sensors Article
A MoS2 Nanosheet-Based Fluorescence Biosensor for Simple and Quantitative Analysis of DNA Methylation Le Xiao 1 , Li Xu 2 , Chuan Gao 1 , Yulin Zhang 1 , Qunfeng Yao 1, * and Guo-Jun Zhang 1, * 1
2
*
School of Laboratory Medicine, Hubei University of Chinese Medicine, 1 Huangjia Lake West Road, Wuhan 430065, China;
[email protected] (L.X.);
[email protected] (C.G.);
[email protected] (Y.Z.) School of Pharmacy, Hubei University of Chinese Medicine, 1 Huangjia Lake West Road, Wuhan 430065, China;
[email protected] Correspondence:
[email protected] (Q.Y.);
[email protected] (G.-J.Z.); Tel.: +86-27-6889-0259 (G.-J.Z.); Fax: +86-27-6889-0071 (G.-J.Z.)
Academic Editor: Alexander Star Received: 4 May 2016; Accepted: 6 July 2016; Published: 22 September 2016
Abstract: MoS2 nanomaterial has unique properties, including innate affinity with ss-DNA and quenching ability for fluorescence dyes. Here, we present the development of a simple fluorescence biosensor based on water-soluble MoS2 nanosheets and restriction endonuclease BstUI for methylation analysis of p16 promoter. The biosensing platform exhibited excellent sensitivity in detecting DNA with a linear range of 100 pM~20 nM and a detection limit of 140 pM. More importantly, our method could distinguish as low as 1% difference in methylation level. Compared with previous methylation analysis, our design is both time saving and simple to operate, avoiding the limitations of PCR-based assays without compromising performance. Keywords: MoS2 ; fluorescence biosensor; homogeneous analysis; DNA methylation
1. Introduction The process of DNA methylation is an essential part of epigenetics that plays critical roles in many biological events, such as gene transcription, X-chromosome inactivation, and genomic imprinting [1–3]. In mammals, DNA methylation occurs mainly in CpG-rich regions, known as CpG islands, which is usually located in the promoter region or near the first exon of transcriptional regulatory genes [4–6]. Aberrant methylation of CpG islands was proven to be closely associated with the occurrence of human diseases, particularly cancers [7,8]. The initiation of a tumor is often accompanied by abnormal rise of methylation levels of CpG islands near tumor suppressor genes, which leads to their inactivation [6–8]. Thus, the change of methylation status in CpG islands is considered to be a promising biomarker for cancer prognosis and diagnosis [9–11]. Due to the great significance of research on DNA methylation status, a variety of biosensors and bioassays have been established for the quantification of gene-specific CpG methylation. Techniques based on bisulfite treatment and polymerase chain reaction (PCR) are the most extensively applied for the detection of methylation status [12–14]. However, these methods involve complicated procedures and require precision instruments. In addition, frequent false positive detection has become their common bottlenecks [15,16]. Lately, some new biosensing technologies without bisulfite or PCR for gene-specific methylation assays have also been developed, such as nanowire field effect transistor (FET) [16], electrochemistry [17], colorimetry [18] and so on. For example, Maki et al. [16] immobilized monoclonal anti-5-methylcytosine antibodies on the nano-FET, which could recognize and bind to methylated target DNA. Dai et al. [17] designed a label-free electrochemical DNA biosensor for quantification of gene-specific methylation, in which the probe was modified on gold electrode and Sensors 2016, 16, 1561; doi:10.3390/s16101561
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methylene blue (MB) was used as the electrochemical indicator. Although these methods each have its advantages, they generally require tedious preparation work, such as sensor surface modification or Sensors 2016, 16, 1561 2 of 10 extra amplification technology. Therefore, it is still necessary to explore convenient methods for the Although these methods each have its advantages, they generally require tedious preparation work, detection of gene-specific methylation. such as sensor surface modification or extra amplification technology. Therefore, it is still necessary Currently, nanomaterials are particularly useful in the field of biosensors due to their unique to explore convenient methods for the detection of gene-specific methylation. optical properties. For example, MoS2 nanosheets exhibit high quenching efficiency to fluorescence Currently, nanomaterials are particularly useful in the field of biosensors due to their unique probes [19–21]. What’sFor more, as anMoS emerging class of alternative graphene-like 2D nanomaterial [22], optical properties. example, 2 nanosheets exhibit high quenching efficiency to fluorescence MoS2probes nanosheets demonstrated their intrinsic abilities 2D to ss-DNA and [22], ds-DNA, [19–21].have What’s more, as an emerging class ofdiscrimination alternative graphene-like nanomaterial with MoS even2 nanosheets better water solubility [19–23]. combination with fluorescent DNA have demonstrated theirThus, intrinsic discrimination abilities to ss-DNA andprobes, ds-DNA,a few MoS2with -based been developed forcombination the detection biomolecules, such as nucleic evenbiosensors better