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Transition Metal Dichalcogenide Nanosheets for Visual Monitoring PCR Rivaling a Real-Time PCR Instrument Liu Wang, Zhicheng Huang, Rui Wang, Yibo Liu, Cheng Qian, Jian Wu, and Juewen Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15746 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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ACS Applied Materials & Interfaces

Transition Metal Dichalcogenide Nanosheets for Visual Monitoring PCR Rivaling a Real-Time PCR Instrument

Liu Wang1,2, Zhicheng Huang2, Rui Wang1, Yibo Liu2, Cheng Qian1, Jian Wu1*, Juewen Liu2*

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College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China Email: [email protected]

2

Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo N2L 3G1, Ontario, Canada Email: [email protected]

1

ACS Paragon Plus Environment

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Abstract Monitoring the progress of polymerase chain reactions (PCR) is of critical importance in bioanalytical chemistry and molecular biology. While real-time PCR thermocyclers are ideal for this purpose, its high cost has limited applications in resource-poor areas. Direct visual detection would be a more attractive alternative. To monitor PCR amplification, DNA staining dyes such as SYBR Green I (SG) are often used. Although these dyes give higher fluorescence when binding to double-stranded (ds) DNA products, they also yield strong background fluorescence in the presence of a high concentration of single-stranded (ss) DNA primers. In this work, we screened various nanomaterials and found that graphene oxide (GO), reduced GO (rGO), molybdenum disulfide (MoS2) and tungsten disulfide (WS2) can quench the fluorescence of nonamplified negative samples while still retain strong fluorescence of positive ones. The signal ratio of positive-over-negative samples was enhanced by around 50-fold in the presence of these materials. In particular, MoS2 and WS2 nearly fully retained the fluorescence of positive samples. The mechanism for MoS2 and WS2 to enhance PCR signaling is attributed to adsorption of both the ssDNA PCR primers and SG with an appropriate strength. MoS2 can also suppress nonspecific amplification caused by excess polymerase. Finally, this method was used to detect extracted transgenic soya GTS 40-3-2 DNA after PCR amplification. Compared with samples without nanomaterials, addition of MoS2 could better distinguish the concentration difference of the template DNA, and the sensitivity of visual detection rivaled that from a real-time PCR instrument.

Keywords:

MoS2;

WS2;

polymerase

chain

reactions;

adsorption;

SYBR

Green

2


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Introduction Polymerase chain reaction (PCR) is an extremely powerful technique, allowing amplification of even a single copy of DNA to infinite. PCR has been widely applied in various analytical fields, including clinical diagnosis, food safety inspection and environmental monitoring.1-2 Because of its importance, efforts have been made to improve PCR. Since about a decade ago, various nanomaterials have been tested to help PCR in terms of sensitivity (e.g. detecting lower copies of templates),3 and specificity (e.g. amplifying only the desired DNA).4 For example, Fan and coworkers reported that gold nanoparticles (AuNPs) enhanced PCR specificity and they proposed a hot-start PCR mechanism.5 Li et al. reported that AuNPs promoted the amplification of GC-rich DNA.6 Zhao et al. proposed that AuNPs could inhibit mismatch extension.7 Later it was proposed the role of AuNPs to be adsorption of excess polymerase, accelerating the dissociation of PCR products, and thermal conduction.8-10 Aside from typical PCR, nanomaterials have also been applied to improve isothermal amplifications (e.g. no temperature cycling).11-12 In addition to AuNPs, many other nanomaterials, such as graphene oxide (GO), reduced GO (rGO),13-14 carbon nanotubes (CNTs),15 magnetic iron oxide nanoparticles,16-17 and CdTe quantum dots18 have also been added in PCR reactions. For PCR reactions, neither under- nor over-amplification is desirable, and many methods have been developed to monitor the progress of PCR. Currently, this can be achieved by using real-time (RT) PCR. However, this instrument and related reagents are both quite expensive. DNA binding dyes such as SYBR Green I (SG) are used for monitoring DNA amplification since they become strongly fluorescent upon binding to double-stranded (ds) DNA products.19-20 However, many such dyes are still quite fluorescent even with ssDNA, especially with a high DNA concentration.21 In the context of PCR, primers are initially at a relatively high 3



