Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic

Apr 17, 2017 - Isothermal amplification is an efficient way to amplify DNA with high accuracy; however, the real-time monitoring for quantification an...
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Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic Acid Amplification Fan Li,† Xiaoguo Liu,† Bin Zhao,† Juan Yan,‡ Qian Li,† Ali Aldalbahi,§ Jiye Shi,∥ Shiping Song,† Chunhai Fan,† and Lihua Wang*,† †

Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ‡ College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, China § Chemistry Department, King Saud University, Riyadh 11451, Saudi Arabia ∥ Kellogg College, University of Oxford, Banbury Road, Oxford OX2 6PN, U.K. S Supporting Information *

ABSTRACT: Isothermal amplification is an efficient way to amplify DNA with high accuracy; however, the real-time monitoring for quantification analysis mostly relied on expensive and precisely designed probes. In the present study, a graphene oxide (GO)-based nanoprobe was used to real-time monitor the isothermal amplification process. The interaction between GO and different DNA structures was systematically investigated, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), DNA 3-helix, and long rolling circle amplification (RCA) and hybridization chain reaction (HCR) products, which existed in one-, two-, and three-dimensional structures. It was found that the high rigid structures exhibited much lower affinity with GO than soft ssDNA, and generally the rigidity was dependent on the length of targets and the hybridization position with probe DNA. On the basis of these results, we successfully monitored HCR amplification process, RCA process, and the enzyme restriction of RCA products with GO nanoprobe; other applications including the detection of the assembly/disassembly of DNA 3-helix structures were also performed. Compared to the widely used end-point detection methods, the GO-based sensing platform is simple, sensitive, cost-effective, and especially in a real-time monitoring mode. We believe such studies can provide comprehensive understandings and evocation on design of GO-based biosensors for broad application in various fields. KEYWORDS: interaction of graphene oxide and DNA, length and position dependent, various DNA structures, real-time detection, isothermal amplification

1. INTRODUCTION DNA amplification is highly important for a wide range of biological applications and ultrasensitive detections in clinical diagnosis.1−3 However, the thermocycling processes in polymerase chain reactions (PCR) indispensably require expensive thermal cyclers and make it inconvenient for pointof-care (POC) analysis, which largely limits its application.1 In contrast to PCR that requires complex thermocycling to mediate template melting, primer annealing, and subsequent extension, isothermal amplification can rapidly and efficiently amplify nucleic acid sequences under simple conditions, especially at a constant temperature.4,5 These methods have recently been utilized to amplify signal and generate diverse output signals for biosensing, including fluorescence, luminescence, colorimetric, and electrochemical signals.6−11 Amplified products also have been extensively exploited to construct versatile nucleic acid nanomaterials, such as higher-ordered nucleic acid nanostructures, nucleic acid templated metal © XXXX American Chemical Society

nanostructures, and nucleic acid hydrogels, demonstrating their promising applications in biomedicine, bioimaging, and biosensing.12−16 Many isothermal amplification techniques are based on DNA replication (e.g., rolling circle amplification, RCA), enzyme-based digestion, or enzyme-free assembly of nucleic acid (e.g., hybridization chain reaction, HCR). However, the detection of isothermal amplification mostly relied on expensive and precisely designed probes, or gel electrophoresis, which generally provides qualitative or semiquantitative data to determine the end point of amplification process.2,3,6,17,18 Some fluorescence detection analyzed the amplification quantitatively with real time, however, which generally uses SYBR Green I or other dyes as DNA intercalators, and the nonspecific binding with all doubleReceived: January 23, 2017 Accepted: April 17, 2017 Published: April 17, 2017 A

DOI: 10.1021/acsami.7b01134 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of Interaction between GO and Different DNA Structures

In this work, the interactions between GO and different DNA structures were systemically investigated, including ssDNA, dsDNA, pdsDNA (partially double-stranded DNA), DNA 3-helix, and long ssDNA and dsDNA structures (Scheme 1). The influence of hybridization position and length of probes on their interaction with GO was also investigated. We then monitored HCR amplification processes, the enzyme restriction process of RCA products, and the assembly and disassembly of DNA 3-helix. We believe such studies can provide comprehensive understandings and evocation on design and optimization of GO-based biosensors.