waterhas solubility [19–23]. Thus, with of fluorescent DNA probes, a few MoS 2 -based biosensors has been developed for the detection of biomolecules, such as nucleic acids acids [19–21], proteins [24], and small molecules [25]. However, such a great biosensing platform has [19–21], proteinsfor [24], and small molecules [25]. However, such a great biosensing platform has yet yet been employed gene-specific CpG methylation analysis. been employed for gene-specific CpG methylation analysis. We herein report a MoS2 -based fluorescence biosensor for methylation analysis of p16 promoter We herein report a MoS2-based fluorescence biosensor for methylation analysis of p16 promoter with easy and quick operation. The mechanism of the sensing system is depicted in Scheme 1. with easy and quick operation. The mechanism of the sensing system is depicted in Scheme 1. A A segment from the promoter of the p16 gene is selected as the investigated target, which includes segment from the promoter of the p16 gene is selected as the investigated target, which includes the the recognition restrictionendonuclease. endonuclease. The FAM-probe is firstly hybridized recognition site site of of BstUI BstUI restriction The FAM-probe (P) (P) is firstly hybridized with with unmethylated and methylated target DNA (T and T ) to form partial duplex (pds-DNA), respectively, 1 2 unmethylated and methylated target DNA (T1 and T2) to form partial duplex (pds-DNA), and then mixed and withthen BstUI. Atwith last,BstUI. MoS2Atnanosheets are addedareinto theinto hybridization solutions. respectively, mixed last, MoS2 nanosheets added the hybridization solutions. a result, the unmethylated pds-DNA (P/T1) at is specific cleaved at specific site 5’-CGCG-3’ and As a result, theAs unmethylated pds-DNA (P/T site 5’-CGCG-3’ and FAM-labeled 1 ) is cleaved FAM-labeled ds-DNA is releasedMeanwhile, to the solution. Meanwhile,pds-DNA the methylated (P/Tby 2) is ds-DNA is released to the solution. the methylated (P/T2pds-DNA ) is adsorbed MoS2 , adsorbed by MoS 2, which lead to quenching of fluorescence. Thus, we provide a straight-forward which lead to quenching of fluorescence. Thus, we provide a straight-forward approach to quantifying approach to quantifying methylated DNA through detection in homogeneous solution. methylated DNA through fluorescence detection in fluorescence homogeneous solution.
Scheme 1. Schematic illustration of of homogeneous analysisofofp16 p16promoter promoter means of Scheme 1. Schematic illustration homogeneousmethylation methylation analysis byby means MoS2ofnanosheets-based biosensor. MoS2 nanosheets-based biosensor.
2. Materials Methods 2. Materials andand Methods 2.1. Materials Reagents 2.1. Materials and and Reagents The restriction endonuclease BstUI was obtained from New England Biolabs (Beverly, MA,
The restriction endonuclease BstUI was obtained from New England Biolabs (Beverly, MA, USA). USA). The layer molybdenum disulfide (MoS2) nanosheets solution (1–8 monolayers, 100–400 nm, The layer molybdenum disulfide (MoS2 ) nanosheets solution (1–8 monolayers, 100–400 nm, 18 mg/L) 18 mg/L) was purchased from Nanjing XFNANO Materials Tech Co. Ltd. (Nanjing, China) and was purchased from Nanjing XFNANO Materials Tech Co. Ltd. (Nanjing, China) andsodium treated by treated by ultrasonic agitation for 3 h before use. Tris(hydroxylmethyl)aminomethane (Tris), ultrasonic agitation h before use. Tris(hydroxylmethyl)aminomethane (Tris), sodium chloride chloride (NaCl) for and3 magnesium chloride (MgCl2) were purchased from Sinopharm Chemical (NaCl) and magnesium chlorideChina). (MgClAll purchased fromwere Sinopharm Chemical Reagent Co. Ltd. Reagent Co. Ltd. (Shanghai, the chemical reagents of analytical reagent grade and 2 ) were used without Ultrapure water used in allreagent experiments fromfurther a (Shanghai, China).further All thepurification. chemical reagents were of analytical gradewas andgenerated used without Milli-Q Direct 8 water purification system (Millipore, Billerica, MA, USA). purification. Ultrapure water used in all experiments was generated from a Milli-Q Direct 8 water Thesystem buffer (Millipore, solutions employed this USA). work were as follows: DNA hybridization buffer and purification Billerica,inMA, BstUI reaction buffer were 1 × CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM The buffer solutions employed in this work were as follows: DNA hybridization buffer and BstUI reaction buffer were 1 × CutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM
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magnesium acetate, 100 µg/mL BSA, pH 7.9, 25 ◦ C). MoS2 quenching and fluorescence detection were performed in buffer containing 20 mM Tris-HCl, 100 mM NaCl and 10 mM MgCl2 (pH 7.4). All DNA sequences used in this work were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and dissolved in ultrapure water to 100 µM stock solution and stored at −20 ◦ C. Gene sequences of probes and targets include methylated and unmethylated were designed according to the promoter region human p16 gene. Non-complementary DNA (N) was from the promoter region of human p53 gene [26]. Detailed sequences are shown in Table 1. Table 1. Used DNA sequences. Oligonucleotides
Gene Sequences (50 –30 )