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concentration (e.g. ~ 0.4-1 µM), and the high background fluorescence strongly limits the room for signal increase. In addition, current RT-PCR-based analysis are mainly restricted to centralized laboratories, while on-site and instant analysis are in a growing demand (e.g. pointof-care testing, POCT). To meet this need, new signaling strategies have been made, such as visual detection based on the aggregation and flocculation of nanoparticles, lateral flow detection, and DNA microarrays.22-24 We reason that nanomaterials might also be useful for monitoring PCR progress. Many nanomaterials preferentially adsorb ssDNA over dsDNA, and different materials adsorb DNA via different mechanisms. For example, GO,25-26 and CNTs,27-28 use π-π stacking and hydrogen bonding. Metallic gold and silver nanoparticles bind DNA bases via strong coordination interactions.29 Metal oxides bind DNA phosphate backbone,30-31 while transition metal dichalcogenides rely on van der Waals force for adsorbing DNA.32-33 In addition, some nanomaterials can also adsorb DNA staining dyes.34-37 Such adsorption was shown to be useful for improving detection, and most previous researchers used carbon-based materials, such as GO,37 and carbon nanoparticles.34 Molybdenum disulfide (MoS2) and tungsten disulfide (WS2) are two important 2D nanomaterials, known as transition metal dichalcogenides. Different from other materials, they adsorb DNA mainly rely on van der Waals force.33 MoS2 and WS2 have already been used to design DNA-based biosensors for detecting adenosine,32 thrombin,38 uranyl ions,39 and miRNA.40 MoS2 was also used to monitor hybridization chain reactions.41 With these progress, MoS2 and WS2 have not yet been used for PCR-related applications. We reason that their unique DNA adsorption properties might make them useful for this purpose. In this work, we screened a few common nanomaterials, and found that MoS2 and WS2 are the most optimal for signaling 4


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PCR progress, allowing visual detection to rival RT-PCR sensitivity. Under certain conditions, they can also improve the specificity of PCR.

Materials and Methods Chemicals. PBR 322 plasmid DNA, low molecular weight DNA ladders and Taq polymerase with Thermopol® Reaction Buffer were purchased from NEW England Biolabs. Primers were from Eurofins Genomics and the sequences are presented in Table S1. Carboxyl GO, MoS2 and WS2 were purchased from ACS Material (Medford, MA). Their TEM micrographs are shown in Figure S1. The rGO sample was prepared following our previous paper,42 and AuNPs (13 nm, ~10 nM) were also synthesized in the lab.43 NiO was from US Research Nanomaterials (Houston, TX), and MnO2 was synthesized following a previous paper.44 SYBRTM Green I (SG) was from Lonza. Ethidium bromide (EB) was obtained from Bio Basic Canada Int. Quanti- iTTM PicoGreen was from Thermo Fisher Scientific. Thiazole Orange (TO), low molecular weight salmon sperm DNA, and CeO2 (~5 nm) nanoparticles were from Sigma-Aldrich. Transgenic soya powder GTS 40-3-2 was purchased from Monsanto Co. Hot Start Taq polymerase and 50 bp DNA ladder were from Takara Bio Inc. PCR amplification. PCR was performed on a T100 Thermal Cycler (Bio-Rad) in 50 µL containing 0.025 U Taq polymerase (NEW England Biolabs), 5 µL Thermopol® reaction buffer, 200 µM each dNTP, 200 nM forward and reverse primer (pBR322, Table S1), and 2.5 µL template DNA. The thermal program was set at 94 °C for 3 min followed by 35 to 45 cycles at 94 °C for 20 s, 55 °C for 30 s and 68 °C for 1 min. For RT-PCR, 1 × SG was also added in the reaction and amplification was performed on a CFX96 Real-Time PCR instrument, with 5