stranded DNA (dsDNA) makes the false positive results possible.19,20 Therefore, a simple, sensitive, and specific realtime monitoring method was required for precise detection of isothermal amplified products. Graphene oxide (GO), a 2D carbon nanocrystal with only one atom thickness, has excellent solubility with carboxylic acid and hydroxyl groups on graphene surface and has been served as a fluorescence superquencher based on its long-range nanoscale energy transfer property.21 It has been demonstrated that ssDNA sequences can be absorbed on the GO surface, and the fluorescence in fluorophore-labeled ssDNA (F-ssDNA) is efficiently quenched because of strong interaction between ssDNA and GO surface, while for dsDNA the interaction with GO is much weaker.22,23 In addition to the π−π stacking interactions of the aromatic nucleobases with the planar GO surface, several other mechanisms have also been suggested to contribute to DNA−GO interaction, e.g., hydrogen bonding, electrostatic, van der Waals, and hydrophobic interactions.24−26 Based on the different GO/ssDNA and GO/dsDNA interaction, a series of biosensors have been developed for detecting DNA, proteins, and other small molecules.22−24,27−30 In addition, introduction of aptamer in biosensor made the detection of various kinds of targets possible due to the high affinity of aptamer with target.30 By employing these GO-based biosensors, background noise could be efficiently decreased, which resulted in excellent sensitivity and selectivity in sample detection and analysis. Most of DNA sensing strategies had been developed with the perfectly complementary DNA sequences (pcDNA) as an ideal model.23,27 In the presence of pcDNA, the Watson−Crick specific hydrogen bonding between pcDNA and F-ssDNA absorbed on GO released FssDNA from GO and resulted in the fluorescence recovery. While for detection of real samples, various molecules including DNA, proteins, and other small molecules existed, which led to much complicated interaction with GO. DNA targets in real samples were also much complicated, and they might be in different lengths, in different forms, and hybridize with fluorescent probes at different positions. The detailed information on the interaction of GO with various kinds of DNA structures was still ambiguous.

2. RESULTS 2.1. Interaction of GO with Various DNA Structures. 2.1.1. Influence of the Length and Position of Hybridization. We first investigated the interaction of GO with different ssDNA and dsDNA structures systematically. The linear dsDNA structure was the mostly used system for investigation on the GO−DNA interaction, which also formed the basis of the DNA biosensors. However, in real sample detection, the dye-tagged probe will meet various DNA targets in different lengths at random positions, which was believed to influence the fluorescence quenching efficiency and the following performance of biosensor. It is still ambiguous whether the position of fluorophore on DNA probe influence the fluorescence quenching effect. Here three F-ssDNA probes of 80 bases were employed, with fluorophore labeled at 5′ end, 3′ end, and middle position of ssDNA, which were termed as 5′probe, 3′-probe, and M-probe, respectively. As shown in Figure S2, the fluorescence of all three probes was quenched by GO efficiently, with only less than 5% fluorescence remained, which implied that these three 80-base ssDNA tightly bond with GO in nanometer level22 and the labeling position of fluorophore did not influence the interaction of probes with GO. In the presence of 30-base targets (T30), comparable fluorescence recovery was obtained for 5′-probe and 3′-probe (Figures S3a and S3b), which further reflected the dye labeling position was not a dominant factor of DNA−GO interaction. While in the case of M-probe with fluorophore labeled at 30th base (counted from 5′ end), T-30 hybridized to different parts resulted in B

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Figure 1. Fluorescence response of (a) 5′-probe, (b) M-probe, and (c) 3′-probe in the presence of different targets on surface of GO nanoprobe. Each sample was incubated for 30 min in 1 mL binding buffer containing 20 nM probe, 20 nM target, and 10 μg mL−1 GO.

Figure 2. Fluorescence response of ssDNA, HCR products, RCA products, and 3-helix in the absence and presence of GO nanoprobe. (a) Schematic presentation of interaction between GO surface and ssDNA and three DNA structures. Comparison of fluorescence quenching by GO of ssDNA with (b) HCR products, (c) RCA products, and (d) 3-helix.