FAM-Probe DNA (P)
FAM-GAC CCC GGG CCG CGG CCG TGG
Unmethylated target DNA (T1 )
AGC AGC ATG GAG CCT TCG GCT GAC TGG CTG GCC ACG GCC GCG GCC CGG GGT C
Methylated target DNA (T2 )
AGC AGC ATG GAG CCT TCG GCT GAC TGG CTG GCC ACG GCmC GmCG GCC CGG GGT C
One base mismatched DNA (M)
AGC AGC ATG GAG CCT TCG GCT GAC TGG CTG GCC ACG GCC TCG GCC CGG GGT C
Non-complementary DNA (N)
CTT GAT ATT CGG CAC ATA GTC CTG GGA GAG ACC GGC GCA CAG AGG AAG AGA A
2.2. Apparatus All fluorescence measurements were carried out on a F-4600 spectrophotometer (Hitachi Co. Ltd., Tokyo, Japan) equipped with a xenon lamp as excitation source. The apparatus for transmission electron microscopy (TEM) was a JEM-2100 (Jeol Ltd., Tokyo, Japan). The UV-vis spectra were obtained using a UV-2550 spectrometer (Shimadzu, Tokyo, Japan). Atomic force microscopy (AFM) image was obtained from a multimode VIII (Veeco, New York, NY, USA). The dynamic light scattering (DLS) was conducted by a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) 2.3. Endonuclease Digestion of Probe/Target DNA 20 nM FAM—labeled probe DNA(P) were hybridized with unmethylated target DNA (T1 , 20 nM) and methylated target DNA (T2 , 20 nM), respectively. The mixture were heated to 95 ◦ C for 10 min, and then slowly cooled down to room temperature to ensure the formation of partial duplex DNAs (pds-DNAs), including the unmethylated pds-DNA (P/T1 ) and the methylated pds-DNA (P/T2 ). Then the pds-DNAs were cleaved by 20 U/mL BstUI endonuclease at 60 ◦ C for 2 h in 50 µL 1 × CutSmart buffer. Finally, the mixture pds-DNAs were mixed with 450 µL solution of 4 µg/mL MoS2 for 10 min, respectively. 2.4. Fluorescence Assays Fluorescence measurements were carried out at room temperature. The emission spectra are measured in the range between 510 and 650 nm for carboxyfluorescein (FAM) with the excitation wavelength set at 495 nm. 2.5. Methylation Assay by Gel Electrophoresis Non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to verify the feasibility of the sensing system. In the gel electrophoresis experiment, 1 µM pds-DNAs (P/T1 or P/T2 ) were prepared and the specimens were treated as follows: (1) unmethylated pds-DNA (P/T1 ); (2) methylated pds-DNA (P/T2 ); (3) unmethylated pds-DNA (P/T2 ) and BstUI; (4) methylated pds-DNA (P/T2 ) and BstUI. The DNA solutions mixed with 1 × loading buffer were loaded on a 15% non-denaturing polyacrylamide gel electrophoresis. The electrophoresis was run at 100 V constant for 105 min in
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1 × TBE running buffer (89 mM Tris-Boric Acid, 2.0 mM EDTA, pH 8.3). Subsequently, the gel was Sensors 16, 1561 bromide for 15 min, and then de-stained in ultrapure water for 15 min. 4 ofFinally, 10 stained by 2016, ethidium electrophoresis images were captured using a Gel Doc XR+ imaging system (Bio-Rad, Hercules, for 105 min in 1 × TBE running buffer (89 mM Tris-Boric Acid, 2.0 mM EDTA, pH 8.3). Subsequently, CA, USA). the gel was stained by ethidium bromide for 15 min, and then de-stained in ultrapure water for 15 min. Finally, electrophoresis images were captured using a Gel Doc XR+ imaging system (Bio-Rad, 3. Results Hercules, CA, USA).
3.1. Characterization of MoS2 3. Results
The MoS2 was characterized and the results are shown in Figure S1. The transmission electron 3.1. Characterization of MoS 2 microscopy (TEM) image showed the stability of MoS2 nanosheets dispersion in aqueous solutions and revealed that was a two-dimensional thinare nanosheet S1A). S1B demonstrated The the MoSMoS 2 was characterized and the results shown in(Figure Figure S1. TheFigure transmission electron 2 microscopy (TEM) image showed the stability of MoS 2 nanosheets dispersion in aqueous solutions that the MoS2 nanosheets possessed their two typical absorption peaks at around 607 and 665 nm and revealed that the spectrum, MoS2 was which a two-dimensional nanosheet (Figureresults S1A). [25]. FigureThe S1BAFM by UV-visible absorption is consistentthin with the reported demonstrated that the MoS 2 nanosheets possessed their two typical absorption peaks at around 607 equipment not mentioned in 2.2 image was also recorded to characterize the size of MoS2 . As shown and 665 nm by UV-visible absorption spectrum, which is consistent with the reported results [25]. The in Figure S1C, the AFM image displayed that the height of the MoS2 sheet was about 2.7 nm thick AFM equipment not mentioned in 2.2 image was also recorded to characterize the size of MoS2. As (inset in Figure S1C), indicating that the MoS2 is a few-layer nanosheet. The characterization of the shown in Figure S1C, the AFM image displayed that the height of the MoS2 sheet was about 2.7 nm MoS2thick nanosheets further confirmedthat by the dynamic scattering (DLS).The Thecharacterization result indicated (inset in was Figure S1C), indicating MoS2 islight a few-layer nanosheet. of that the lateral dimensions of most of MoS nanosheets were about 100 to 400 nm, suggesting that 2 the MoS2 nanosheets was further confirmed by dynamic light scattering (DLS). The result indicated the nanosheets a high polydispersity that thehad lateral dimensions of most [27,28]. of MoS2 nanosheets were about 100 to 400 nm, suggesting that the nanosheets had a high polydispersity [27,28].
