Article Cite This: Langmuir XXXX, XXX, XXX−XXX
pubs.acs.org/Langmuir
Fluorescent DNA Probing Nanoscale MnO2: Adsorption, Dissolution by Thiol, and Nanozyme Activity Liu Wang,†,‡ Zhicheng Huang,‡ Yibo Liu,‡ Jian Wu,*,† and Juewen Liu*,‡ †
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo N2L 3G1, Ontario, Canada
‡
S Supporting Information *
ABSTRACT: Manganese dioxide (MnO2) is an interesting material due to its excellent biocompatibility and magnetic properties. Adsorption of DNA to MnO2 is potentially of interest for drug delivery and sensing applications. However, little fundamental understanding is known about their interactions. In this work, carboxyfluorescein (FAM)-labeled DNA oligonucleotides were used to explore the effect of salt concentration, pH, and DNA sequence and length for adsorption by MnO2, and comparisons were made with graphene oxide (GO). The DNA desorbs from MnO2 by free inorganic phosphate, while it desorbs from GO by adenosine and urea. Therefore, DNA is mainly adsorbed on MnO2 through its phosphate backbone, and DNA has a stronger affinity on MnO2 than on GO based on a salt-shock assay. At the same time, DNA was used to study the effect of thiol containing compounds on the dissolution of MnO2. Adsorbed DNA was released from MnO2 after its dissolution by thiol, but not from other metal oxides with lower solubility such as CeO2, TiO2, and Fe3O4. DNAfunctionalized MnO2 was then used for detecting glutathione (GSH) with a detection limit of 383 nM. Finally, DNA was found to inhibit the peroxidase-like activity of MnO2. This study has offered many fundamental insights into the interaction between MnO2 and two important biomolecules: DNA and thiol-containing compounds.
■
INTRODUCTION Attaching DNA to inorganic nanoparticles has enabled a broad range of applications from directed materials assembly1−4 and biosensor development5−10 to drug delivery.11 These applications have in turn stimulated fundamental biointerface research related to DNA. While most attention has focused on gold nanoparticles12,13 and carbon-based materials such as graphene oxide (GO),14−17 metal oxides have also attracted more and more recent attention.18−24 Metal oxides encompass a diverse range of useful materials. Among them, MnO2 is a very interesting one for its excellent biocompatibility, magnetic properties, and solubility in biological fluids, leading to many unique applications. For example, MnO2 was recently used as a delivery vehicle for RNA-cleaving DNAzymes.25,26 At the same time, it serves as a source of intracellular Mn2+ ions after dissolution by intracellular glutathione (GSH) to activate the DNAzyme. Its fluorescence quenching property was also demonstrated to be useful for biosensor development,27−29 and fluorescent DNA was adsorbed on MnO2 for detecting nucleic acids and other analytes.30−34 The complex was also used for making new materials.35 The magnetic property of Mn2+ allowed its use as a MRI contrast agent, and cellular uptake of MnO2 was facilitated by an adsorbed aptamer.36 The electric capacitance of MnO2 makes it a good candidate for fabricating electrode materials, and DNA can also play a role in this regard.37−39 While various applications of DNA-functionalized MnO2 have been demonstrated, our fundamental understanding of © XXXX American Chemical Society
this system is still quite limited in terms of adsorption mechanism. Two aspects of MnO2 are particularly interesting to us: its interaction with DNA and its dissolution by thiolcontaining compounds such as GSH. Herein, we employed fluorescent DNA oligonucleotides as probes for studying surface science of MnO2. Comparisons with GO and other metal oxides were made when appropriate to gain further insights.
■
MATERIALS AND METHODS
Chemicals. All of the DNA samples were from Integrated DNA Technologies (Coralville, IA), and their sequences are in Table S1. Carboxyl GO was from ACS Material (Medford, MA). CeO2 (5 nm) and Fe3O4 (50 nm) nanoparticles were from Sigma-Aldrich, while TiO2 (30 nm) was from US Research Nanomaterials (Houston, TX). Sodium chloride, magnesium chloride, urea, adenosine free base, and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonate (HEPES) were from Mandel Scientific (Guelph, Ontario, Canada). Hydrogen peroxide, reduced GSH, D-fructose, dextrose, cysteine, sodium phosphate monobasic, tetramethylammonium hydroxide, 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and manganese chloride tetrahydrate were from Sigma-Aldrich. Milli-Q water was used for all the experiments. Preparation of MnO2. MnO2 nanobelts were prepared according to a previous paper.40 Briefly, 20 mL of a mixed aqueous solution of Received: November 2, 2017 Revised: January 22, 2018 Published: February 19, 2018 A
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. TEM micrographs of (A) MnO2 and (B) GO used in this work. Scale bars = 100 nm. (C) ζ-potential of our MnO2 as a function of pH. (D) ζ-potential of MnO2 and GO in 10 mM HEPES buffer (pH 7.5). 20 nM FAM-T15 was mixed with 2 μL of MnO2 (final concentration 20 μg/mL) in buffer (10 mM HEPES, pH 7.5 with 300 mM NaCl). After incubation for 2 h, the mixture was centrifuged before 2 μL of T15 (400 nM) was added. Detection of GSH. To detect GSH, 25 nM FAM-A15 was incubated with 20 μg/mL MnO2 in buffer (10 mM HEPES, pH 7.5, 300 mM NaCl). After incubation for 2 h, the mixture was centrifuged, and the precipitant was dispersed in the same buffer. Then, 4 μL of GSH was added into 96 μL of the DNA/MnO2 complex. Peroxidase-like Activity Assays. In a typical assay, 2 μL of TMB or ABTS (50 mM) in pure DMSO was added into 100 μL of MnO2 (final concentration 3.2 μg/mL) with or without A15 or T15 DNA (200 nM) in an acetate buffer (10 mM, pH 5) followed by a quick mixing. Afterward, 2 μL of H2O2 (0.5 M) was added immediately to initiate the reaction. The UV−vis absorption spectra were measured using a spectrometer (Agilent 8453A) after 20 min.