C

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Figure 3. Analysis of HCR products. (a) Schematic illustration of HCR reaction on GO surface. (b) Fluorescence response of HCR reaction solution in the absence and presence of 100 nM initiator. (c) Time-dependent response of HCR reaction solution. (d) Fluorescence analysis and (e) 20% PAGE gel analysis of HCR reaction solutions in the presence of different amounts of initiators. Lane 1: 50 bp marker. Lanes 2−9: HCR reaction solutions with the initiator/(H1.H2) ratio of 0, 10, 3, 1, 0.3, 0.1, 0.03, and 0.01, respectively. Lane 10: initiator. HCR reactions were carried out in reaction buffer containing 10 nM H1 and H2 with 10 μg mL−1 GO and in reaction buffer containing 1 μM H1 and H2 for fluorescence detection and gel analysis, respectively, at 37 °C. λex = 494 nm.

comparable fluorescence recovery (Figure S3c), indicating the distance between fluorophore and GO rather than ssDNA tail absorbed on GO played dominant role in fluorescence recovery.31 We then challenged these three probes to detect DNA targets of different lengths. As shown in Figure 1a, in the presence of DNA targets of 30, 60, and 80 bases (named as T30, T60, and T80, respectively), the fluorescence of 5′-probe (80 bases) was recovered significantly, which suggested that the fluorophore was liberated from the GO surface after hybridization of DNA targets with the probe. The longest pcDNA T80 led to the highest fluorescence recovery. For the 5′-probe, the fluorescence recovery after adding T30 reached 82% of T80, indicating that 10 nm (approximate length of 30 nucleotides) of dsDNA was long enough to pull fluorophore out of GO surface. Similarly, the fluorescence recovered by T60 target (about 20 nm) was 98%, slightly lower than T-80 and much higher than T-30. These results suggested the fluorescence recovery increased with complementary lengths of DNA targets,31 which was attributed to the great repelling force between dsDNA and GO. Meanwhile, the ssDNA parts were generally considered to be adsorbed on GO surface due to their strong binding affinity to GO, but the long distance (more than 10 nm) between the fluorophore and GO resulted in their separation. Therefore, the presence of ssDNA tail only exhibited slight influence on the fluorescence recovery after target addition. As expected, the fluorescence recovery of 3′probe labeled at 3′-end after hybridization with T30 reached

94% of T80 (Figure 1c), which was comparable with that of 5′probe (Figure 1a). For the M-probe with fluorophore labeled at 30th base (counted from 5′ end), a similar trend of fluorescence recovery was observed and the longer target usually led to the higher fluorescence recovery. Difference of the fluorescence recovery was observed; for example, the fluorescence recovery of Mprobe induced by T30 reached 41% of T80, which was much lower than the case of 5′-probe (Figure 1b). The relative low fluorescence recovery was associated with the short distance between fluorophore and GO, which was induced by high affinity of its neighboring ssDNA with GO. The fluorescence recovery induced by T60 reached 87% of T80, which was higher than the case of T30 and comparable with the case of 5′probe/T30 (82%), implying the distance between fluorophore and GO plays a dominant role in the fluorescence recovery while the labeling position of fluorophore showed a minor effect. 2.1.2. Interaction of GO with Complex DNA Nanostructures. On the basis of the well understanding on the interaction of ssDNA/dsDNA with GO, we challenged the interaction of GO with much complicated DNA nanostructures which generally existed in real systems. Long HCR products, random RCA products, and a 3-helix DNA nanostructure were employed as mimics of different dimensions of nanostructures (Figure 2). The long HCR products were considered as linear 1D dsDNA structures containing repeated units, which were about 480 bp and about 160 nm in length. In the presence of GO, D

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Figure 4. Analysis of enzyme digested RCA products. (a) Schematic illustration of the “turn-off” and “turn-on” fluorescence detection of digested RCA products using GO nanoprobe. (b) 1% agarose gel analysis of RCA products. (c) Fluorescence emission spectra of RCA products and digested RCA products in the presence of GO nanoprobe. Enzyme restriction time: 2 h. (d) Time-dependent fluorescence response of enzyme restriction solution of RCA products. Enzyme restriction reactions were carried out in 40 μL reaction buffer containing 10 U HhaI at 37 °C. λex = 494 nm.

