilizing high concentrations of graphene nanosheets in an aqueous medium. A direct electrochemical DNA sensor was constructed using the prepared Gr-FMNS nanocomposite for the electrochemical detection, without label or any exterior indicator, as shown in Scheme 1. Gr-FMNS shows not only high conductivity, but also electrochemical activity that is superior to the self-signal in previously reported DNA sensors.10,22 In order to better research the sensing performance of our proposed DNA sensor, the comparison between using traditional electrochemical probe and using self-signal was fully conducted. The results indicate that the proposed DNA sensing platform displays fine selectivity, high sensitivity, good stability and reproducibility using either [Fe(CN)6]3-/4- probe or the self-signal of Gr-FMNS. And by contrast, using the self-signal as response signal for DNA detection displays the same level of detection limit, and the higher sensitivity (the slope of linear equation).



2. EXPERIMENTAL

2.1. Chemicals and apparatus

Graphene powder (prepared by chemical oxide-thermal reduction method) was purchased from Jining Leader Nano Technology Co., Ltd. (Jining, China). FMNS and tris(hydroxymethyl)aminomethane (Tris) were provided by Sigma-Aldrich Co. (USA). K3[Fe(CN)6]), K4[Fe(CN)6] and other reagents used in this experiment were of analytical grad and obtained from some common chemical reagent Co. in China. 0.1 M phosphate buffer solution (PBS) (pH 7.0) was prepared with NaH2PO4/Na2HPO4. Ultrapure water produced by an ultrapure water system (AWL-1002-P, Aquapro International Co.) were was used for the preparation of all aqueous solutions. The 23-base synthetic probe DNA (pDNA), its complementary DNA (cDNA, the tlh gene sequence of Vibrio, target DNA), non-complementary DNA, single-base mismatched, and three-base mismatched DNA were same as the previous report.23 The detection of DNA in real samples was carried out in DNA extracts that obtained from Vibrio parahaemolyticus tainted oysters through the TIANamp Bacterial DNA Kit (TIANGEN Biotech (Beijing) Co., Ltd., China). The stock solution, rinsing solution and hybridization solution of DNA were prepared according to our previous report and stored at 4 °C.23 The electrochemical measurement system and three-electrode system were used as same as previously reported.24 Ultrasonic operation was conducted with a 500 W ultrasonic cleaning apparatus (KQ-500B, Kunshan ultrasonic instruments Co., Ltd.,


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China). Scanning electron microscopy (SEM) (JSM-6700F, JEOL, Japan) and transmission electron microscopy (TEM) (JEM-2100, JEOL, Japan) were used for the morphological characterization. Fourier Transform Infrared (FTIR) spectra and UV-vis spectra were respectively recorded on a Bruke Tensor 27 Spectrometer and a Hitachi UH5300 double-beam spectrophotometer.

2.2 Preparation of various modified electrodes

Carbon paste electrode (CPE) was fabricated used the reported method.25 The Gr-FMNS nanocomposite was prepared by a modified reported procedure.21 Briefly, 10 mg of graphene and a certain amount of FMNS (the optimal amount in this work was 5 mg) were dispersed in 10 mL of ultrapure water, then the mixture was sonicated for 7 h at room temperature after violent shaking to get a homogeneous dispersion. In order to remove excess FMNS that not adsorbed on graphene, the Gr-FMNS nanocomposite was sedimentated via 20 min of centrifugation at 14000 rpm, and then was re-suspended in ultrapure water through sonication for 2 min. After two cycles of re-suspension and sedimentation process, the precipitate was re-dispersed into 50 mL of ultrapure water to obtain the Gr-FMNS aqueous dispersion, in which the graphene concentration was about 0.2 mg mL-1. It is important to note that FMNS is a light-sensitive chemical, so the preparation and storage of FMNS and Gr-FMNS dispersions were in dark condition. Gr-FMNS modified CPE (Gr-FMNS/CPE) was prepared by a simple drop-coating. Briefly, the prepared Gr-FMNS dispersion was sonicated for 2 min before use, then 20 µL of the homogeneous dispersion was slowly



dripped on the tip of the freshly polished CPE and dried naturally at room temperature. As comparison, graphene modified CPE (Gr/CPE) was obtained by dripping 0.2 mg mL-1 graphene aqueous dispersion that directly sonicated for 7 h without the addition of FMNS on the tip of CPE. Similarly, FMNS modified CPE (FMNS/CPE) was prepared by dripping 0.2 mg mL-1 FMNS aqueous solution.

