Yilan Chu,
Bin Cai,
Ye Ma,
Minggang Zhao,
Zhizhen Ye* and
Jingyun Huang*
School of Materials Science and Engineering, Zhejiang University of China, Hangzhou, 310027, China. E-mail: huangjy@zju.edu.cn; yezz@zju.edu.cn
First published on 12th February 2016
By integrating thin-layer molybdenum disulfide (MoS2) and graphene through a hydrothermal process and an ultrasonic method, a label-free, amplification-free and ultrasensitive circulating tumor DNA electrochemical sensor was made. Compared with other methods, this preparation process was simpler and the electrochemical properties were enhanced. Using a differential pulse voltammetry test, this sensor can detect trace amounts of DNA in the range from 10−16 M to 10−13 M. Compared with other methods that are used to detect the same circulating tumor DNA, this sensor has an obvious advantage in sensitivity, cost and simplicity because it removes the need for labelling processes and amplifiers.
Nowadays gene diagnosis and therapy attract more and more attention because of its application in many aspects such as tumor detection, forensic investigation and environmental monitoring.3–6 Among the methods for gene detection, the electrochemical DNA sensor aiming at the detection of oligonucleotide sequences is a promising and convenient way to detect specific gene sequences, especially at low physiological levels. There are some other methods based on optical and chemiluminescence properties that have been widely used in the modern life. However, these methods usually have some shortcomings. For example, the chemiluminescence DNA biosensor needs to label the oligonucleotide probes that increases the cost and complexity. Compared with these methods, electrochemical DNA biosensor is label-free and enzyme-free which provides convenience. The complexity is reduced and the test time could be greatly shortened.7–9 Because of these merits, electrochemical biosensors have received more attention and been applied widely in DNA detection.
In DNA detection, circulating tumor DNA (ctDNA) detection is a new and prospering application direction because of its value in tumor prognosis and treatment.10 Circulating tumor DNA could be used as a biomarker. It is a double stranded DNA which has tumor-specific sequence mutations and its existence could be a proof of specific cancer. ctDNA could be found in the cell-free fraction of blood which means the detection just need a sample of blood instead of tissue biopsy. This feature could reduce the risk of patients and provide convenience. The traditional biopsy which is widely used for cancer prognostic procedure is inadequate according to the result made by Charles Swanton. The result shows that biopsy may miss mutations away in a small range that might affect the judgment of illness.11 Besides, the information provided by biopsies is static and could not reflect real-time dynamic of the tumor. On the contrary, the ctDNA detection can monitor the evolution and offer more comprehensive data of tumor. It can help doctors know about therapeutic effect of present treatment and resistance evolution.12 However, concentrations of ctDNA is quite low and hard to detect. The amounts of ctDNA are typically low and extremely variable. Usually ctDNA makes up barely 1–0.01% of the circulating DNA in blood. When people have very advanced cancers, the ctDNA come from tumors and the concentration would increase. For this reason, early sequencing technologies were unable to detect the ctDNA and the sensitivity of the ctDNA sensor is very important.12
In order to improve detection limit, many efforts have been made, such as surface plasmon resonance (SPR) biosensors.10,13,14 In 2014, Huang et al. combine MoS2 with multi-walled carbon nanotubes to improve the electronic conductivity and electrochemical activity, then the composites were further combined with gold nanoparticles to immobilize DNA on the composites' surface via Au–S bonds self-assembly. By enzyme multiple signal amplification, the DNA biosensor could achieve sub-femtomolar DNA detection, that would improve the limit of detection.
In this contribution, an ultra-sensitive label-free electrochemical biosensor for detecting ctDNA was made. To improve the electronic conductivity and electrochemical activity, MoS2 was integrated with graphene by hydrothermal method, which was further ultrasonicated for 6 h to get the MoS2/graphene nanosheets, and probe DNA could be directly immobilized on the nanosheets by van der Waals force between nucleobases of single stranded DNA (ssDNA) and the basal plane of nano MoS2.16 Then K3[Fe(CN)6] was used as the electroactive indicator to monitor the changes happened on the electrode surface. The changes caused by DNA immobilization and hybridization were detected by directly monitoring the differential pulse voltammetric (DPV) response of the guanine bases. Compared with other biosensors, such a sensor is quite convenient and cheap because there is no fluorophore labelling and enzyme amplification step. In addition, the high sensitivity is also a remarkable advantage.10
Graphene oxide suspension (2 mg mL−1, dissolved in deionized water) was purchased from Tanmei Sinocarbon Materials Technology Company (Taiyuan, China). The bulk MoS2, L-cysteine, NaOH, KCl, K3[Fe(CN)6], N,N-dimethylformamide (DMF) and Na2MoO4·2H2O were purchased from Tianjin Ba Si Fu Reagent Company (Tianjin, China).
The DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technological Co. Ltd (Shanghai, China). Their base sequences are listed below:
Probe DNA (pDNA): 5′-AGT GAT TTT AGA GAG-3′;
Complementary DNA (cDNA): 5′-TCA CTA AAA TCT CTC-3′;
One-mid-base mismatch ssDNA (1MTDNA): 5′-TCA CTA ATA TCT CT-3′;
Two-mid-bases mismatch ssDNA (2MTDNA): 5′-TCA CAA ATA TCT CT-3′;
Non-complementary ssDNA (ncDNA): 5′-CAC TCC GCG CTA ACT-3′.
Then transfer the residue into 100 mL deionized water, stir the mixture for about 1 h and followed by ultrasonication for 6 h and get a homogenous suspension of nano MoS2/graphene composites (0.01 M). The GCE was sequentially polished with 0.3 and 0.05 μm alumina slurries and rinsed with acetone, 0.5 M water dilute nitric acid for 1 min, respectively. Drip 40.0 μL of the suspension on the polished GCE surface and dried naturally at room temperature. Then the composites modified GCE was prepared. For comparison, the same concentration of MoS2 homogenous suspension was also prepared and tested.
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Fig. 2 Morphology (A) SEM images of MoS2/graphene (1![]() ![]() ![]() ![]() ![]() ![]() |
Fig. 3A is the XRD patterns of the MoS2/graphene (1:
1) composite and pure MoS2. From the pattern we can see that diffraction peaks of the MoS2/graphene composite show at 2θ = 15°, 33°, 40° and 59°, which corresponds to (002), (100), (103), and (110) planes of MoS2 (JCPDS no. 37-1492), respectively.19 The pattern of MoS2/graphene composites is similar with the pattern of pure MoS2 material. One reason of this phenomenon is that graphene usually does not have obvious and particular peak in XRD pattern. And all the peak widths are wide, which shows that the composites have poorly crystalline due to amorphism of the composites. The amorphism of the composites was attributed to the hydrothermal process used to incorporate the MoS2 nanosheets and graphene. Because the graphene inhibited the growth of the layered MoS2 crystalin the hydrothermal process.15
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Fig. 3 (A) XRD patterns of MoS2 nanosheets and MoS2/graphene (1![]() ![]() ![]() ![]() |
For further confirming the prepared material and the structural and electronic properties, the Raman spectrum of the MoS2/graphene composites was recorded (Fig. 3B). The Raman spectrum showed two strong bands at 1342 cm−1 that was caused by sp3-hybridized carbon and the G bands at 1586 cm−1 that was because of the E2g zone center mode of the graphene. And the other bands at 357.3 cm−1 and 409.2 cm−1 were the characteristic bands of MoS2 which aroused from the E2g and A1g peaks respectively.20 Above-mentioned peaks could prove the presence of graphene and MoS2 in the composites, which was consistent with the results in the XRD diffraction studies.
Fig. 4 is the TEM image of the MoS2/graphene (1:
1) composites which were ultrasound exfoliated for 6 h. From the Fig. 4A, we can see that the composites are thin sheets morphologies and have large specific surface area which were in agreement with the SEM images. The images showed the composites had thin layer structure and the layers folded and tangled together. From Fig. 4B, graphene inserted in the layered MoS2 nanosheets and the MoS2 served as the substrate. This was because in the preparation process, MoS2 sheets served as the substrate for the nucleation and growth of graphene and turned to a layered structure. The presence of the graphene disturbed the growth of the layered structure, and the 6 h exfoliation promoted the separation of the layered structure of the composites, too. These factors directly influence the structure of the composites, which turned the material to the few-layer MoS2/graphene composites. Such a thin-layer structure remarkably increased the specific surface area and the recombination of the MoS2 and the graphene enhanced the electrical conductivity, which promoted the upgrade of the electrochemical performances of the MoS2/graphene composites. Also the BET tests showed that the specific surface area of 2D MoS2/graphene composites is 12.42 m2 g−1 and the specific surface area of bulk MoS2 is 3.62 m2 g−1, that proved this analysis.
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Fig. 4 (A and B) TEM images of MoS2/graphene (1![]() ![]() |
Fig. 5 is the TEM surface scanning spectrum diagram scanned by energy dispersive spectrometer (EDS). Fig. 5A displays the K-spectrum of the carbon, Fig. 5B displays the K-spectrum of the molybdenum and Fig. 5C displays the K-spectrum of the sulfide. These figures display the distribution of three elements in the same area. The bright areas of the spectra are almost the same and coincident, which shows that the graphene and MoS2 were relatively well distributed in the composites nanosheets.
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Fig. 5 EDS images of MoS2/graphene (1![]() ![]() |
As the concentration of the electrolyte was the same, the higher peak current confirmed that the composites modified GCE had good electrochemical activity. The CV curve of the pDNA modified GCE showed significantly difference with the other two curves as the pDNA adsorbed on the electrode surface and changed its electrical conductivity. The figure showed that the anodic peak current value of the probe DNA immobilized MoS2/graphene composites modified GCE had an obvious increase compared with the probe DNA immobilized bulk MoS2 modified GCE, while the peak current of the composites modified GCE was quite close with the bulk MoS2 modified GCE.
From the formula ‘i = FAC*0(D0b)1/2π1/2χ(σb)’. The current is directly proportional to C0 (initial concentration of reactants) and ν1/2 (scanning speed). The scanning speed is fixed and the initial concentration of reactants is proportional to the weight of reactants. So the weight of reactants (MoS2) is involved in the decrease/increase in conductivity. As the pure bulk MoS2 material weighed more, this phenomenon showed that the nano MoS2/graphene composites had better electrical conductivity than the bulk MoS2 material which meant that compounded with graphene improved the conductivity of the nano MoS2 material. The decrease of the probe DNA immobilized bulk MoS2 modified GCE showed that bulk MoS2 could not effectively adsorbed probe DNA which means that the electrode cannot gather K3[Fe(CN)6] because of its poor specific surface area. And graphene and similar 2D structure materials have nucleobase–graphene inter-molecular π–π stacking interactions with ssDNA, that is also beneficial to immobilize ssDNA on the 2D MoS2/graphene composites.21 And this phenomenon also proved that the probe DNA has been immobilized on the nano MoS2/graphene composites modified electrode surface.
Fig. 6B is the Nyquist diagram of nano MoS2/graphene modified GCE and bulk MoS2 modified GCE. The interface electron-transfer resistance (Ret) was the diameter of arc in the high-frequency section of the curve and the figure displays composites had lower Ret than the bulk MoS2 owing to the better electric conductivity. Ret (composites) = 172 ohm, Ret (MoS2) = 225.5 ohm. The EIS experiments were in good accordance with the DPV experiments presented later on.
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Fig. 7 DPV plots of K3[Fe(CN)6] at pDNA immobilized MoS2/graphene composites modified GCE before and after immobilization. |
The DPV plots (Fig. 7) showed the signals of K3[Fe(CN)6] at probe DNA immobilized MoS2/graphene composites modified GCE before and after pDNA immobilization. From the plots we can see that after the pDNA immobilized on the modified GCE, the DPV anodic peak current value had an increase and it was caused by more [Fe(CN)6]3− gathering together around the probe DNA immobilized electrode surface. From this we can know that probe DNA had been successfully bounded to the MoS2/graphene composites modified GCE surface. Xinxing Wang and his team had made a work proving that the positively charged guanine and adenine could not only adsorb on the nano MoS2 surface via the van der Waals force, but also through electrostatic adsorption.7 Before this experiment, another probe DNA (5′-CGA CAG TGG TCC CAA AGA-3′) which had been used and in that experiment the concentration of the K3[Fe(CN)6] was one half of this experiment while the oxidation peak current value of DPV signals was the same. Comparing two probe DNA, the biggest difference was the base number (5′-AGT GAT TTT AGA GAG-3′ and 5′-CGA CAG TGG TCC CAA AGA-3′). It also implied that the MoS2/graphene composites could immobilize the probe DNA because of the van der Waals bond between the probe DNA and the nanosheets.
Fig. 8A shows the DPV signals of the nano MoS2/graphene composites modified GCE after different concentration (1.0 × 10−17 M to 1.0 × 10−12 M, 20 μL) cDNA hybridizing with the pDNA and probe DNA immobilized MoS2/graphene composites modified GCE. The DPV experiment was made in the 1.0 M KCl solution containing 0.2 M K3[Fe(CN)6]. We can see that the DPV signal of oxidation peak current value had an obvious decrease which illustrates that the amount of remaining pDNA had decreased, because the dsDNA formed by the hybridization of pDNA and cDNA had been released from the electrode surface by the electrochemical pretreatment (−0.7 V, 300 s). From the change of the DPV signals we can see that with the hybridization the oxidation peak current value decreased fast. It shows an opposite tend with that in pDNA immobilization, which proves that the amount of pDNA has decreased and the hybridization has successfully conducted.
From the DPV plots we can find that with the hybridization conducting the oxidation peak current value decreases, and the difference (namely ΔIpc) between the oxidation peak current value of K3[Fe(CN)6] could be used as the measurement signal. As the plot of 10−12 M and 10−17 M deflected too much, the linear relationship used the data from 10−16 M to 10−13 M. And from the data we can conclude a linear relationship between ΔIpc and the concentration of the target (cDNA) from 1.0 × 10−16 M to 1.0 × 10−13 M, and the R square is 0.985, the regression equation is −ΔIpc/A = 1.142log
C/M − 22.761. The limit of detection (LOD) is 10−17 M and 10−12 M is the value at which the response starts reaching a plateau. Fig. 8B is figure of the linear fit. And this detection limit is the remarkable sub-femtomolar LOD of our assay compares well with that of similar magnitude previously reported by other with the same E542K gene detection (50 fM) and similar gene detection (miRNA-21, 100 aM).10,22 These results show that the nano MoS2/graphene composites modified GCE has high electrochemical activity and has an ultrahigh sensitivity for the DNA biosensing.
Fig. 9 compares three percentages of ΔIpc/Ipc in different ssDNA hybridization with the probe DNA including one-middle-base mismatched DNA (1MT DNA), two-middle-bases-mismatched DNA (2MT DNA) and non-complementary DNA (ncDNA) sequence.
From the figure we can see that the highest ΔIpc is the complementary DNA sequence followed by 1MT DNA, 2MT DNA and the non-complementary DNA successively. Compared with the ΔIpc of the complementary DNA, the signal decrease of the non-complementary DNA was much lower and could be neglected. Although the selectivity of 1MT DNA is not so good, other mismatched DNA sequences can be easily distinguished. From this result we can conclude that the specificity of the DNA sensor is high selectivity and could distinguish most mismatched DNA sequences.
This journal is © The Royal Society of Chemistry 2016 |