31% of the fluorescence of HCR products remained, which was much higher than the case of 3-′probe that only 1% of fluorescence remained, implying the HCR products had a similar interaction behavior with GO as dsDNA (Figure 2a). The long ssDNA products of RCA can cover the length range from several hundred nanometers up to a few micrometers and form complex structures like a loose ball like of string.32−34 Many bases located inside of the structure and thus could not interact with GO surface. As a consequence, 22% of the fluorescence intensity of RCA retained (Figure 2b), exhibiting a lower quenching efficiency of GO compared to ssDNA, demonstrating RCA products are complex structures of coiling type ssDNA rather than long linear strands. An 80 bp 3-helix DNA nanostructure with a length, width, and thickness of 27, 7.1, and 2.4 nm, respectively, was considered as a mimic of 2D structure. As shown in Figure 2c, 38% of the fluorescence intensity of 3-helix remained while only 5% remained for the 5′-probe, indicating the interaction of 3-helix with GO was dramatically weaker. This is in agreement with the prediction that 3-helix would not be strongly adsorbed on GO due to negative charges of dsDNA. Therefore, all of these three DNA structures had strong rigid characteristics and exhibited weak interaction with GO. The characteristic interaction of GO with highly ordered DNA structures implied that we could establish similar sensing strategies for monitor the formation and the degradation of complicated DNA structures. 2.2. Real-Time Monitoring of Isothermal Amplification Process. 2.2.1. Detection of HCR Process. The HCR process is a simple and efficient process to generate a large quantity of long dsDNA structures, which generally performed at a constant temperature and no enzyme was needed. A typical HCR usually employs two hairpin species (H1 and H2) and one initiator strand (Scheme S1) to produce long DNA

structure with controllable length. In our design, each hairpin contained a stem of 18 base pairs enclosing a 6-mer loop, together with an additional 6-mer sticky end labeled with fluorophore (Scheme S1). In the presence of GO, the fluorescent ssDNA sticky terminal strands adsorbed on GO surface (Figure 3a), and their fluorescence was efficiently quenched, therefore a low fluorescence intensity marked at the initial status of HCR process (Figure 3b). The introduction of the initiator strand can trigger a chain reaction of hybridization events, and the long hybridization DNA strand products with periodical fluorophore units led to a much lower rate of migration (Figure 3e). As shown in Figure 3c, the fluorescence intensity of HCR solution increased along with the time prolonged and reached a plateau after being incubated 4.5 h (Figure S4), implying the HCR products were long enough for separation from GO surface. Taking all of these results together, we concluded that real time HCR process could be monitored by using GO as probes, which is less timeconsuming and more convenient than DNA gel electrophoresis. Notably, the concentration ratio of initiator strand to H1 and H2 plays a key role to control the length of HCR products, and the excess of initiator strands usually led to formation of shorter HCR products. Therefore, a feasible concentration ratio of initiator to H1 and H2 was important for HCR process. Here the GO-based sensor was used to conveniently optimize initiator concentration with a real-time course. Since all the HCR products contain the sticky end (ssDNA tail), the shorter HCR products had stronger affinity with GO and was supposed to result in lower fluorescence recovery. As expected, with the increased initiator concentration, the fluorescence intensity of HCR solution in the presence of GO increased and reached the highest level at a concentration ratio of initiator to H1 and H2 at 0.1 and then decreased (Figure 3d and Figure S5). Native polyacrylamide gel electrophoresis (PAGE) was used to verify E

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Figure 5. Analysis of 3-helix. (a) Schematic illustration of the formation and decomposition of DNA 3-helix in the presence of GO nanoprobe. (b) 6% PAGE gel analysis of 3-helix. (c) Fluorescence detection of 3-helix and decomposed 3-helix in the presence of GO nanoprobe. Fluorescence response of 3-helix during the (d) decomposition and (e) formation process. Reactions were carried out in 1 × PBS buffer containing 20 μg mL−1 GO and 20 nM 3-helix DNA structure. λex = 545 nm.