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fluorescent signal collected at the end of each cycle. Primers for each experiment are indicated in the figure caption. Fluorescence detection. 50 µL PCR products containing 1 × SG was added with 1.5 µL GO (500 µM) and mixed thoroughly. For visual observation, a 470 nm LED (Safe ImagerTM 2.0 Blue Light Transilluminator, Invitrogen) was used after 5 min. A SpectraMax® M3 multi-mode microplate reader was used for quantitatively measuring fluorescence with excitation at 497 nm and emission at 520 nm. Adsorption of SG with nanomaterials. To study the adsorption of SG, a 50 µL PCR mixture was prepared containing all the reagents except for the primers. Then, 1.5 µL of nanomaterials (final concentrations in the caption of the data figure) was added. After 15 min incubation, the solution was centrifuged at 15000 rpm for 10 min to precipitate the nanomaterials, and the supernatant fluorescence was detected after adding 200 nM of each primer. This fluorescence allows us to quantify the remaining SG in the supernatant. To study the effect of dsDNA competing with nanomaterials for SG, the precipitant adsorbed with SG was dispersed in the PCR buffer containing 700 µg/mL salmon sperm DNA for fluorescence measurement. Comparison of different dyes. To compare different dyes, 1: 400 Quanti- iTTM Picogreen dsDNA reagent (Ex: 480 nm; Em: 520 nm), 1 × SG (Ex: 497 nm; Em: 520 nm), 1 µM TO (Ex: 512 nm; Em: 530 nm), and 4 µg/mL EB (Ex: 285 nm; Em: 605 nm) was respectively added into 50 µL PCR products. Visual detection of the stained DNA with PicoGreen, SG and TO was performed with the 470 nm LED imager, while EB was with a compact UV lamp (254 nm excitation).

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Agarose gel electrophoresis. 10 µL PCR products were mixed with 2 µL 6 × loading buffer and run with 2 % agarose gel for 50 min in 1 × TBE buffer. The gel was stained with EB and then photographed with a ChemiDocTM MP imaging system (Bio-Rad). Detection of real samples. Soya powder (30 mg) was used for DNA extraction using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing). The obtained DNA was dissolved in 200 µL TE buffer. The PCR amplification protocol was almost the same as descried above except the annealing temperature was set at 65.8 °C and the CaMV 35S primer set was used (Table S1).

Results and Discussion Screen of nanomaterials. Given that many nanomaterials can adsorb DNA and DNA staining dyes, we started with an initial screen to identify optimal materials that can help monitor PCR progress. We reason that the material should have a certain affinity for DNA primers to reduce background fluorescence due to free primers. DNA adsorption by nanomaterials has been extensively studied, but most of those experiments were performed in simple buffers with a covalent fluorophore label on DNA. Here, our adsorption reaction was performed in PCR buffers, where a high concentration of dNTP, Mg2+ and other additives were present. In addition, a common DNA staining dye for PCR, SYBR Green I (SG), was employed. Various nanomaterials were respectively mixed with the ssDNA primer, and the fluorescence of the samples was monitored (Figure 1A). The control tube was the free DNA without any nanomaterial, and a strong green fluorescence was observed (e.g. the background fluorescence). GO, rGO, MoS2 and WS2 fully quenched the fluorescence, suggesting efficient DNA and/or dye adsorption, while the other nanomaterials failed to quench. It is interesting to 7