3.2. Feasilbility of the Assay
3.2. Feasilbility of the Assay
The principle of the method was outlined in Scheme 1. In this system, BstUI specifically cleaves the principle of the method (P/T was outlined in Scheme 1. In this system, BstUI specifically cleaves residue ofThe unmethylated pds-DNA 1 ) containing the human methylation specific site 5’-CGCG-3’, the residue of unmethylated pds-DNA (P/T 1) containing the human methylation specific site and a FAM-labeled complementary DNA is released, while the methylated pds-DNA (P/T2 ) could not 5’-CGCG-3’, and a FAM-labeled complementary DNA is released, while the methylated pds-DNA be digested by BstUI [29,30]. To validate the methylation-sensitive cleavage process of BstUI, the PAGE (P/T2) could not be digested by BstUI [29,30]. To validate the methylation-sensitive cleavage process test was performed. As shown in Figure 1A, in the absence of BstUI, both the bands of unmethylated of BstUI, the PAGE test was performed. As shown in Figure 1A, in the absence of BstUI, both the pds-DNA pds-DNA (P/T , line 2) were identical. When the BstUI was 1 , line 1) and methylated bands(P/T of unmethylated pds-DNA (P/T 1, line 1) and 2methylated pds-DNA (P/T2, line 2) were added into pds-DNAs solutions, the band situation unmethylated 1 , line 3) was identical. When the BstUI was added into pds-DNAsof solutions, the bandpds-DNA situation of(P/T unmethylated lowerpds-DNA than those of 1the above-mentioned cases, showing the unmethylated was the cleaved (P/T , line 3) was lower than those of the that above-mentioned cases,pds-DNA showing that into shorter chainspds-DNA by BstUIwas endonuclease. unmethylated cleaved into shorter chains by BstUI endonuclease.
Figure 1. (A) Gel image about the methylation-sensitive cleaved process of BstUI in non-denaturing
Figure 1. (A) Gel image about the methylation-sensitive cleaved process of BstUI in non-denaturing polyacrylamide gel electrophoresis. Lane M: DNA marker; Lane 1: unmethylated pds-DNA (P/T1); polyacrylamide gel electrophoresis. Lane M: DNA marker; Lane 1: unmethylated pds-DNA (P/T1 ); Lane 2: methylated pds-DNA (P/T2); Lane 3: unmethylated pds-DNA (P/T1) + BstUI; Lane 4: Lane methylated 2: methylated pds-DNA (P/T2 );(B) Lane 3: unmethylated pds-DNA (P/T1 ) detection + BstUI; and Lane 4: pds-DNA (P/T2) + BstUI. Fluorescence emission spectra of the DNA methylated pds-DNA (P/T ) + BstUI. (B) Fluorescence emission spectra of the DNA detection 2 methylation analysis strategy under different conditions: (1) unmethylated pds-DNA (P/T1); (2) and methylation analysis (P/T strategy different conditions: pds-DNA (P/T1 ); 2); (3) under unmethylated pds-DNA (P/T1)(1) + unmethylated BstUI + MoS2; (4) methylated methylated pds-DNA (2) methylated pds-DNA (P/T pds-DNA (P/T 2) + BstUI + MoS . unmethylated pds-DNA (P/T1 ) + BstUI + MoS2 ; (4) methylated 2 ); 2(3) pds-DNA (P/T2 ) + BstUI + MoS2 .