tetramethylammonium hydroxide (0.6 M) and H2O2 (3 wt %) were added into a MnCl2 solution (0.3 M, 10 mL) within 15 s. The mixture was stirred vigorously overnight in open air at room temperature. The as-prepared MnO2 was centrifuged at 2000 rpm for 20 min and washed with copious amounts of distilled water and methanol. After that, the sample was dried at 60 °C. Then, 10 mg of the bulk sample was dispersed in 20 mL of water and ultrasonicated for 4 h to obtain the MnO2 nanobelt. ζ-Potential, X-ray Diffraction (XRD), and Transmission Electron Microscopy (TEM). The ζ-potential of MnO2 (30 μg/ mL) was measured using a Zetasizer Nano ZS90 (Malvern) at 25 °C. To obtain pH-dependent ζ-potential, HCl was added to adjust the pH of MnO2 dispersion in Milli-Q water (pH 2−9). Note the initial pH of our MnO2 dispersion was 9. Powder XRD was performed using a PANalytical Empyrean X-ray diffractometer with Cu Kα radiation (λ = 1.789 01 Å). TEM was performed on a Philips CM10 microscope. The TEM samples were prepared by dropping MnO2 dispersion (100 μg/ mL) on a copper grid followed by drying in air. DNA Adsorption. The kinetics of DNA adsorption was studied by adding 2 μL of MnO2 nanobelts or GO into 98 μL of solution containing FAM-A15 (20 nM) in buffer (10 mM HEPES, pH 7.5, 300 mM NaCl) at 25 °C. The fluorescence before adding MnO2 was measured to be the initial intensity. Several different salt concentrations were also tested by fixing MnO2 at 20 μg/mL. DNA Desorption. To study DNA desorption by lowing salt concentration, FAM-A15 (20 nM, 98 μL) was mixed with 2 μL of MnO2 (final concentration 10 μg/mL) in buffer (10 mM HEPES, pH 7.5, 300 mM NaCl and 1 mM MgCl2). After incubation for 2 h, the mixture was centrifuged. The precipitant was then dispersed in 100 μL of HEPES (10 mM, pH 7.5) containing different concentrations of NaCl. To explore DNA desorption in 4 M urea, the precipitant was first dispersed in 50 μL of buffer (10 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM MgCl2). Then, another 50 μL of buffer or 8 M urea in the buffer was added. To explore DNA desorption in phosphate or adenosine, 98 μL of FAM-A15 (20 nM) was incubated with 2 μL of MnO2 or GO (final concentration 20 μg/mL) in 10 mM HEPES, pH 7.5, with 100 mM NaCl. Then, 2 μL of phosphate (100 mM) or adenosine (50 mM) was added 2 h later. All of the experiments were performed in triplicates. To study DNA desorption by DNA, 98 μL of
■
RESULTS AND DISCUSSION MnO2 as a Negatively Charged Nanobelt. Manganese oxide refers to a few different materials including Mn2O3, MnO2, and MnO.41 Among them, MnO2 has been most studied for interfacing with biomolecules. We synthesized MnO2 nanobelts based on a literature reported method.40 The resulting material was characterized by transmission electron microscopy (TEM) showing an average length of ∼200−500 nm and a width of ∼10 nm (Figure 1A). Powder X-ray diffraction (XRD) confirmed the successful preparation of crystalline MnO2 (Figure S1, Supporting Information). Since DNA is negatively charged, electrostatic interactions are likely to be critical for affecting its adsorption. We then measured the ζ-potential of MnO2 as a function of pH (Figure 1C), and its surface remained negatively charged until pH 2.42 Therefore, we expect a charge repulsion between DNA and MnO2. In this study, we compared MnO2 with GO for a few experiments, and our GO appeared as nanosheets (Figure 1B). At neutral pH used for most of our experiments, MnO2 and GO had a similar ζ-potential (Figure 1D). B
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 2. (A) A scheme showing DNA adsorption on MnO2 with quenched fluorescence and then DNA desorbed from MnO2 with fluorescence recovery by adding various chemical reagents. Dissolution of MnO2 is achieved by adding GSH. (B) Fluorescence quenching of FAM-A15 by adding 10 μg/mL of MnO2 or various concentrations of GO in 10 mM HEPES, pH 7.5, with 100 mM NaCl. Adsorption kinetics of 20 nM FAM-A15 DNA by 20 μg/mL of (C) MnO2 and (D) GO as a function of NaCl concentration in 10 mM HEPES, pH 7.5. (E) FAM-A15 DNA adsorption rate constants and (F) relative binding site density on MnO2 in different concentrations of NaCl by fitting the data with a double-exponential equation: y = A1 exp(−k1t) + A2 exp(−k2t) + C, where k1 and k2 are the two adsorption rate constants and A1 and A2 are the relative DNA population undergoing each kinetic route.