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note that CeO2 did not quench the fluorescence here, since it could strongly adsorb DNA and quench fluorescence.45-46 Metal oxides such as CeO2, MnO2 and NiO adsorb DNA mainly through its phosphate backbone.30 We suspected that the high concentration of dNTP in the PCR buffer (e.g. 200 µM each dNTP) inhibited DNA adsorption. Indeed, fluorescence was immediately quenched by these materials once dNTP was removed from the solution (Figure 1B). Previous studies showed that GO and rGO adsorb DNA through base stacking and hydrogen bonding,25-26, 47 while DNA is adsorbed on MoS2 and WS2 via van der Waals force.33 DNA as a polymer can compete favorably with other small molecules in this system, including dNTP. AuNPs can also adsorb DNA, and here we attributed its failure in quenching fluorescence to 1) low concentration of AuNPs; and 2) competition from the dNTP and other reagents in the system. We only used 2 nM AuNPs, and it can only adsorb around 40 nM ssDNA even in pure buffer. With a strong color, we did not try to increase the concentration of AuNPs in this study. Overall, in the PCR buffer, GO, rGO, MoS2 and WS2 are potentially useful to decrease background fluorescence from SG binding to PCR primers. Interestingly, all these are 2D nanomaterials. As a critical reagent in molecular biology, SG has been extensively studied for its interaction with DNA.48 SG is a cationic dye and it can bind to both ss and dsDNA, and the fluorescence of the ssDNA complex is ~ 11-fold lower. With increasing of DNA concentration, this difference in fluorescence is decreased.49 When the ratio between SG and DNA base pair is below 0.15, the fluorescence barely increased, and SG intercalation is the main interaction mechanism. Further addition of SG results in its binding to DNA surface leading to strong fluorescence enhancement. Therefore, nanomaterials do not need to fully adsorb SG or DNA. As long as the dye is adsorbed to make the ratio low, fluorescence can be sufficiently suppressed. 8


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Figure 1. Fluorescence photographs of 200 nM pBR322-FP2 and 200 nM pBR322-RP2 primers mixed with 1 × SG dye (~ 2 µM) in the presence of various nanomaterials in 1 × Thermopol® reaction buffer (A) with 200 µM of each dNTP; (B) without dNTP. The concentrations of the nanomaterials were: 15 µg/mL GO; 15 µg/mL rGO; 60 µg/mL CeO2; 2 nM AuNPs; 40 µg/mL MoS2; 40 µg/mL WS2; 140 µg/mL MnO2; 200 µg/mL NiO. Samples without nanomaterials were performed as a control, and the excitation wavelength was 470 nm. (C) A scheme of enhancing PCR signaling using nanomaterials. Without adding MoS2 or WS2, a high fluorescence is observed even before PCR due to ssDNA primer binding to SG. This background is fully eliminated by these materials while the fluorescence of the dsDNA products is less affected.

Effect of MoS2 on PCR products. The above experiments suggested the feasibility of using nanomaterials to suppress background fluorescence. To be practically useful, the nanomaterial should retain the fluorescence of dsDNA products after PCR. Among the four 2D nanomaterials, 9



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we used MoS2 as an example to test it. We performed two PCR reactions in parallel, one without adding any template DNA as a negative control (NTC), while the other contained 0.88 pM template as a positive control (PTC). After every 5 PCR cycles, an aliquot of the sample was stained by SG without or with MoS2. Without MoS2 (Figure 2A), the NTC samples all showed a quite strong and stable background fluorescence, and the difference between NTC and PTC was observed only after 35 and more cycles.

Figure 2. Visual detection of the progress of PCR amplification at every 5 cycles with 1 × SG (~ 2 µM) as an indicator (A) in the absence of MoS2; and (B) after adding 35 µg/mL MoS2 to reduce the background fluorescence. Note MoS2 was added after the PCR reaction. (C) The fluorescent ratio of positive controls to negative controls at every five cycles. All of the reactions were performed with 200 nM pBR322-FP3 and 200 nM pBR322-RP3 primers.

In the presence of MoS2 (Figure 2B), the fluorescence of the NTC samples all disappeared, while that of the PTC samples also dropped significantly. The visual difference appeared much larger for the MoS2 containing group due to its low background. The fluorescence ratio of PTC to NTC was enhanced from 3.8 to 27.4 in the presence of MoS2 10


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(Figure 2C). Therefore, MoS2 can increase the monitoring of PCR progress by reducing the background for both visual and instrument based detection. This effect is schematically presented in Figure 1C. We also performed the same experiment by adding GO. A similar effect was observed, although GO quenched the dsDNA product more (Figure S2). Generality to different dyes. Various DNA staining dyes can be used for PCR. To test if the background suppression can be applied to other dyes, we then also tested PicoGreen, TO and EB. For all of these dyes, addition of MoS2 decreased the fluorescent signal of both NTC and PTC (Figure 3A). We then quantitatively measured the fluorescence (Figure 3B) and calculated the ratio of PTC to NTC (Figure 3C). The best performance was achieved with SG, followed by PicoGreen, while the effect on TO was not obvious. TO does not bind dsDNA very strongly but is a G-quadruplex staining dye, which may explain its low fluorescence.50 EB already had a quite large difference and further adding MoS2 did not produce an obvious enhancement either. The highest enhancement was for SG reaching ~30. Again, we repeated the experiment with GO, and a similar trend was observed (Figure S3). EB is a strong carcinogen, and thus we chose to use SG for most of the experiments in this work.