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Nevertheless, there was no change in the band of methylated pds-DNA (P/T2, line 4), suggesting that thethere cleavage of BstUI blocked pds-DNA by methylation. time, Nevertheless, was no changeendonuclease in the band ofwas methylated (P/T2 , At linethe 4),same suggesting little difference of fluorescence intensities between unmethylated pds-DNA (P/T 1, Figure 1B curve 1) that the cleavage of BstUI endonuclease was blocked by methylation. At the same time, little difference and methylatedintensities pds-DNA (P/T2, unmethylated Figure 1B curve 2) was Upon addition of MoS2 of fluorescence between pds-DNA (P/Tobserved. 1 , Figure 1B curve 1) and methylated nanosheets, the methylated pds-DNA (P/T 2) containing the single-stranded was tightly adsorbed by pds-DNA (P/T2 , Figure 1B curve 2) was observed. Upon addition of MoS2 nanosheets, the methylated MoS 2 nanosheets and the fluorescence intensity of P/T2 was sharply quenched (Figure 1B curve 4). pds-DNA (P/T2 ) containing the single-stranded was tightly adsorbed by MoS2 nanosheets and But unmethylated pds-DNA by BstUI just slightly interacted nanosheet in the fluorescence intensity of(P/T P/T1)2 cleaved was sharply quenched (Figure 1B curvewith 4). MoS But 2unmethylated the form of totally complementary DNA, and exhibited a little lower fluorescence signal than in pds-DNA (P/T1 ) cleaved by BstUI just slightly interacted with MoS2 nanosheet in the form of that totally the absence of MoS 2 (Figure 1B curve 3). The accordance of fluorescence spectroscopy experiment complementary DNA, and exhibited a little lower fluorescence signal than that in the absence of MoS2 with the aforementioned gel analysis successfully demonstrates that with the the designed sensing (Figure 1B curve 3). The accordance of fluorescence spectroscopy experiment aforementioned system is feasible. gel analysis successfully demonstrates that the designed sensing system is feasible. 3.3. 3.3. Optimization Optimization of of Assay Assay Conditions Conditions To To obtain the best sensing performance, performance, the the optimal optimal conditions conditions of of experimental experimental parameters, parameters, including MoS22concentration, concentration, concentration of BstUI endonuclease and cleavage time, were including MoS concentration of BstUI endonuclease and cleavage time, were evaluated evaluated by comparing the relative fluorescence change. The relative fluorescence change by comparing the relative fluorescence change. The relative fluorescence change was expressed aswas the expressed as the signal difference value ΔF. signal difference value ∆F. ΔF ∆F == FF11 −−FF2,2 ,where whereFF1 1and andFF2 2are arefluorescence fluorescenceintensities intensitiesofof the the system system where where the the primer primer FAM-probe are hybridized with its complementary unmethylated (T 1 ) and methylated FAM-probe are hybridized with its complementary unmethylated (T1 ) and methylated (T (T22)) target target DNA, DNA, respectively. respectively. The MoS22 concentration concentrationplayed playeda adecisive decisive role in distinguishing between the partial duplex The MoS role in distinguishing between the partial duplex DNA DNA (pds-DNA) and the double-stranded DNA As shown Figure 2, range in the (pds-DNA) and the double-stranded DNA (ds-DNA). As(ds-DNA). shown in Figure 2, in the in concentration of concentration of MoS 2 atincreased 1.0–4.0 µg/mL, the ΔFasincreased significantly as 2the concentration of MoS2 at 1.0–4.0range µg/mL, the ∆F significantly the concentration of MoS increased. However, MoS 2 increased. However, when the concentration 2 exceeded 4.0 µg/mL indicating or higher, that the ΔF when the concentration of MoS 4.0 µg/mLoforMoS higher, the ∆F decreased, the 2 exceeded decreased, indicating that the MoS2cause of high concentration wouldeffect causeonanthe excessive quenching MoS2 of high concentration would an excessive quenching cleavage-produced effect on the double-stranded cleavage-produced FAM-labeled DNA.a concentration According to of theMoS above FAM-labeled DNA. According double-stranded to the above results, 2 at results, a concentration of MoS 2 at 4.0 µg/mL was selected for the following analysis experiments. 4.0 µg/mL was selected for the following analysis experiments.