DNA Is Adsorbed More Strongly on MnO2. We chose to compare MnO2 and GO since they may have different adsorption mechanisms. GO interacts with DNA bases through π−π stacking and hydrogen bonding,17 while MnO2 is unlikely to have such mechanisms. For a fair comparison, we need to ensure that MnO2 and GO were used at a concentration with a similar DNA adsorption capacity. We followed DNA adsorption using the scheme in Figure 2A (the first step) based on fluorescence quenching. With 20 nM carboxyfluorescein-labeled A15 DNA (FAMA15), we added a final of 10 μg/mL of MnO2 nanobelt and fluorescence was quenched by ∼60% in 2 h, indicating DNA adsorption (Figure 2B, black thick line). We then performed the same experiment by adding various concentrations of GO (Figure 2B, thin lines). A higher concentration of GO induced more fluorescence quenching, and it took ∼7 μg/mL GO to achieve the same extent of quenching as that by 10 μg/mL MnO2. Therefore, these two materials had a similar adsorption capacity. Since manganese is a lot heavier than carbon, and our MnO2 is not a single-layered material, the specific surface area of our MnO2 should be smaller. To confirm this, we used methylene blue (MB) as a probe to measure the surface area. Under the same condition, the specific surface of GO was around twice of that of MnO2 (Figure S2). The fact that they had a similar DNA adsorption capacity suggested that DNA was adsorbed by MnO2 with a higher affinity, resulting in a higher density of DNA on MnO2 than on GO. Note that the adsorption capacities measured here were not the ultimate capacity of these materials since DNA adsorption can be promoted by adding salt (vide infra). We measured DNA adsorption here under a buffer close to physiological ionic strength as a starting point. Based on the specific area measured using MB as a probe, each DNA occupied 691 nm2 of MnO2 (Figure S2), although the radius of gyration of a 15-mer DNA is only 3.2 nm.43 This suggested that the DNA was very sparsely adsorbed on MnO2. Since MB is positively charged, GO is negatively charged, and DNA is negatively charged, using MB estimated surface area to calculate DNA footprint could
result in an overestimation. At the same time, this estimation suggested that electrostatic repulsion between neighboring DNA molecules and between DNA and MnO2 could be important for affecting DNA adsorption capacity. A similar observation was also reported on gold nanoparticle surfaces.44 As a control, we added MnO2 and GO into a free fluorescein solution (no DNA, Figures S3 and S4), where very little fluorescence quenching was observed. We also incubated FAMA15 with MnCl2 and MnO2 (Figure S5), where quenching was observed only with MnO2. These experiments indicated that the observed quenching could indeed be attributed to DNA adsorption instead of artifacts caused by dissolved metal ions or pure fluorescein dye adsorption. The above study already confirmed that MnO2 can adsorb DNA, and its affinity might be even stronger than that of GO. We then explored the adsorption mechanism. Since both DNA and MnO2 are negatively charged, they should experience an electrostatic repulsion. Therefore, salt might be required to screen the charge repulsion for DNA adsorption. To test this, we followed DNA adsorption as a function of NaCl concentration at pH 7.5. The FAM-A15 DNA was respectively dispersed in buffers with various NaCl concentrations from 0 to 300 mM, and the same concentration of MnO2 was added to each sample (Figure 2C). It is interesting to note that even without NaCl, the DNA was still adsorbed by MnO2. Raising the salt concentration to just 10 mM significantly accelerated the adsorption rate. With more than 100 mM NaCl, full adsorption was achieved in 5 min. We then tested GO under the same conditions (Figure 2D). With up to 10 mM NaCl, no adsorption took place, suggesting a stronger repulsion relative to its attraction force. With 50 mM NaCl, a very slow adsorption occurred. Electrostatic repulsion is a long-ranged force. Salt is often required to screen the charge repulsion to allow shorter-ranged attraction forces to take place. Since both materials have a similar ζ-potential (Figure 1D), they should also have a similar repulsive force against DNA. We reason the better adsorption on MnO2 is due to its stronger attraction force. C
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 3. Desorption kinetics of FAM-A15 DNA from (A) MnO2 and (B) GO by reducing salt concentration. Adsorption kinetics of (C) FAMlabeled 15-mer homo-DNA of different sequences and (D) FAM-labeled poly-A DNA of different lengths.