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Figure 3. (A) Photographs showing the fluorescence of PCR products without and with 35 µg/mL MoS2 in the presence of different DNA staining dyes. N: negative control; P: positive control. (B) Fluorescent values of the PTC and NTC samples in the presence of different dyes without and with 35 µg/mL MoS2. (C) The fluorescent ratio of PTC to NTC by using different dyes without and with 35 µg/mL MoS2. All of the reactions were performed for 45 cycles with 200 nM pBR322-FP3 and 200 nM pBR322-RP3 primers.

Further screening of 2D nanomaterials. Based on our initial screening, a few other 2D nanomaterials, like rGO, MoS2 and WS2 can also quench the fluorescence. We further compared MoS2 and GO in the above experiments and MoS2 appeared to perform better. To systematically compare these nanomaterials for PCR signaling, we then studied all these four 2D materials. All 12


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of these nanomaterials fully quenched the fluorescence of the NTC samples (Figure 4A), which was consistent with the non-amplified samples in Figure 1A. Interestingly, the fluorescence of the PTC samples varied among these materials. GO displayed the weakest fluorescence, while MoS2 and WS2 showed much stronger fluorescence. Therefore, the fluorescence from the dsDNA product was less affected by MoS2 and WS2. We then quantitatively measured the fluorescence of PCR products by adding different concentrations of these nanomaterials (Figure 4B-4E). With increasing concentration of the nanomaterials, the fluorescence of both the NTC and PTC samples decreased. With sufficiently high nanomaterial concentrations, the background fluorescence of NTC was nearly zero (red bars). When the ratio of the PTC to NTC is plotted (Figure 4F-4I), all the materials showed a similar concentration-dependent enhancement. At high materials concentrations, this ratio all improved from below 5 to around 50. For visual detection, a high positive fluorescence signal is desirable. The PTC fluorescence almost linearly decreased with GO and rGO, and this was consistent with that they can also adsorb dsDNA.51-53 On the other hand, MoS2 and WS2 showed little effect on the PTC samples. Given the stable and strong signal of the PTC samples, we reason that MoS2 and WS2 appear to be better candidates for this purpose than GO and rGO.

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Figure 4. (A) Fluorescence photographs of the NTC and PTC samples with 15 µg/mL GO, 10 µg/mL rGO, 30 µg/mL MoS2 and 30 µg/mL WS2. PCR products without any nanomaterials were performed as control. N: NTC; P: PTC. Fluorescence intensity of the PTC and NTC samples in the presence of various concentrations of (B) GO, (C) rGO, (D) MoS2 and (E) WS2. Fluorescence ratio of PTC over NTC with different concentrations of (F) GO, (G) rGO, (H) MoS2 and (I) WS2. All of the amplifications were performed for 35 cycles with pBR322-FP2 and -RP2 as primers.

Mechanism of improved signaling. Given the improved PCR signaling by MoS2 and WS2, we then studied the reason behind it. The main improvement was from the decreased background fluorescence due to SG binding to ssDNA primers. This could be attributed to the adsorption of 14