Figure fluorescence change change(∆F (∆F == FF11 −−FF2)2 )on Figure 2. 2. Dependence Dependence of the relative fluorescence ondifferent differentconcentrations concentrations of of MoS22nanosheets. nanosheets.Error Errorbars barsrepresent representthe thestandard standard deviation deviation of of three three experiments. experiments. MoS
In In addition, addition, the the concentration concentration of of BstUI BstUI endonuclease endonuclease and and cleavage cleavage time time also also affect affect the the sensing sensing system. As can be seen in Figure S2A, as the concentration of BstUI increased, the ΔF was enhanced system. As can be seen in Figure S2A, as the concentration of BstUI increased, the ∆F was enhanced and and reached a maximum 20 U/mL. Similarly, as depicted in Figure the ΔF increased and reached a maximum at 20 at U/mL. Similarly, as depicted in Figure S2B, theS2B, ∆F increased and reached reached plateauwhen phase when the cleavage h. As a20result, U/mLendonuclease BstUI endonuclease a plateaua phase the cleavage time wastime 2 h. was As a2result, U/mL20BstUI and 2 h and 2 h cleavage time were selected as the optimum conditions of BstUI for theexperiments. following cleavage time were selected as the optimum conditions of BstUI for the following analysis analysis experiments. 3.4. Kinetic Behavior 3.4. Kinetic Behavior The kinetic behavior of MoS2 fluorescence quenching was investigated as well by monitoring The kinetic behavior 2 fluorescence was the investigated as well by monitoring the fluorescence intensity of as MoS a function of time. quenching Figure 3 shows fluorescence quenching of P/T1 the fluorescence intensity as a function of time. Figure 3 shows the fluorescence quenching of P/T1
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(curve red) and P/T2 (curve blank) after digested in the presence of MoS2 as a function of incubation (curve red) and P/T blank) after in the of of incubation (curve and P/T22(curve (curve blank) afterdigested digested thepresence presence of MoS MoS22 as asawas a function function time.red) Upon addition of MoS 2 (4 µg/mL) into theinsolutions, the quenching found of to incubation be very fast. time. Upon addition of MoS 2 (4 µg/mL) into the solutions, the quenching was found to be very fast. time. Upon addition of MoS (4 µg/mL) into the solutions, the quenching was found to very for fast.10 2 intensity of P/T1 decreased slightly and reached a stablebe After that, the fluorescence signal After that, the fluorescence intensity of P/T 1 decreased slightly and reached a stable signal for 10 After the fluorescence intensity intensity of P/T1 decreased slightly and reached stable 10almost min. min.that, Similarly, the fluorescence of P/T2 decreased sharply in athe firstsignal 30 s for and min. Similarly, the fluorescence intensity of P/T2 decreased sharply in30 the firstalmost 30 s and almost Similarly, the fluorescence intensity of P/T decreased sharply in the first s and reached 2 other words, the relative fluorescence change of boththe reached the equilibrium within 10 min. In P/T1 reached the equilibrium within 10 min. In other words, the relative fluorescence change bothP/T P/T1 equilibrium within 10 min. In other words, the relative change of both P/Tof1 and 2 and P/T2 achieved maximum when the quenching timefluorescence was at 10 min. and P/T2 achieved maximum the quenching achieved maximum when thewhen quenching time wastime at 10was min.at 10 min.
Figure 3. The kinetic behavior of FAM-probe fluorescence quenching the presence of2 MoS with Figure 3. The kinetic behavior of FAM-probe fluorescence quenching in theinpresence of MoS with2the Figure 3. The kinetic behavior of FAM-probe fluorescence quenching in the presence of MoS2 with the unmethylated (red) and methylated (blank), respectively. unmethylated target target (red) and methylated target target (blank), respectively. the unmethylated target (red) and methylated target (blank), respectively.
3.5. Detection Target DNA 3.5. Detection of of Target DNA 3.5. Detection of Target DNA Firstly, based high fluorescence quenching property and discrimination ability between Firstly, based onon thethe high fluorescence quenching property and discrimination ability between Firstly, based on the high fluorescence quenching property and discrimination ability between ss-DNA and ds-DNA of MoS 2 [19], the unmethylated target DNA (T 1 ) (a fragment of P16 promoter) ss-DNA and ds-DNA of MoS [19], the unmethylated target DNA (T ) (a fragment of P16 promoter) ss-DNA and ds-DNA of MoS22 [19], the unmethylated target DNA (T11) (a fragment of P16 promoter) could detected in homogeneous this homogeneous sensing Figure 4A exhibited the fluorescence could be be detected in this sensing system.system. Figure 4A exhibited the fluorescence spectra could be detected in this homogeneous sensing system. Figure 4A exhibited the fluorescence spectra of 20 nM FAM-probe DNA (P) in the presence of different concentrations of unmethylated of 20 nM FAM-probe DNA (P) in the presence of different concentrations of unmethylated target DNA spectra of 20 nM FAM-probe DNA (P) in the presence of different concentrations of unmethylated (TnM 1) from 0 to 40optimized nM underconditions. the optimized conditions. It is noted that fluorescence (T1target ) fromDNA 0 to 40 under the It is noted that fluorescence could still increasecould as target DNA (T1) from 0 to 40 nM under the optimized conditions. It is noted that fluorescence could still increase as the concentration of T 1 exceeded that of FAM-probe DNA (P). That’s because that the concentration of T1 exceeded that of FAM-probe DNA (P). That’s because that the redundant T1 has still increase as the concentration of T1 exceeded that of FAM-probe DNA (P). That’s because that the redundant T 1 has a stronger interaction with MoS 2 compared to the cleaved double-stranded a stronger interaction with MoS2 compared to the cleaved double-stranded DNA of P/T1 , and replaces the redundant T1 has a stronger interaction with MoS2 compared to the cleaved double-stranded of P/T1, and replaces the few cleaved ds-DNA on MoS2 [19]. theDNA few adsorbed cleaved ds-DNA onadsorbed MoS2 [19]. DNA of P/T1, and replaces the few adsorbed cleaved ds-DNA on MoS2 [19].