To obtain further confirmation, we next designed a “saltshock” release study. We first adsorbed the DNA at a high salt concentration. At this moment, the energy barrier preventing adsorption was eliminated for both MnO2 and GO. We then lowered the salt concentration and monitored fluorescence increase indicative of DNA desorption. In this case, a lot more DNA released from GO (Figure 3B) than from MnO2 (Figure 3A), confirming that MnO2 adsorbed the DNA more tightly than GO did. On GO, when the salt concentration was reduced, the electrostatic repulsion started to dominate. After this qualitative observation, we then fitted the kinetic traces in Figure 2C. These traces could not be fitted with a single exponential model, but they were fitted well with a double-exponential model, suggesting two types of DNA adsorption kinetics: one faster and the other slower. We call the sites associated with faster adsorption to be site 1 and those with slower adsorption to be site 2. With more salt, both rates increased (Figure 2E), but the population of the faster adsorbing rate increased with salt while the population with lower rate decreased (Figure 2F). Note the data were all collected with our nanoscale MnO2 samples, and we only obtained an average behavior of all of their available crystal surfaces in the sample. Different crystal surfaces might have different DNA interaction strength (e.g., whether the Mn centers are exposed or not). To have further detailed understanding of each crystal surface, bulk single crystal samples are needed, and this is beyond the scope of this study. Our MnO2 was crystalline with a long-range order, and its surface should be quite homogeneous on the scale of a short DNA oligonucleotide (i.e., a few nanometers). We reason that the two different adsorption kinetics are unlikely to be assigned to different surface features, since the relative size of these two was a function of salt concentration. We propose that the fast binding population was due to the initial adsorption. The initially adsorbed DNA created more charge repulsion against further incoming DNA nearby. With more salt, the effective
area of charge repulsion was decreased, and the surface could accommodate more initially adsorbed DNA without feeling the repulsion from its neighbors. Effect of DNA Length and Sequence. To have a full understanding of DNA adsorption, we then used a few FAMlabeled 15-mer homo-DNAs and compared their adsorption kinetics (Figure 3C). All of these DNAs showed very fast adsorption kinetics in the presence of 300 mM NaCl, and they all reached equilibrium at a similar rate. The extent of quenching, however, was slightly different. Most quenching occurred with the A15 and T15 (over 95%) followed by C15, while the G15 DNA showed the lowest quenching efficiency of ∼70%. This might be related to the formation of secondary structures, such as G-quadruplex, which could reduce adsorption affinity. This also suggests that DNA adsorption on MnO2 might be less dependent on its base interaction since poly-A, T, and C DNA all showed a similar adsorption profile. We then studied the effect of DNA length using FAMlabeled poly-A DNA of 5-, 10-, 15-, and 30-mer (Figure 3D). The quenching was stronger with longer DNA until the 15-mer was reached. This can be explained by polyvalent interactions, and each nucleotide in the DNA may contribute to adsorption. Further increasing the length to 30 might shield the FAM label by the longer DNA backbone (e.g., the fluorophore might not be directly adsorbed). Therefore, for a very long DNA, it is unlikely that each nucleotide is adsorbed. For most of the work in this study, we used 15-mer poly-A and poly-T DNA to achieve fast adsorption and efficient fluorescence quenching. Adsorption Mechanism Probed by DNA Desorption. By studying DNA adsorption, we learned about adsorption capacity, strength, and kinetics, but we lack understanding on the type of attractive force responsible for adsorption. For this, we then studied DNA desorption. For example, hydrogen bonding is known to be important for GO,45 and urea can be used to disrupt hydrogen bonding. To test for hydrogen bonding, the adsorbed FAM-A15 was exposed to 4 M urea in a D
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 4. FAM-A15 desorption from (A) MnO2 and (B) GO induced by 4 M urea (added at 10 min) in a high salt buffer (10 mM HEPES, pH 7.5 with 300 mM NaCl and 1 mM MgCl2). Samples added with the buffer alone without urea were included as a control. DNA desorption from MnO2 and GO after adding (C) 2 mM phosphate and (D) 1 mM adenosine. (E) FAM-labeled T15 desorption from MnO2 and GO by the nonlabeled T15 DNA.
Figure 5. Fluorescence recovery of FAM-A15 DNA adsorbed on (A) MnO2, (B) CeO2, (C) TiO2, (D) Fe3O4, and (E) GO after adding various compounds at 10 min. The buffer was 10 mM HEPES, pH 7.5, with 300 mM NaCl. (F) A photograph of dispersed 100 μg/mL MnO2 in water and in 10 mM GSH. In GSH, the color of MnO2 disappeared, indicating its dissolution.