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primers, SG, or both by the added nanomaterials. Using MoS2 as an example, nearly 93% of the SG dye and 88% of DNA primers were adsorbed under our experimental condition (Figure 5A). Therefore, the effect was likely from adsorption of both. As the PCR reaction proceeds, ssDNA primers were gradually extended to dsDNA products, and the dsDNA can bind SG more strongly. To understand the competition, we designed the following experiment shown in Figure 5B. We incubated MoS2 with SG and then removed the excess SG after centrifugation. The precipitant was dispersed in the PCR buffer with 400 nM primers or with 700 µg/mL salmon sperm DNA. No fluorescence was observed with the DNA primer added sample, suggesting that most of the SG was still associated with MoS2. With the salmon sperm DNA, a strong fluorescence was observed. Since the SG quantum yield is different with dsDNA and ssDNA, we performed a further centrifugation and collected the supernatant, which contained desorbed SG and the added DNA. We then added the salmon sperm DNA to the ssDNA primer sample, and the ssDNA primer to the salmon sperm sample, such that each sample should contain roughly the same DNA composition. Still, only the sample initially incubated with the salmon sperm DNA showed strong fluorescence, suggesting that SG has a much higher affinity to dsDNA than to MoS2, leading to SG transferred from MoS2 to the dsDNA. In Figure 1 we screened a few nanomaterials for DNA adsorption, and concluded that the four 2D materials retained DNA adsorption in the PCR buffer. Here, we also studied their ability to adsorb SG (Figure 5C). Over 90% SG was adsorbed by GO, rGO, MoS2 and WS2. The metal oxides, even at a much higher concentration, were ineffective for adsorbing SG. Metal oxides adsorb DNA via its phosphate backbone, but SG does not have a favorable mechanism for adsorption by oxides. AuNPs were used at a low concentration of 2 nM since AuNPs have an 15



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intense color and they can strongly affect our visual detection at high concentrations. The lack of sufficient SG adsorption by AuNPs is attributed to the insufficient AuNPs and with more AuNPs, SG could still be effectively adsorbed.54-56 To better compare the 2D materials for adsorbing SG, we then lowered their concentration to 5 µg/mL. All the materials still showed over 50% adsorption (Figure 5D), and rGO had the highest capacity, suggesting that hydrophobic and π-π stacking interactions could be important for SG. GO adsorbs DNA also via hydrogen bonding.26, 57 To understand hydrogen bonding for SG adsorption, we pre-adsorbed the dye on MoS2 and GO, and then 4 M urea was added to wash the samples. The dye released from GO was more than doubled of that from MoS2 (Figure 5E), suggesting that hydrogen bonding is also an important force for SG adsorption on GO. Finally, we compared the adsorption of dsDNA by MoS2 and GO. In the PCR buffer, we mixed salmon sperm DNA with 35 µg/mL MoS2 or 15 and 35 µg/mL GO. Then the samples were centrifuged and the supernatant containing non-adsorbed DNA was stained by SG (Figure 5F). It appears that MoS2 fully adsorbed the DNA while the adsorption by GO was much less. MoS2 could exert a stronger van der Waals force for dsDNA, while GO has strong electrostatic repulsion that made the adsorption more difficult. Therefore, the better signaling cannot be explained by the lack of dsDNA adsorption by MoS2. Overall, it appears that MoS2 is efficient at adsorbing both the SG dye and dsDNA. Therefore, we believe its weaker adsorption of the dsDNA/SG complex relative to that for GO could be the main reason for its excellent signaling property.

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Based on our observation, MoS2 and WS2 behaved very similarly for this application, and we used MoS2 for most of our studies. This similarly is understandable since the surface of both materials are sulfur atoms and the metal centers are sandwiched and do not interact directly with DNA or dye. Both materials could only use van der Waals force as the main interaction mechanism, explaining the similarity between them. MoS2 and WS2 have the appropriate adsorption strength, allowing the best signaling performance.

Figure 5. (A) Fraction of adsorbed 1 × SG (~ 2 µM) and 200 nM pBR322-FP2 and 200 nM pBR322-RP2 primers by 30 µg/mL MoS2. (B) Photographs of MoS2 adsorbed with SG in the presence of 400 nM primers, or 700 µg/mL salmon sperm DNA. After centrifugation, the supernatant was added with more DNA to make sure that each sample had roughly the same DNA composition. (C) Fraction of adsorbed 1 × SG (~ 2 µM) by 15 µg/mL GO; 15 µg/mL rGO; 60 µg/mL CeO2; 2 nM AuNPs; 40 µg/mL MoS2; 40 µg/mL WS2; 140 µg/mL MnO2; 200 µg/mL NiO. (D) Fraction of adsorbed SG by 5 µg/mL GO, rGO, MoS2 and WS2. (E) Fraction of preadsorbed SG washed off from the MoS2 and GO by 4 M urea in 1 × Thermopol® reaction buffer containing 200 µM each dNTP. (F) Using 1 × SG (~ 2 µM) to detect the supernatant 17



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fluorescence of salmon sperm DNA (10 µg/mL) after incubating the DNA with MoS2 and GO for 15 min and then centrifugation to remove the nanomaterials and adsorbed DNA.