2.3 DNA immobilization, hybridization and electrochemical measurements

The immobilization of pDNA was carried out by dripping 20 µL of Tris-HCl buffer solution containing 1.0 µM pDNA onto the surface of Gr-FMNS/CPE, and drying it naturally at room temperature. The obtained electrode was rinsed with ultrapure water to remove unfixed oligonucleotides. The obtained ssDNA modified Gr-FMNS/CPE was denoted as ssDNA/Gr-FMNS/CPE. 20 µL of 2 × SSC buffer solution containing 1.0 µM cDNA was dripped on the tip of ssDNA/Gr-FMNS/CPE to realize hybridization. In order to eliminate non-specific adsorption of cDNA, the obtained electrode was rinsed thoroughly using 0.2% SDS solution. The obtained dsDNA modified Gr-FMNS/CPE was denoted by dsDNA/Gr-FMNS/CPE. Furthermore, the hybridization between ssDNA/Gr-FMNS/CPE and ncDNA, single-base mismatched or three-base mismatched DNA was performed via the same procedure. Cyclic voltammogram (CV) measurements were conducted in 0.1 M PBS (pH 7.0), the potential range and scan rate were set as -1.0 ~ 0 V and 0.1 V s-1, respectively. While in 1.0 mM [Fe(CN)6]3-/4- solution containing 0.1 M KCl, the potential was set between -0.3 and 0.6 V. Differential pulse voltammetry (DPV) tests were carried out


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in 0.1 M PBS (pH 7.0), and the initial potential, final potential, amplitude, pulse width and pulse period were set as -1.0 V, 0 V, 50 mV, 0.1 s and 0.2 s, respectively. And in 1.0 mM [Fe(CN)6]3-/4- solution containing 0.1 M KCl, the DPV parameters were same as that in PBS except the scan range from -0.3 to 0.6 V. The parameters of electrochemical impedance spectroscopy (EIS) in 1.0 mM [Fe(CN)6]3-/4- solution containing 0.1 M KCl were set as follows: the potential, frequency range, and amplitude were 0.2 V, 106 ~ 0.01 Hz, and 5.0 mV, respectively.



3. RESULTS AND DISCUSSION

3.1. Characterization of Gr-FMNS nanocomposite

The stability of graphene and Gr-FMNS in aqueous dispersions was investigated. Figure 1A shows the photographs of prepared graphene, FMNS and Gr-FMNS aqueous dispersions after 1 h. It can be seen that the aqueous dispersion of sole graphene is very instable (left tube), which has agglomerated due to the few hydrophilic groups. The FMNS aqueous dispersion (mid tube) presents a yellow transparent solution, which attribute to the hydrophilic phosphorylated alcohol (ribitol) moiety of FMNS molecular (Figure 1B).26 It is also found that the Gr-FMNS aqueous dispersion (right tube) is a homogeneous and stable disperse system without any agglomerations. It is because the FMNS is an amphiphilic molecule that not only possessing hydrophilic moiety but a tricyclic heteronuclear organic ring (dimethylated isoalloxazine) (Figure 1B).

21

It has been confirmed that the isoalloxazine moiety of

FMNS molecule has a strong binding affinity to graphene sidewalls, thus FMNS molecule can be intensely adsorbed onto the basal plane of graphene through π-π stacking interaction.27 On the other hand, the hydrophilic phosphate group with negative charge in aqueous phase could be sure colloidal stability to graphene through electrostatic repulsion.28 The UV-vis absorption spectra of FMNS aqueous solution and Gr-FMNS aqueous dispersion after removing free FMNS are shown in Figure 1C. Four strong absorption bands at about 445, 374, 266, and 222 nm are present in the UV-vis


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spectrum of FMNS (curve a), which are the typical absorption bands of FMNS in water.21,29 The UV-vis spectrum of Gr-FMNS in aqueous dispersion after purification (curve b) shows one strong absorption band at about 270 nm, which could be attributed to the π-plasmon resonance in graphitic materials.21,30,31 Besides, other inconspicuous absorption bands are corresponding to that of FMNS, demonstrating the coupling of graphene and FMNS, which was further confirmed by FTIR spectra (see Supporting Information, Section S1). Moreover, UV-vis spectra also proved that the ultrasonication treatment in dark condition and at room temperature could keep the stability of FMNS (see Supporting Information, Section S2). The morphological characterization of graphene and Gr-FMNS were performed by SEM and TEM, as displayed in Figure 2. The large lumps could be seen in the SEM image of graphene (Figure 2A), indicating the graphene nanosheets were instable in aqueous solution and could easily agglomerate with each other to form bulk graphene. However, as shown in Figure 2B, Gr-FMNS displays wrinkled and crumpled sheet-like structures, with lateral dimensions of several micrometers and very thin thickness. Moreover, the TEM image can further confirmed thin-layered nanosheet structures of Gr-FMNS (Figure 2C).

3.2. Electrochemical behavior of modified electrodes

CV measurements were conducted to research the electrochemical performances of various modified electrodes (bare CPE, Gr/CPE and Gr-FMNS/CPE). From Figure 3A, it is found various electrodes show different CV signals in 1.0 mM [Fe(CN)6]3-/4-



containing 0.1 M KCl. The CV curve shows no anodic or cathodic peak for bare CPE (curve a), implying the low conductivity and small electrode effective surface. One anodic and one cathodic peak were present for Gr/CPE (curve b) owing to the large specific surface area and good conductivity of graphene. However, after the modification of FMNS on CPE, the CV result (curve c) shows no redox peaks and the smaller current change compared with that of bare CPE, owning to the very low conductivity of FMNS. And as expected, compared with Gr/CPE, an obvious increase in peak currents was recorded at Gr-FMNS/CPE (curve d), indicating the superior electrochemical performance of Gr-FMNS/CPE. The lower current response of Gr/CPE than that of Gr-FMNS/CPE may be because instability and agglomeration of graphene nanosheets in aqueous media caused hindered electron transfer. The electroactive surface areas of different electrodes were calculated and compared for the electrochemical characterization of modified electrodes (see Supporting Information, Section S3 for details). Table S1 shows that Gr-FMNS/CPE has the largest electroactive surface area, which exactly confirmed the above result. Besides, the modification process of the electrode was also investigated by EIS technique, as shown in Figure S3. The EIS data are corresponding with CV results, which proves, once again, the consistency of our experimental results. Figure 3B shows the CVs recorded in 0.1 M PBS (pH 7.0) at above modified electrodes. It is found that the CVs of Gr/CPE (curve b) as well as CPE (curve a) displays nearly no anodic or cathodic peaks. Being different from bare CPE and Gr/CPE, a couple of reversible redox peaks at about -0.43 and -0.50 V appears in the CV curve of FMNS/CPE (curve c),


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corresponding with oxidation/reduction processes of FMNS.21 The CV curve of Gr-FMNS/CPE (curve d) desplays a couple of more apparent redox peaks and much larger peak currents than that of FMNS/CPE (curve c), which is because the exist of the high-conductive graphene with large surface area vastly promotes the electron transfer between FMNS molecule and electrode. Furthermore, this result also demonstrated the successful combination of FMNS and graphene. The Gr-FMNS nanocomposite was optimized through varying the weight ratio of graphene to FMNS (20:1, 10:1, 5:1, 2:1, 1:1 and 1:2) in the preparation process. The electrochemical

behaviors

of

the

CPEs

modified

with

above

Gr-FMNS

nanocomposites with different weight ratio were investigated in two kind of solution: 1.0 mM [Fe(CN)6]3-/4- or 0.1 M PBS (pH 7.0), as displayed in Figure 3C and D. Figure 3C shows the largest peak currents were observed at Gr-FMNS (2:1)/CPE in [Fe(CN)6]3-/4- solution (green solid line). It may be because the skimpy FMNS couldn’t optimally stabilize the graphene aqueous solution, while the excess of FMNS onto the surface of graphene would slightly decrease the conductivity of graphene. Figure 3D shows the peak currents increases with increasing the weight ratio of FMNS in PBS, but the increase becomes insignificant after the weight ratio reaches to 2:1 (green solid line). That may be because sufficient FMNS has been adsorbed on graphene, and further increasing the amount of FMNS can’t increase the adsorbed amount. Therefore, Gr-FMNS(2:1)/CPE was selected. The comparison of response signal before and after immobilization of pDNA and hybridization with target DNA were respectively investigated using [Fe(CN)6]3-/4-