Figure Figure4. 4. (A) (A)Fluorescence Fluorescencespectra spectraofofthe thehybridization hybridizationofof2020nM nMprobe probeDNA DNAwith withvarious various Figure 4. (A) Fluorescence spectra of2, the hybridization of4020 nM(B) probe DNA relationship with various concentrations of T (0, 0.1, 0.2, 0.5, 1, 5, 10, 15, 20, 30 and nM); The linear 1 of F concentrations of T1 (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30 and 40 nM); (B) The linear relationship of 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30 and 40 nM);The (B) inset The linear relationship of F concentrations of T1 (0, 0.1, F (Y) shows linear responses (Y) with withvarious variousDNA DNAconcentrations concentrations(X) (X)ranging rangingfrom from0 0toto 40nM. nM. The inset shows linear responses (Y) withconcentrations various DNA concentrations (X) ranging from 0 to bars 40 nM. The inset linear responses to to low of of T1Tranging from 0 to 2 nM. Error represent theshows standard deviation of low concentrations 1 ranging from 0 to 2 nM. Error bars represent the standard deviation of to low concentrations of T 1 ranging from 0 to 2 nM. Error bars represent the standard deviation of three experiments. three experiments. three experiments.
According to the relative calibration curve (Figure 4B), the MoS2-based DNA sensor revealed a According to the relative calibration curve (Figure 4B), the MoS2-based DNA sensor revealed a linear response in the range from 0 to 20 nM with the calibration equation is Y = 5.81X + 14.75 (Y linear response in the range from 0 to 20 nM with the calibration equation is Y = 5.81X + 14.75 (Y
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According to the relative calibration curve (Figure 4B), the MoS2 -based DNA sensor revealed of 10 in the range from 0 to 20 nM with the calibration equation is Y = 5.81X + 714.75 (Y represents the fluorescence intensity and X represents T1 concentrations, R2 = 0.9946). The detection represents the fluorescence intensity and X represents T1 concentrations, R2 = 0.9946). The detection limit is 140 pM obtained in terms of 3 times deviation of blank sample, which is at the same magnitude limit is 140 pM obtained in terms of 3 times deviation of blank sample, which is at the same with the previously reported nanomaterials-based fluorescent assay [20,31]. magnitude with the previously reported nanomaterials-based fluorescent assay [20,31]. In order to investigate the specificity of detection of target DNA, one base mismatched DNA In order to investigate the specificity of detection of target DNA, one base mismatched DNA (M), non-complementary DNA (N), and unmethylated target DNA (T1 ) were employed, respectively. (M), non-complementary DNA (N), and unmethylated target DNA (T1) were employed, It was seen that the unmethylated DNA (T1 , a) possessed the highest fluorescence response (Figure S3). respectively. It was seen that the unmethylated DNA (T1, a) possessed the highest fluorescence In contrast, the fluorescence intensity of M (b) and N (c) decreased greatly, only a slightly higher than response (Figure S3). In contrast, the fluorescence intensity of M (b) and N (c) decreased greatly, that of blank control (d). The results indicate that the MoS2 nanosheet-based fluorescence biosensor only a slightly higher than that of blank control (d). The results indicate that the MoS2 has a satisfactory specificity. nanosheet-based fluorescence biosensor has a satisfactory specificity. Sensors 2016, 16, 1561 a linear response
3.6. Quantitative Analysis of DNA Methylation 3.6. Quantitative Analysis of DNA Methylation Promoter region of p16 is known to be differentially methylated in a variety of physiological Promoter region of p16 to to becheck differentially methylated in a variety of physiological states. Therefore, we use it as is anknown example the efficacy of our sensor in quantitiative analysis states. Therefore, we use it as an example to check the efficacy of our sensor in quantitiative analysis of DNA methylation. We prepared 20 nM probe DNA (P) to hybridize with the artificial mixtures of DNA methylation. We prepared 20 nM probe target DNA DNA (P) toathybridize with the artificial mixtures consisting of 20 nM methylated and unmethylated different ratios, 0, 5%, 10%, 20%, 30%, consisting of 20 nM methylated and unmethylated target DNA at different ratios, 0, 5%, 10%, 20%, 40%, 60%, 80%, and 100% methylated target DNA. The methylation level of target DNA at 5’-CGCG-3’ 30%, 60%, 80%, and methylated target DNA. The methylation level of target DNA at could40%, be evaluated from the100% formula: 5’-CGCG-3’ could be evaluated from the formula: Methylation levels levels % = % M = /M (U M) × × (1) Methylation / (U++ M) 100100 % % (1)
Under the optimized conditions, experiments experiments were were carried carried out by adding the mixtures with increasing methylation levels into the proposed MoS22-based sensing system to examine whether the relative fluorescence fluorescencechange change(∆F, (ΔF, F, where F1 Fand are fluorescence intensities of the ∆FΔF = F=1 –F1F,–where F1 and are Ffluorescence intensities of the system systemthe where theFAM-probe primer FAM-probe is hybridized with the unmethylated DNA (T1) and where primer is hybridized with the unmethylated DNA (T1 ) and mixtures of mixtures different of different methylation target DNA (T), respectively) be used forlevel methylation methylation proportions proportions target DNA (T), respectively) could be usedcould for methylation analysis. level analysis. As presented in Figure 5, the relative fluorescence change (ΔF) increased with the As presented in Figure 5, the relative fluorescence change (∆F) increased with the increasing DNA increasing correlation in the DNA methylation level methylationDNA level,methylation indicating a level, linear indicating correlation ainlinear the DNA methylation level range from 0 to 100%. range from 0 to 100%. Theiscalibration Y = 2.14X +∆F 8.09 (Y Xrepresents ΔFmethylation and X represents The calibration equation Y = 2.14Xequation + 8.09 (Yisrepresents and represents level, methylation R2 = 0.9956). limit was calculated toLOD be 1.0% LOD = 3 σ/S R2 = 0.9956). level, The detection limitThe wasdetection calculated to be 1.0% based on = 3 based σ/S (σon is the standard (σ is the standard deviation S is the slope of the calibration graph, n = 3). The deviation of the intercept andof S isthe theintercept slope of and the calibration graph, n = 3). The result was close to that result was close toreported that of the previouslylevel reported methylation level analysis [5,14]. of the previously methylation analysis [5,14].
Figure 5. The linear linear relationship relationship between between ∆F ∆F (Y) (Y) and and methylation methylation level level (X) (X) ranging ranging from from 0% 0% to to 100% 100% Figure 5. The (5%, 10%, 15%, 20%, 30%, 40%, 60%, 80%, and 100%). Error bars represent the standard deviation (5%, 10%, 15%, 20%, 30%, 40%, 60%, 80%, and 100%). Error bars represent the standard deviation of of three experiments. three experiments.
The reproducibility of the proposed biosensing system is essential for an assay’s protential in practical application. The reproducibility was assessed by calculating the relative standard deviations (RSD) and measured in different days at the identical experimental conditions. The RSD (n = 3) with the DNA detection were 4.4%, 2.0%, 1.3% at 0.5 nM, 5 nM and 20 nM Unmethylated
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The reproducibility of the proposed biosensing system is essential for an assay’s protential in practical application. The reproducibility was assessed by calculating the relative standard deviations (RSD) and measured in different days at the identical experimental conditions. The RSD (n = 3) with the DNA detection were 4.4%, 2.0%, 1.3% at 0.5 nM, 5 nM and 20 nM Unmethylated DNA (T1 ), respectively. And the RSD (n = 3) with the methylation analysis were 7.2%, 5.1%, 2.7% at the DNA methylation level of 20%, 60% and 100%, respectively. These results demonstrate that the proposed biosensing system possesses optimistic reproducibility. 4. Conclusions In summary, we developed a simple nanobiosensing platform for DNA-methylation analysis with MoS2 nanosheets and demonstrated its efficacy on p16 promoter in homogenous solution. Unlike conventional DNA-methylation detection technologies, our method does not rely on bisulfite and PCR, providing a straightforward fluorescence readout of methylation level within 3 h. Without any amplification strategy, the assay could distinguish as low as 1% methylation level in the mixtures with excellent reproducibility. The biosensing platform could also be used to detect DNA with a detection limit of 140 pM with high sensitivity. Thus, a homogeneous quantitative analysis of DNA methylation was provided with short-time, easy operation, as well as relative good sensitivity. The results indicate that such nanobiosensor is promising in nucleic acid detection, particularly quantitative analysis of DNA methylation at the point of care. Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/16/10/1561/s1, Figure S1: Characterization of the MoS2 nanosheets. (A) TEM image of MoS2 ; (B) UV-visible absorption spectrum of MoS2 ; (C) AFM image and height profile (inset) of MoS2 ; (D) The lateral dimension distribution of MoS2 . Figure S2: Effect of different BstUI endonuclease concentrations (A) and cleavage reaction time (B) on ∆F. Error bars show the standard deviation of three experiments. Figure S3: The specificity of detection of target DNA. Fluorescence emission spectra in the presence of (a) complementary unmethylated target DNA (T1 ), (b) one-base mismatched DNA (M), (c) noncomplementary DNA (N) and (d) blank. The inset shows the histogram corresponds to the fluorescence spectra in fluorescence emission spectra. Error bars represent the standard deviation of three experiments. Acknowledgments: This work was supported by the National Natural Science Foundation of China (Nos. 21275040 and 21475034). Author Contributions: Guo-Jun Zhang and Qunfeng Yao designed the study. Le Xiao conducted the whole experiments. Le Xiao, Li Xu and Guo-Jun Zhang wrote the manuscript. Chuan Gao conducted the experiments related to electrophoresis. Yulin Zhang performed the fluorescent experiments. All the authors reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations MoS2 pds-DNA ss-DNA ds-DNA Tris NaCl MgCl2 TEM AFM DLS UV PAGE
molybdenum disulfide partial duplex-deoxyribonucleic acid single stranded-deoxyribonucleic acid double stranded-deoxyribonucleic acid Tris(hydroxylmethyl)aminom-ethan sodium chloride magnesium chloride transmission electron microscopy atomic force microscopy dynamic light scattering ultraviolet polyacrylamide gel electrophoresis
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