Based on the above experiments and discussion, we summarized the mechanism of signal enhancement in Figure 1C. SG can bind to a high concentration of free ssDNA primers and enhance its fluorescence. This fluorescence is significant at the initial stage of PCR since both have a high concentration. With MoS2, both are adsorbed and the background is close to zero. After PCR, dsDNA products are produced and such dsDNA was adsorbed very weakly. The dye binds to dsDNA more strongly than its adsorption on MoS2, retaining its strong fluorescence. MoS2 can also eliminate non-specific PCR amplification due to excessed polymerase. For the current method to suppress background signal, the nanomaterials were added after PCR. We then tested if this method could also be useful during RT-PCR, and a few nanomaterials were directly to the RT-PCR buffer before PCR. However, the PCR amplification was completely inhibited by 30 µg/mL MoS2 (Figure 6A), and no amplicon was detected by fluorescence or by gel electrophoresis (inset). Previous studies have shown that nanomaterials such as GO and rGO can enhance PCR specificity.13-14,

58

To understand the inhibition of PCR by MoS2, we incubated the Taq

polymerase with MoS2 and performed five PCR cycles. We then centrifuged the samples to precipitate MoS2, and took the supernatant for PCR (Figure 6B). Amplification was also inhibited with increasing of MoS2 concentration, suggesting that the polymerase might be adsorbed by MoS2. When the incubation was performed in the presence of BSA as a competing protein, the amplification was gradually recovered (Figure 6C). Therefore, the inhibition of 18


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MoS2 to real-time PCR was mainly attributed to the adsorption of Taq polymerase. BSA alone could also improve PCR specificity (Figure S4),59 but here its main function was to block the MoS2 surface and to prevent the polymerase from being adsorbed. In the absence of MoS2, we observed higher molecular weight non-specific products after increasing the Taq polymerase by ten-fold (Figure 6D, the lane marked with 0). Under this condition, MoS2 was able to eliminate non-specific amplification caused by the excess polymerase. The more MoS2 added, the lower non-specific amplification products (Figure 6D and see Figure S5 for quantification), and this can be explained by the adsorption of excess of polymerase by MoS2. We also used a different polymerase MoS2 could also eliminate its nonspecific amplification at different polymerase concentrations (Figure S6). In addition, we tested one more PCR system with different template sequences reaching the same conclusion (Figure S7). Similar observations were also reported previously with AuNPs and other nanomaterials.8, 60 Overall, MoS2 should be added after PCR reactions for the purpose of improving signaling.

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Figure 6. (A) PCR progress monitored using a real-time PCR instrument without and with 30 µg/mL MoS2. Inset: a gel micrograph of the final PCR product. (B) Taq polymerase (0.025 U) was incubated with different concentrations of MoS2. After 5 PCR cycles in 1 × Thermopol® Reaction buffer, the supernatant was used for PCR after centrifugation and the PCR was inhibited by MoS2 in a concentration dependent manner. (C) The same reaction as in (B) with 8 µg/mL MoS2 but also with different concentrations of BSA. (D) Using MoS2 to enhance the specificity of PCR in the presence of 0.25 U Taq polymerase (10-fold of the normal PCR condition) and 0.88 pM pBR322 DNA as template. All of the reactions were performed for 35 cycles with pBR322-FP2 and pBR322-RP2 as primers. In (B-D), Lane M: low molecular weight DNA ladder.