the performance of the HCR process (Figure 3e), and the results were consistent with the fluorescence results obtained using GO-based sensor. When the concentration ratio of initiator to H1 and H2 was 0.1, significant amounts of the corresponding products could not migrate out of the sample loading hole (Figure 3e, lane 7), suggesting the remarkable length of HCR products. With higher ratios, HCR products of different lengths were formed (lanes 3, 4, 5, and 6), which were shorter than in the case of ratio 0.1, due to the limited numbers of H1/H2 assigned to each initiator. Therefore, the results obtained by using GO sensor indicated that 0.1 was the best the concentration ratio of initiator to H1 and H2, which resulted in the release of more fluorescent ssDNA from GO surface and the formation of longer dsDNA products, compared to other concentration ratios. 2.2.2. Detection of RCA Process and Enzyme Restriction of RCA Products. In the RCA process, the primer prolonged along a circular oligonucleotide sequence in the presence of DNA polymerase, generating a long single-stranded DNA chain containing periodic repeats of the sequence coded by the circular oligonucleotide. The addition of fluorophore-labeled dUTP resulting in amplification products tagged with fluorophores in every periodic repeat fragment, which can be visualized by the naked eye under UV light without staining (Figure 4b). AFM imaging results further confirmed the formation of long ssDNA with length to micrometer scale (Figure S6). The increase of fluorescence intensity of reaction solution compared to ssDNA also implied the formation of RCA products and the weaker absorption of fluorescent RCA products on GO. By adding short ssDNA (60 bases) which can hybridize with the periodic repeat fragments of fluorescent RCA products, the observed fluorescence intensity increased 0.3 times (Figure S7), indicating the RCA products were not released but still adsorbed on GO, which also suggested the rigidity of RCA products was improved through hybridization of ssDNA with the periodic repeat fragments.

We further studied the degradation process of large RCA products into short pieces with the GO nanoprobe. The enzyme restriction sites (5′-GCGC-3′) of RCA products can be recognized and digested by HhaI, resulting in the formation of repeated fragments (Figure 4a). After treatment, the fluorescence of RCA products was greatly declined, suggesting the original complex structure was digested into shorter sequences of ssDNA which had much stronger interaction with GO surface (Figure 4c). Worth noting, the above sensing strategy was performed in a turn-off mode; besides that, another turn-on sensor was also developed to monitor the digestion process (Figure 4a). This turn-on type was developed through introduction of a 60-mer ssDNA probe to hybridize ssDNA pieces and form perfectly complementary dsDNA, thus leading to a significant fluorescence increase. For the real-time monitoring, the RCA products was split into small pieces after the addition of enzyme and dispersed in aqueous solution, which hybridized with probe with high efficiency and released from GO surface. Figure 4d demonstrated that the observed fluorescence increased with time prolonging (Figure S8), indicating more and more short ssDNA were produced by enzyme restriction and released from GO surface by hybridization with ssDNA probe. The higher contrast made the performance of the turn-on sensor (Figure 4d) better than the turn-off one (Figure 4c) due to the higher signal-to-noise ratio (2.6 at 2 h). Therefore, the enzyme restriction process of RCA products can be monitored using both two types of GO-based probes. 2.2.3. Detection of Assembly and Disassembly of DNA 3Helix Structure. This GO-based nanoprobe also can be used to investigate the assembly and disassembly process of various kinds of high ordered DNA nanostructure. Here we monitored the assembly of a 3-helix DNA structure, which was composed from three dsDNA through cross-linking mode (Figure 5a). A typical assembly procedure was performed through a denaturing and annealing process by heat treatment, and all DNA denatured at high temperature and then slowly annealed F

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accumulation or quenching, which generally requires dual labeling of fluorophore and quencher on one hairpin or labeling of fluorophore and quencher on two hairpins separately. In a multiplexing biological sample, the nonspecific interaction of hairpin with nontarget molecules to form by products will increase the background noise. Meanwhile, since the initiators are tethered in close proximity to the newly formed dsDNA, the opening of the hairpin is reversible to some extent, which will also influence the fluorescence signal. The interaction of HCR products with GO, which has ultrahigh fluorescence quenching ability, is dramatically different from the one of byproducts with GO. Therefore, using GO-based sensors will not only allow very flexible sequence design but also decrease the signal of nonspecific hairpin binding, allowing a simple, straightforward, and high signal-to-background fluorescence detection. For another isothermal amplification process, RCA generates large and mega base fragments of repeated sequences which fold into micrometer-sized random coils in solution in the presence of a circular DNA template.32,34,40 The detection of enzyme restriction process of RCA products was mainly focused on end point detection, rather than real-time monitoring, including gel electrophoresis and fluorescence or color metric detection assisted by nanoparticles.12,13,41−43 The detection of RCA products was difficult because they were too large to migrate from the sample hole in gel electrophoresis. AFM imaging can provide information on lengths of RCA products;16,32 however, the equipment is expensive, and this technique requires expertise. The GO-based sensor provided a convenient way to detect the products formation, and the results well coincided with that of gel electrophoresis and AFM. Using GO-based sensors also allows the cost-effective real-time monitoring of enzyme restriction process of RCA products with high sensitive and selectivity, which performed well in both turn-off and turn-on modes.