probe and self-signal (in PBS). Figure 4A shows the CVs of Gr-FMNS/CPE, ssDNA/Gr-FMNS/CPE and dsDNA/Gr-FMNS/CPE in 1 mM [Fe(CN)6]3-/4containing 0.1 M KCl. It could be observed that, after the immobilization of pDNA on Gr-FMNS/CPE (curve b), the current response decreased than that before immobilization (curve a). This is because the negative charged DNA was adsorbed onto the basal plane of graphene by π-π stacking interaction, which generated a repulsion interaction with [Fe(CN)6]3-/4- and a steric hindrance effect on the surface of electrode, as a result, the electron transfer was blocked.32 At the same time, this result also confirmed that the pDNA was successfully immobilized onto the surface of electrode. As expected, the signal was further reduced after the hybridization between ssDNA and cDNA (curve c), due to the larger electro-negativity and steric hindrance effect of dsDNA, which further inhibited the electron transfer.33 Accordingly, the electroactive surface areas of ssDNA/Gr-FMNS/CPE and dsDNA/Gr-FMNS/CPE were decreased in turn compared with Gr-FMNS/CPE (Table S1). Furthermore, the CVs of these electrodes in 0.1 M PBS (pH 7.0) adopting self-signal show the similar results, which can be observed from Figure 4B. As reported, ssDNA acts as flexible and random coils, which could alter the conformation and block the electron transfer.34 Moreover, dsDNA could form an electron-transfer as well as mass-transfer blocking layer on the surface of electrode, which further inhibited the electrode reaction.35 Consequently, after the modification of ssDNA and dsDNA, the electron exchange rate for FMNS reaction on the electrode surface was decreased. The larger hindrance effect made the lower electron exchange rate, and the current signal to be


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weaker accordingly.

3.3 Selectivity, sensitivity of DNA hybridization detection

Selectivity is one of the most significant parameters for DNA detection. In this research work, the selectivity of the DNA sensing platform was explored through the hybridization between ssDNA/Gr-FMNS/CPE and mismatched DNA sequences. Figure 5A and B show the comparison of DPV signals obtained from ssDNA/Gr-FMNS/CPE hybridized with cDNA (curve b), single-base mismatched (curve c), three-base mismatched (curve d) and non-complementary (curve e) DNA, respectively. It can be seen that the current responses are very different from each other both in [Fe(CN)6]3-/4- and PBS, implying our sensing platform can distinguish the cDNA from non-complementary single-base mismatched, and three-base mismatched DNA. Therefore, the proposed DNA sensing platform shows good selectivity for the DNA detection no matter using [Fe(CN)6]3-/4- probe or self-signal. In order to further investigate and contrast the detection capability of Gr-FMNS/CPE for DNA hybridization, a series of concentrations of target DNA were measured by comparably using [Fe(CN)6]3-/4- electrochemical probe and self-signal (in PBS). Figure 5C shows the DPVs for the detection of various concentrations of cDNA using [Fe(CN)6]3-/4-. It was evident that the anodic peak current (Ipa) decreased with the augment of the cDNA concentration (C). The peak current linearly reduced with lg (C/M) ranging from -16 to -6, as shown in Figure 5E. The linear regression equation was Ipa (10-5 A) = -0.1514 lg(C / M) + 0.171, R2 = 0.9976. The detection



limit of the DNA sensing platform was calculated to be 7.4 × 10-17 M (S/N = 3).36 As comparison, the detection of various concentrations of cDNA (from 0 to 1.0 µM) was also investigated in PBS (pH 7.0) by DPV. Figure 5D shows that the anodic peak current decreases with the increase of cDNA concentration. A linear relationship between Ipa and lg (C/M) is displayed in Figure 5F. And the linear regression equation was Ipa (10-5 A) = -0.3278 lg(C / M) - 0.6815, (R2 = 0.9963). The detection limit was 8.3 × 10-17 M (S/N = 3). From above results, it can be seen that the proposed DNA sensing platform based on Gr-FMNS nanocomposite exhibits ascendant performance with wide linear ranges and low detection limits compared with previously reported label-free electrochemical DNA sensors (Table S2). Moreover, using self-signal as response signal for DNA detection showed the same level of detection limit, and the higher sensitivity (the slope of linear equation).

3.4 Reproducibility and stability of DNA detection

To investigate the reproducibility, eight same prepared ssDNA/Gr-FMNS/CPEs were respectively measured by DPV in 1 mM [Fe(CN)6]3-/4- solution and 0.1 M PBS (pH 7.0) after hybridizing with 1.0 × 10-6 M target DNA. Just using the reported research method,37 the intra-day and inter-day relative standard deviations (RSDs) were 4.3% ~ 8.1% and 4.5% ~ 8.7% (n = 8) by using [Fe(CN)6]3-/4- indicator, while 3.6% ~ 7.5% and 4.0 % ~ 7.8% by using self-signal, which shows the acceptable reproducibility for DNA detection. The stability of our proposed DNA sensing platform was also studied


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using a long-term storage detection. After two weeks’ storage at 4 °C, the current signals still retained 91.1% and 90.4% of the initial values using [Fe(CN)6]3-/4- probe and the self-signal, respectively. The above results indicated that the DNA sensing platform possessed good stability no matter using [Fe(CN)6]3-/4- probe or the self-signal.

3.5 DNA detection in real samples

The DNA detection in real samples was performed in DNA extracts. The concrete operating steps were addressed in the Supporting Information (Section S5). The results show that our prepared electrochemical DNA sensing platform could be applied to real DNA samples detection (Figure S4).



4. CONCLUSIONS

In summary, we have prepared a biomolecule functionalized graphene nanohybrid by simultaneously sonicating graphene and the FMNS. The FMNS not only serve as an amphiphilic molecule to obtain highly stable graphene in aqueous medium, but also possesses good electrochemical activity. The prepared Gr-FMNS nanocomposite shows high conductivity as well as high and stable electrochemical activity, which was used to design a direct electrochemical DNA sensing platform. The sensing performance of the proposed DNA platform between using traditional probe and using self-signal was fully contrasted. The results indicate that the proposed DNA sensing platform possesses a high sensitivity and selectivity for DNA detection using either classic [Fe(CN)6]3-/4- or the self-signal of the Gr/FMNS. And using the self-signal as response signal for DNA detection displays the same level of detection limit, and the higher sensitivity compared with that using [Fe(CN)6]3-/4- indicator.


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ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (Nos. 41476083, 21675092, 51525903, 21275084), 863 program (No. 2015AA034404), and Aoshan Talents Outstanding Scientist Program Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2017ASTCP-OS09).



ASSOCIATED CONTENT Supporting Information FTIR characterization of FTIR spectra of FMNS, graphene and Gr-FMNS. UV-vis spectra of FMNS solution. Electroactive surface areas and EIS characterization of the various electrodes. Comparison of sensing performance with previously reported work. The detail operational processes of DNA extraction and the results of DNA detection in real samples.


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Scheme 1. Schematic representation of preparation of highly stable Gr-FMNS aqueous dispersion for direct DNA detection.



Figure 1. (A) Digital photograph of the aqueous dispersions. From left to right: 0.2 mg mL-1 graphene aqueous dispersion, 0.1 mg mL-1 FMNS aqueous solution, and Gr-FMNS aqueous dispersion (graphene content was about 0.2 mg mL-1); (B) molecular structural formula of FMNS; (C) UV-Vis absorption spectra of (a) FMNS aqueous solution and (b) Gr-FMNS aqueous dispersion.


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Figure 2. SEM images of (A) graphene and (B) Gr-FMNS, (C) TEM image of Gr-FMNS.



Figure 3. CV curves recorded at (a) bare CPE, (b) Gr/CPE, (c) FMNS/CPE and (d) Gr-FMNS/CPE in (A) 1.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl and (B) 0.1 M PBS (pH 7.0); CV curves at different Gr-FMNS/CPEs prepared with varying ratio of graphene to FMNS (20:1, red solid star; 10:1, dark yellow solid triangle; 5:1, navy frame triangle; 2:1, green solid line; 1:1, wine solid square; 1:2, blue frame square) in (C) 1.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl and (D) 0.1 M PBS (pH 7.0).


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Figure 4. CV plots recorded at (a) Gr-FMNS/CPE, (b) ssDNA/Gr-FMNS/CPE, and (c) dsDNA/Gr-FMNS/CPE in (A) 1.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl and (B) 0.1 M PBS (pH 7.0).



Figure 5. DPV curves of ssDNA/Gr-FMNS/CPE (a), dsDNA/Gr-FMNS/CPE (b), and ssDNA/Gr-FMNS/CPE hybridized with single-base mismatched DNA (c), three-base mismatched DNA (d) and non-complementary DNA (e) in (A) 1.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl and (B) 0.1 M PBS (pH 7.0); DPV curves for the detection of cDNA with different concentrations (10-6, 10-8, 10-10, 10-12, 10-14, 10-16, and 0 M) in (C) 1.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl and (D) 0.1 M PBS (pH 7.0); the corresponding calibration plot of Ipa and -lgC in (E) 1.0 mM [Fe(CN)6]3-/4- containing 0.1 M KCl and (F) 0.1 M PBS (pH 7.0).


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TOC


Direct Electrochemical Vibrio DNA Sensing Adopting Highly Stable

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