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Detection in a real sample. To test if MoS2 could be employed for real samples for enhancing PCR signaling, we chose transgenic soya GTS 40-3-2 with CaMV 35S as the target. Its genomic DNA was extracted from the soya powder purchased from Monsanto and serially diluted by tenfold. After amplification for 35 PCR cycles, the products were added with a final concentration of 40 µg/mL MoS2 and detected with a 470 nm LED excitation in a dark room (Figure 7A). Both the negative and positive samples have high fluorescence. With 40 µg/mL MoS2, the fluorescence of the negative sample was fully suppressed, while the fluorescence of the positive ones increased with the template concentration. Therefore, we could better discriminate the negative and positive samples, and such visual observation could even achieve semi-quantification. To quantify the fold of fluorescence enhancement, we measured the fluorescence intensity by a microplate reader (Figure 7B). Without MoS2, the fluorescence of all of the samples was over 1000, while the highest fluorescence was around 2000. With 40 µg/mL MoS2, the fluorescence of the negative control dropped to < 100, while the highest fluorescence intensity for the positive samples exceeded 1200. Therefore, the signal-to-background ratio was enhanced from ~ 2-fold to ~ 13-fold (Figure 7C). We also measured the same samples using realtime PCR (Figure 7D). After 35 cycles, the sample with 104-fold dilution just started to show signal, while its Ct value could not be accurately determined (Figure S8). For the 105-fold diluted sample, its signal cannot be distinguished from the negative control. Therefore, the detection limit of the RT-PCR was about 104-fold dilution, and it was consistent with the visual detection with MoS2 in Figure 7A.

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Figure 7. Detection of the CaMV 35S gene from the GTS 40-3-2 soya powder by 10 × serial diluting the extracted DNA. (A) Fluorescence photographs of samples amplified by 35 PCR cycles with 1 × SG (~ 2 µM) as an indicator without and with 40 µg/mL MoS2. -5 to -1 in the xaxis means 105 to 101-fold dilution. (B) Fluorescence intensities of the PCR products in (A). (C) Fluorescent ratio of the PCR products of various dilutions compared to the negative control. (D) The real-time PCR amplification plot of the samples.

Conclusions In conclusion, we have screened eight kinds of nanomaterials (MoS2, WS2, GO, rGO, CeO2, MnO2, NiO, and AuNP) for enhancing the signaling of PCR reaction in the presence of DNA staining dyes. The four 2D nanomaterials were effective for this purpose. They suppressed the 22


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background fluorescence signal due to a high concentration of free primers and DNA binding dye. By introducing these nanomaterials into PCR products, the fluorescence ratio of positive samples to negative samples were increased from around 3.5-fold to above 50-fold. MoS2 and WS2 provided even better signaling properties than GO or rGO since these dichalcogenides did not adsorb much dsDNA products and their samples had much higher fluorescent intensity. Both ssDNA primers and SG dye were adsorbed by these materials under the PCR condition. Since dsDNA has a higher affinity to the dyes, it can outcompete the nanomaterials for SG binding. MoS2 also eliminated the non-specific amplification caused by excess Taq polymerase. Finally, we detected the extracted DNA from transgenic GTS 40-3-2 soya sample using MoS2 and its sensitivity rivaled that of RT-PCR. We expect this easy and cost-effective method to be employed for other DNA amplification methods and bring convenience to resource-limited areas.

Acknowledgement Funding for this work was from The Natural Sciences and Engineering Research Council of Canada (NSERC). L. Wang was supported by National Natural Science Foundation of China (No. 31571918) and the Program of Supporting Graduate Students Studying Abroad by Zhejiang University to visit the University of Waterloo to perform this research.

Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: xxxxxxx.

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DNA primers used in this work (Table S1); TEM micrographs of GO, MoS2 and WS2 (Figure S1); using GO to enhance the fluorescent signal of PCR products (Figure S2) and with different dyes (Figure S3); using BSA to enhance the specificity (Figure S4); quantification of PCR specificity by adding MoS2 (Figure S5); using MoS2 to enhance PCR specificity (Figure S6); and the threshold cycle of RT-PCR for DNA from transgenic soya powder GTS 40-3-2 (Figure S7). (PDF)

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