to form well-defined structure (Figure 5b). The GO-based sensing strategy could monitor the whole procedure with a significant fluorescence change. Through heating the 3-helix/ GO solution from 25 to 90 °C, a gradual fluorescence decrease was observed, indicating the fluorophore-labeled 3-helix structure denatured under heating and consequently the loosened structure was absorbed on GO (Figure 5d). A reverse process was performed by cooling this DNA/GO solution from 90 °C back to 25 °C; as shown in Figure 5e, a fluorescence recovery was observed, and the great fluorescence increase occurred at 54 °C, indicating the departure of F-ssDNA from GO and the following assembly of 3-helix. This observation demonstrated that the DNA base pairing effect overcame the interaction of ssDNA with GO surface to form 3-helix structure. Therefore, the GO-based sensor provided a real-time monitoring of assembly and disassembly of DNA 3-helix structure, which might be extended to other complex DNA structures in various fields.

3. DISCUSSION GO-based sensor has been widely used in various fields, especially in environmental protection, biology, and clinic diagnosis, etc. Several groups have investigated the interaction of DNA and GO, however, which generally focused on short ssDNA probes ranging from 15 to 30 bases.22,27,35,36 How the long DNA interacted with GO was still ambiguous,25,37 and in fact it was very important for real sample detection because of their complexity. In general, the complicated biological samples generally contain different DNA structures in various lengths, and the longer DNA sequence in hundreds to thousands nanometers required long ssDNA probe to ensure the specificity and accuracy. In our work, we used a long ssDNA sequence (80 bases) as a probe and investigated its hybridizing behavior with different targets of various lengths and labeling positions. We found that high fluorescence intensity was usually remained when the distance between dye and GO was long enough, and the longer targets also led to a higher fluorescence recovery. This observation is consistent with the previous work of Liu’s group, that the fluorescence quenching by GO was progressively decreased for the longer target.31 We further investigated the interaction of GO with DNA structures in 1D, 2D, and 3D forms and found all of them had similar interaction of dsDNA with GO. HCR products and 3-helix were mainly composed of dsDNA and exhibit GO−dsDNA interaction; therefore, they were employed as 1D and 2D structures, respectively. The RCA products were considered as a 3D structure since they existed in a loose ball-like form in hundreds nanometer to micrometer scale and had a weak interaction with GO since most of nucleotides were packaged inside of the structure and hardly approached GO surface. It was found out generally the higher dimension of structure leads to the lower interaction with GO. Based on good understanding of the interaction of DNA structures with GO, GO-based probes were designed to achieve the real-time monitoring of several important isothermal amplification process, including HCR and RCA process. HCR introduced by Dirks and Pierce is a well-known strategy of enzyme-free isothermal amplification method.3 By integrating initiators into a variety of molecular probes, HCR amplification has been applied to the detection of diverse classes of targets, including nucleic acids, proteins, small molecules, and cells.3,10,38,39 To monitor a typical hairpin-based HCR process, the most well-known strategy is monitoring the fluorescence

4. CONCLUSION The interaction of different DNA structures with GO surface was investigated systematically, including ssDNA, dsDNA, 3helix, HCR, and RCA products. It was found out the interaction of ssDNA with GO is significantly stronger than dsDNA, and the base paring of two complementary ssDNA strands can overcome the strong interaction of ssDNA with GO. The fluorophore-labeling position has a minor effect on DNA/GO interaction; however, the dye located near the dsDNA part generally leads to a high fluorescence. It was also found that the longer target led to higher fluorescence recovery because of the longer dsDNA part in hybridized products. We further investigated the interaction of GO with other DNA structures in 1D, 2D, and 3D forms and found all of them had similar interaction of dsDNA with GO based on ratios of duplex in DNA structures. On the basis of this, we successfully monitored real-time assembly and disassembly of 3-helix structures, isothermal amplification process of HCR and RCA and the enzyme restriction process of RCA products by detecting fluorescence intensity of reaction solution in the presence of GO. Compared to the widely used traditional methods for detecting isothermal amplification process, including gel electrophoresis, atomic force microscopy, and fluorescence change that require dual labeling of probes, the GO-based sensing platform is simple, sensitive, and cost-effective. In addition, we can monitor the enzyme degradation of RCA products with GO-based sensor in both fluorescence turn-off G

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and turn-on modes. The GO-based sensors provide a simple, cost-effective, and highly specific method to monitor various kinds of biological events, and our conclusion and application attempt may be valuable for design and optimization of sensors and devices based on DNA and GO.