high salt buffer, and ∼25% DNA rapidly desorbed from GO (Figure 4B), consistent with the literature report.45 On the other hand, only 2% of DNA was desorbed from MnO2 with the same concentration of urea compared to the control sample without urea (Figure 4A), suggesting that hydrogen bonding is not important for DNA adsorption by MnO2. The main structural features of DNA are its nucleobases and the phosphate backbone. To pinpoint the chemical groups on DNA for adsorption, we next designed a few competition assays. We added 2 mM inorganic phosphate or 1 mM adenosine to probe adsorption by the DNA phosphate
backbone and nucleobases, respectively. Adding phosphate immediately released nearly all the DNA from MnO2, but not from GO (Figure 4C). On the other hand, adding adenosine had almost no impact on MnO2, but it quickly desorbed a fraction of DNA from GO (Figure 4D). This study indicates that DNA is adsorbed on MnO2 mainly via its phosphate while on GO via its bases. To further test the overall adsorption stability, we then used a T15 DNA (with both phosphate and nucleobase) to achieve desorption (Figure 4E). The signal from MnO2 was less than half of that from GO, also confirming a higher DNA adsorption affinity on MnO2. Taking all the data E
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 6. (A) Kinetics of fluorescent recovery of FAM-A15 adsorbed on MnO2 in the presence of different concentrations of GSH due to dissolution of MnO2. (B) Relative fluorescence enhancement in different concentrations of GSH at 30 min. (C) A linearly fitted relationship between the fluorescence enhancement and the concentration of GSH in the low GSH concentration region. Chromogenic reaction of oxidation of (D) TMB and (E) ABTS by 10 mM H2O2 in the presence of naked MnO2 and A15 or T15 DNA coated MnO2.
also tested GO, and no fluorescence was observed by adding GSH either (Figure 5E), further confirming its different GSH interaction mechanisms from MnO2. Detection of GSH. The increased fluorescence specific to GSH or other thiol-containing compounds might be useful for their detection. We titrated different concentrations of GSH to the FAM-DNA/MnO2 conjugate and observed a gradual fluorescence increase (Figure 6A). The fluorescence enhancement at 30 min was used to quantify GSH (Figure 6B), and the dynamic range was up to ∼100 μM GSH. We took the initial enhancement at low GSH concentrations, measured its slope, and calculated the detection limit to be 380 nM GSH based on 3σ/slope, where σ is the standard deviation of background in the absence of GSH (Figure 6C). This detection limit is comparable with other sensors based on GSH dissolution. DNA has the advantage of being cost-effective, and the sensitivity might also be tuned by varying the length and sequence of DNA.49,50 Peroxidase-like Nanozyme Activity of MnO2. MnO2 was reported to be a peroxidase-mimicking nanozyme that can oxidize substrates in the presence of H2O2.51 Because of the very high adsorption affinity of DNA, we want to test if DNA can be used to modulate the catalytic activity of MnO2. When A15 or T15 DNA was adsorbed on MnO2, the activity was significantly suppressed for the oxidation of both cationic TMB (Figure 6D) and anionic ABTS substrates (Figure 6E). We studied the nanozyme activity at pH 5 since this was the most optimal condition for activity. Since most of our above studies were performed at pH 7.5, we compared DNA adsorption at these two pH’s. More DNA was adsorbed by MnO2 at pH 5, which might even more effectively prevent MnO2 from contacting with TMB and ABTS (Figure S6, Supporting Information). It is interesting that for Fe3O4 nanozyme the peroxidation of TMB was accelerated by about 10-fold by DNA, while the reaction of ABTS was inhibited.52 In that case,
together, MnO2 and GO had a similar negative charge density, and DNA should experience a similar repulsive force. Therefore, the coordination by the DNA phosphate on MnO2 must be stronger than the π−π stacking and hydrogen bonding on GO to explain the stronger DNA adsorption on the former. Previous studies have shown that free inorganic phosphate ions can form strong inner-sphere coordination on MnO2,46 and it is likely that the DNA phosphate can also have strong coordination. MnO2 Interaction with GSH Probed by DNA. After understanding DNA adsorption by MnO2, we then used DNA as a probe to study other reactions of MnO2. For example, MnO2 has been commonly used to detect glutathione (GSH) based on its dissolution. Previously, a sensor was designed by using MnO2 to quench the fluorescence of graphene quantum dots, and the fluorescence was restored after dissolving MnO2 with GSH (Figure 2A, reaction 3).47 Deng et al. used MnO2 as a quencher for lanthanide-doped upconversion nanoparticles for the same purpose.48 Here, we want to use DNA to further study this reaction. The fluorescence of our FAM-labeled DNA was very low when it was adsorbed by MnO2. After adding GSH, the fluorescence was recovered attributable to the dissolution of MnO2 (Figure 5A). We confirmed dissolution by visual observation (Figure 5F). Cysteine also showed a similar rate of fluorescence enhancement, suggesting that it was the thiol group that had the dissolution effect. As a control, we added a few sugar molecules, and they did not produce any signal change. We also tested other metal oxide nanoparticles including CeO2, TiO2, and Fe3O4, each adsorbed with the FAM-A15 DNA (Figure 5B−D). In all these cases, very little signal increase was observed. In this regard, the uniqueness of MnO2 is likely due to the thiophilicity of Mn2+ leading to the dissolution of MnO2. It is likely that the other oxides were not dissolved by GSH. We F
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Langmuir
■
electrostatic interactions between the substrates and DNA were used to explain the observation. The fact that DNA inhibited both substrates on MnO2 suggested a different strength of DNA adsorption, and DNA is likely to be adsorbed more tightly on MnO2.
CONCLUSIONS MnO2 is an important material for its excellent biocompatibility, magnetic properties, and fluorescence quenching ability. Interfacing MnO 2 with DNA has enabled its further applications in biosensor development, targeted nucleic acid delivery, and imaging. In this work, we focused on the fundamental aspects of this system and performed systematic studies to understand biointerfacial properties of MnO2 for DNA adsorption and the dissolution of MnO2 by adding GSH. The effects of salt concentration, urea, DNA length, and DNA sequence have been systematically studied. We concluded very strong adsorption of DNA via its phosphate backbone interaction with MnO2, leading to poor DNA-induced DNA desorption. At the same time, the adsorbed DNA can block the surface accessibility of MnO2, leading to inhibited peroxidase like activity. We also studied the effect of GSH on MnO2 and compared it with a few other metal oxides and GO, confirming that MnO2 was the only dissolved material by GSH likely due to the thiophilicity of manganese. This has allowed us to detect GSH or other thiol-containing compounds using DNA as a sensitive probe. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b03797. DNA sequences employed in this work (Table S1); XRD spectrum of MnO2 (Figure S1); surface area measurement of MnO2 and GO using methylene blue (Figure S2); the effect of MnO2 (Figure S3) and GO (Figure S4) on the fluorescence intensity of free fluorescein and FAM-A15; the fluorescence of FAM-DNA quenched by MnCl2 and MnO2 (Figure S5); and the percentage of DNA adsorbed at pH 5 and pH 7.5 (Figure S6) (PDF)
■
REFERENCES
(1) Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Programmable Materials and the Nature of the DNA Bond. Science 2015, 347 (6224), 1260901. (2) Lu, Y.; Liu, J. Smart Nanomaterials Inspired by Biology: Dynamic Assembly of Error-Free Nanomaterials in Response to Multiple Chemical and Biological Stimuli. Acc. Chem. Res. 2007, 40 (5), 315− 323. (3) Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-Guided Crystallization of Colloidal Nanoparticles. Nature 2008, 451 (7178), 549−552. (4) Lan, X.; Wang, Q. B. DNA-Programmed Self-Assembly of Photonic Nanoarchitectures. NPG Asia Mater. 2014, 6 (4), e97. (5) Rosi, N. L.; Mirkin, C. A. Nanostructures in Biodiagnostics. Chem. Rev. 2005, 105 (4), 1547−1562. (6) Liu, J.; Cao, Z.; Lu, Y. Functional Nucleic Acid Sensors. Chem. Rev. 2009, 109 (5), 1948−1998. (7) Wang, H.; Yang, R. H.; Yang, L.; Tan, W. H. Nucleic Acid Conjugated Nanomaterials for Enhanced Molecular Recognition. ACS Nano 2009, 3 (9), 2451−2460. (8) Zhao, W.; Brook, M. A.; Li, Y. Design of Gold NanoparticleBased Colorimetric Biosensing Assays. ChemBioChem 2008, 9 (15), 2363−2371. (9) Song, S. P.; Qin, Y.; He, Y.; Huang, Q.; Fan, C. H.; Chen, H. Y. Functional Nanoprobes for Ultrasensitive Detection of Biomolecules. Chem. Soc. Rev. 2010, 39 (11), 4234−4243. (10) Katz, E.; Willner, I. Nanobiotechnology: Integrated Nanoparticle-Biomolecule Hybrid Systems: Synthesis, Properties, and Applications. Angew. Chem., Int. Ed. 2004, 43 (45), 6042−6108. (11) Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A. Gold Nanoparticles for Biology and Medicine. Angew. Chem., Int. Ed. 2010, 49 (19), 3280−3294. (12) Liu, J. Adsorption of DNA onto Gold Nanoparticles and Graphene Oxide: Surface Science and Applications. Phys. Chem. Chem. Phys. 2012, 14, 10485−10496. (13) Carnerero, J. M.; Jimenez-Ruiz, A.; Castillo, P. M.; Prado-Gotor, R. Covalent and Non-Covalent DNA−Gold-Nanoparticle Interactions: New Avenues of Research. ChemPhysChem 2017, 18 (1), 17− 33. (14) Wu, M.; Kempaiah, R.; Huang, P.-J. J.; Maheshwari, V.; Liu, J. Adsorption and Desorption of DNA on Graphene Oxide Studied by Fluorescently Labeled Oligonucleotides. Langmuir 2011, 27 (6), 2731−2738. (15) Park, J. S.; Goo, N.-I.; Kim, D.-E. Mechanism of DNA Adsorption and Desorption on Graphene Oxide. Langmuir 2014, 30 (42), 12587−12595. (16) Tang, Z. W.; Wu, H.; Cort, J. R.; Buchko, G. W.; Zhang, Y. Y.; Shao, Y. Y.; Aksay, I. A.; Liu, J.; Lin, Y. H. Constraint of DNA on Functionalized Graphene Improves Its Biostability and Specificity. Small 2010, 6 (11), 1205−1209. (17) Liu, B.; Salgado, S.; Maheshwari, V.; Liu, J. DNA Adsorbed on Graphene and Graphene Oxide: Fundamental Interactions, Desorption and Applications. Curr. Opin. Colloid Interface Sci. 2016, 26, 41− 49. (18) Ma, L.; Liu, B.; Huang Po-Jung, J.; Zhang, X.; Liu, J. DNA Adsorption by ZnO Nanoparticles near Its Solubility Limit: Implications for DNA Fluorescence Quenching and DNAzyme Activity Assays. Langmuir 2016, 32, 5672−5680. (19) Liu, B.; Liu, J. DNA Adsorption by Indium Tin Oxide Nanoparticles. Langmuir 2015, 31 (1), 371−377. (20) Liu, B.; Liu, J. Comprehensive Screen of Metal Oxide Nanoparticles for DNA Adsorption, Fluorescence Quenching, and Anion Discrimination. ACS Appl. Mater. Interfaces 2015, 7 (44), 24833−24838. (21) Zhang, X.; Wang, F.; Liu, B.; Kelly, E. Y.; Servos, M. R.; Liu, J. Adsorption of DNA Oligonucleotides by Titanium Dioxide Nanoparticles. Langmuir 2014, 30 (3), 839−845.
■
■
Article
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (J.W.). *E-mail:
[email protected] (J.L.). ORCID
Jian Wu: 0000-0003-4518-7027 Juewen Liu: 0000-0001-5918-9336 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS Funding for this work is 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. The authors thank Q. Pang for help in the XRD experiment. G
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir (22) Liu, B.; Liu, J. DNA Adsorption by Magnetic Iron Oxide Nanoparticles and Its Application for Arsenate Detection. Chem. Commun. 2014, 50 (62), 8568−8570. (23) Bülbül, G.; Hayat, A.; Andreescu, S. ssDNA-Functionalized Nanoceria: A Redox-Active Aptaswitch for Biomolecular Recognition. Adv. Healthcare Mater. 2016, 5 (7), 822−828. (24) Liu, J.; Liu, B.; Ma, L.; Huang, Z.; Hu, H.; Wu, P. Janus DNA Orthogonal Adsorption of Graphene Oxide and Metal Oxide Nanoparticles Enabling Stable Sensing in Serum. Mater. Horiz. 2018, 5, 65−69. (25) Fan, H.; Zhao, Z.; Yan, G.; Zhang, X.; Yang, C.; Meng, H.; Chen, Z.; Liu, H.; Tan, W. A Smart DNAzyme−MnO2 Nanosystem for Efficient Gene Silencing. Angew. Chem. 2015, 127, 4883−4887. (26) Chen, F.; Bai, M.; Cao, K.; Zhao, Y.; Wei, J.; Zhao, Y. X. Fabricating MnO2 Nanozymes as Intracellular Catalytic DNA Circuit Generators for Versatile Imaging of Base-Excision Repair in Living Cells. Adv. Funct. Mater. 2017, 27 (45), 1702748. (27) He, D.; He, X.; Wang, K.; Yang, X.; Yang, X.; Li, X.; Zou, Z. Nanometer-Sized Manganese Oxide-Quenched Fluorescent Oligonucleotides: An Effective Sensing Platform for Probing Biomolecular Interactions. Chem. Commun. 2014, 50 (75), 11049−11052. (28) Qi, L.; Yan, Z.; Huo, Y.; Hai, X.-M.; Zhang, Z.-Q. MnO2 Nanosheet-Assisted Ligand-DNA Interaction-Based Fluorescence Polarization Biosensor for the Detection of Ag+ Ions. Biosens. Bioelectron. 2017, 87, 566−571. (29) Tian, R.; Jiang, H.; Wang, G. MnO2 Nanosheet-Based Heparin and OSCS Fluorescent Biosensor with Lowered Background and Amplified Hybridization Chain Reaction. RSC Adv. 2016, 6 (92), 89803−89809. (30) Liu, Z. M.; Li, Z. J.; Shen, G. L.; Yu, R. Q. Label-Free Detection of DNA Hybridization Based on MnO2 Nanoparticles. Anal. Lett. 2009, 42 (18), 3046−3057. (31) Prathap, M. U. A.; Srivastava, R.; Satpati, B. Simultaneous Detection of Guanine, Adenine, Thymine, and Cytosine at Polyaniline/MnO2 Modified Electrode. Electrochim. Acta 2013, 114, 285−295. (32) Sultan, S. C.; Kara, P.; Anik, U.; Ozsoz, M. Label and Indicator Free Electrochemical Nanobiosensing of DNA Hybridization Based on MnO2 Nanomaterial Modified GCPE. RSC Adv. 2014, 4 (75), 39691− 39696. (33) Ouyang, W. J.; Liu, Z. H.; Zhang, G. F.; Chen, Z.; Guo, L. H.; Lin, Z. Y.; Qiu, B.; Chen, G. N. Enzyme-Free Fluorescent Biosensor for miRNA-21 Detection Based on MnO2 Nanosheets and Catalytic Hairpin Assembly Amplification. Anal. Methods 2016, 8 (48), 8492− 8497. (34) Ou, M.; Huang, J.; Yang, X. H.; Quan, K.; Yang, Y. J.; Xie, N. L.; Wang, K. M. MnO2 Nanosheet Mediated “DD-A″ FRET Binary Probes for Sensitive Detection of Intracellular mRNA. Chem. Sci. 2017, 8 (1), 668−673. (35) Ede, S. R.; Anantharaj, S.; Nithiyanantham, U.; Kundu, S. DNAEncapsulated Chain and Wire-Like Beta-MnO2 Organosol for Oxidative Polymerization of Pyrrole to Polypyrrole. Phys. Chem. Chem. Phys. 2015, 17 (7), 5474−5484. (36) Zhao, Z.; Fan, H.; Zhou, G.; Bai, H.; Liang, H.; Wang, R.; Zhang, X.; Tan, W. Activatable Fluorescence/MRI Bimodal Platform for Tumor Cell Imaging Via MnO2 Nanosheet−Aptamer Nanoprobe. J. Am. Chem. Soc. 2014, 136 (32), 11220−11223. (37) Ede, S. R.; Ramadoss, A.; Anantharaj, S.; Nithiyanantham, U.; Kundu, S. Enhanced Catalytic and Supercapacitor Activities of DNA Encapsulated Beta-MnO2 Nanomaterials. Phys. Chem. Chem. Phys. 2014, 16 (39), 21846−21859. (38) Guo, C. X.; Chitre, A. A.; Lu, X. DNA-Assisted Assembly of Carbon Nanotubes and MnO2 Nanospheres as Electrodes for HighPerformance Asymmetric Supercapacitors. Phys. Chem. Chem. Phys. 2014, 16 (10), 4672−4678. (39) Chen, Z.; Lu, M. Target-Responsive Aptamer Release from Manganese Dioxide Nanosheets for Electrochemical Sensing of Cocaine with Target Recycling Amplification. Talanta 2016, 160, 444−448.
(40) Peng, L.; Peng, X.; Liu, B.; Wu, C.; Xie, Y.; Yu, G. Ultrathin Two-Dimensional MnO2/Graphene Hybrid Nanostructures for HighPerformance, Flexible Planar Supercapacitors. Nano Lett. 2013, 13 (5), 2151−2157. (41) Remucal, C. K.; Ginder-Vogel, M. A Critical Review of the Reactivity of Manganese Oxides with Organic Contaminants. Environ. Sci. Process Impacts 2014, 16 (6), 1247−1266. (42) Kosmulski, M. Isoelectric Points and Points of Zero Charge of Metal (Hydr)Oxides: 50 Years after Parks’ Review. Adv. Colloid Interface Sci. 2016, 238, 1−61. (43) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Persistence Length of Single-Stranded DNA. Macromolecules 1997, 30 (19), 5763−5765. (44) Zhang, X.; Servos, M. R.; Liu, J. Surface Science of DNA Adsorption onto Citrate-Capped Gold Nanoparticles. Langmuir 2012, 28, 3896−3902. (45) Park, J. S.; Na, H.-K.; Min, D.-H.; Kim, D.-E. Desorption of Single-Stranded Nucleic Acids from Graphene Oxide by Disruption of Hydrogen Bonding. Analyst 2013, 138 (6), 1745−1749. (46) Yao, W.; Millero, F. J. Adsorption of Phosphate on Manganese Dioxide in Seawater. Environ. Sci. Technol. 1996, 30 (2), 536−541. (47) Yan, X.; Song, Y.; Zhu, C.; Song, J.; Du, D.; Su, X.; Lin, Y. Graphene Quantum Dot-MnO2 Nanosheet Based Optical Sensing Platform: A Sensitive Fluorescence “Turn Off-On” Nanosensor for Glutathione Detection and Intracellular Imaging. ACS Appl. Mater. Interfaces 2016, 8 (34), 21990−21996. (48) Deng, R.; Xie, X.; Vendrell, M.; Chang, Y.-T.; Liu, X. Intracellular Glutathione Detection Using MnO2-Nanosheet-Modified Upconversion Nanoparticles. J. Am. Chem. Soc. 2011, 133 (50), 20168−20171. (49) Yu, T.; Liu, B.; Liu, J. Adsorption of Selenite and Selenate by Metal Oxides Studied with Fluorescent DNA Probes for Analytical Application. J. Anal. Test. 2017, 1 (1), 2. (50) Lopez, A.; Zhang, Y.; Liu, J. Tuning DNA Adsorption Affinity and Density on Metal Oxide and Phosphate for Improved Arsenate Detection. J. Colloid Interface Sci. 2017, 493, 249−256. (51) Liu, J.; Meng, L. J.; Fei, Z. F.; Dyson, P. J.; Jing, X. N.; Liu, X. MnO2 Nanosheets as an Artificial Enzyme to Mimic Oxidase for Rapid and Sensitive Detection of Glutathione. Biosens. Bioelectron. 2017, 90, 69−74. (52) Liu, B.; Liu, J. Accelerating Peroxidase Mimicking Nanozymes Using DNA. Nanoscale 2015, 7 (33), 13831−13835.
H
DOI: 10.1021/acs.langmuir.7b03797 Langmuir XXXX, XXX, XXX−XXX