AUTHOR INFORMATION

Corresponding Author

*(L.W.) E-mail: [email protected]. ORCID

Bin Zhao: 0000-0002-0987-4586 Shiping Song: 0000-0002-0791-8012 Chunhai Fan: 0000-0002-7171-7338 Lihua Wang: 0000-0002-6198-7561

5. EXPERIMENTAL SECTION 5.1. Materials. Graphite powder and all other chemical reagents (analytical grade) were purchased from China National Pharmaceutical Group Corporation (Shanghai, China). All chemicals were used without further purification. Milli-Q water was used for all the experiments. DNA oligonucleotides (Table S1) were purchased from TAKARA (Dalian, China). Restriction enzyme Hhal (HhaI), fluorescein-12-dUTP, and dNTP mixture were purchased from New England Biolabs Inc. (NEB). Phi29 DNA polymerase, T4 DNA ligase, and T4 DNA polymerase were purchased from Fermentas (Ontario, Canada). 5.2. Instruments. Fluorescence spectroscopy was performed on a Hitachi F-4500 fluorescence spectrophotometer and a Cary Eclipse fluorescence spectrophotometer. AFM measurements were carried out on a Nanoscope IIIa (Digital Instrument, USA) under tapping mode, with a Pt/Ti coated tip (force constant of 4 N/m, MicroMasch). 5.3. Hybridization Chain Reaction (HCR). Reaction solution containing H1 and H2 (0.3 M NaCl, 10 mM PB, pH 7.4) were heated for 5 min at 95 °C to anneal hairpins, and then the temperature was slowly cooled down to room temperature for 1 h. Solutions of initiator strand were added to allow the hybridization chain reaction for 6 h at 37 °C. Products were analyzed using 20% PAGE gel electrophoresis and fluorescence assay. For gel analysis, the final reaction solution contained 1 μM H1 and H2, while for fluorescence assay, the final reaction solution contained 10 nM H1 and H2 in the presence of different concentrations of initiator. Ratio of initiator/(H1.H2): 0, 0.01, 0.03, 0.1, 0.3, 1, and 3. More details are shown in the Supporting Information. 5.4. Rolling Cycle Amplification (RCA). Reaction solutions containing 300 nM circular DNA template, 300 nM RCA primer, 50 μM fluorescein-12-dUTP, 50 μM dATP, 50 μM dCTP, 50 μM dGTP, and 20 U phi29 DNA polymerase (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% Tween 20, and 1 mM dithiothreitol (DTT)) were incubated at 30 °C overnight and then inactivated at 65 °C for 10 min. The RCA products were characterized by 1% agarose gel electrophoresis and AFM imaging. The enzyme restriction was carried out by adding 10 U HhaI restriction enzyme to a 40 μL reaction solution followed by incubation at 37 °C. More details are shown in the Supporting Information. 5.5. Assembly of 3-Helix DNA Structure. Reaction solution containing 2 μM DNA strands (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, and 125 mM magnesium acetate) was slowly cooled from 90 °C to room temperature. Products were analyzed using 6% PAGE gel electrophoresis and fluorescence assay. For the fluorescence quenching assay, the fluorescence of solution was directly measured by a Hitachi F-4500 fluorescence spectrophotometer after mixing the 3-helix DNA structure with GO. For the temperature-dependent fluorescence assay, the sample solution was heated from 25 to 90 °C and then cooled down from 90 to 25 °C using temperature controlling equipment with recording the fluorescence signal. More details are shown in the Supporting Information.

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Present Address

F.L.: Department of Biochemistry and Biomedical Sciences, Health Science Centre, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1. Author Contributions

F.L. and X.L. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Grants 21675167, 21373260, 21227804, and 21329501) and the Youth Innovation Promotion Association CAS.

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01134. DNA sequences, GO preparation, analysis protocol, Figures S1−S8 (PDF) H

DOI: 10.1021/acsami.7b01134 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

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DOI: 10.1021/acsami.7b01134 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX