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Continuously Tunable Nucleotide/Lanthanide Coordination Nanoparticles for DNA Adsorption and Sensing

Metal–organic coordination polymers (CPs) have attracted great research interest because they are easy to prepare, porous, flexible in composition, and designable in structure. Their applications in biosensor development, drug delivery, and catalysis have been explored. Lanthanides and nucleotides can form interesting CPs, although most previous works have focused on a single type of metal ligand. In this work, we explored mixed nucleotides and studied their DNA adsorption properties using fluorescently labeled oligonucleotides. Adenosine monophosphate (AMP) and guanosine monophosphate (GMP) formed negatively charged CP nanoparticles with most lanthanides, and thus a salt was required to adsorb negatively charged DNA. DNA adsorption was faster and reached a higher capacity with lighter lanthanides. Desorption of pre-adsorbed DNA by inorganic phosphates, urea, proteins, surfactants, and competing DNA was successively carried out. The results suggested the importance of the DNA phosphate backbone, although hydrogen bonding and DNA bases also contributed to adsorption. The AMP CPs adsorbed DNA more strongly than the GMP ones, and using mixtures of AMP and GMP, continuous tuning of DNA adsorption affinity was achieved. Such CPs were also used as a sensor for DNA detection based on the different affinities of single- and double-stranded DNA, and a detection limit of 0.9 nM target DNA was achieved. Instead of tuning DNA adsorption by varying the length and sequence of DNA, the composition of CPs can also be controlled to achieve this goal.


■ INTRODUCTION
Materials that can adsorb DNA are of great analytical and biomedical importance for applications such as biosensor development, 1−4 delivery of nucleic acids, 5,6 and stimuliresponsive materials. 7−9 To date, many types of inorganic nanomaterials have been tested for this purpose, including metal nanoparticles, 10−12 carbon-based nanomaterials, 9,13−16 transition metal dichalcogenides, 17 and metal oxides. 18 They interact with DNA through base chemisorption, π−π stacking, hydrogen bonding, and van der Waals forces, or by binding to the phosphate backbone of DNA. 19−21 Although DNA adsorption affinity can be tuned by varying the length and sequence of DNA, 22−25 it is more difficult to tune the interaction by continuously varying the properties of nanomaterials. From materials design standpoint, it is interesting and potentially useful to have materials with continuously tunable affinities for DNA adsorption.
Other intermolecular interactions, such as DNA base pairing interactions and metal coordination, were less explored for DNA adsorption. In this regard, nucleotide-coordinated materials are capable of harnessing such interactions. 26,27 Coordination polymer (CP) nanoparticles made of metal ions and organic ligands may offer an opportunity to design such materials. Nucleotides are excellent metal ligands, with both nucleobases and phosphate groups for metal binding. Nishiyabu et al. first reported coordination polymer (CP) nanoparticles formed from various lanthanide (Ln) ions and purine nucleotides. 28, 29 Various applications of such CPs in loading guest molecules and sensing have been explored. 28−31 Under certain conditions, such materials can also form hydrogels. 32 By using other metal ions, such as Zn 2+ , 33 Fe 3+ , 34 Au 3+ , 35−37 and Cu 2+ , 38 coordination materials forming hydrogels, coating materials, and nanozymes have also been demonstrated.
Lanthanides (Ln) contain 15 elements and they can form CPs with various nucleotides. 28, 39 We have recently performed a systematic titration of lanthanides to nucleotides and found a continuous thermodynamic trend. 40 We hypothesize that it might be possible to continuously tune the interaction strength between DNA and lanthanide containing CPs by varying the lanthanide and/or nucleotides. In this work, we wanted to achieve two goals: (1) a careful study of DNA adsorption by such CPs in terms of material composition and interaction forces; and (2) tuning DNA adsorption affinity. Finally, preliminary analytical applications of such materials were demonstrated.

■ RESULTS AND DISCUSSION
Nucleotide-Coordinated Lanthanide Nanoparticles. Our CPs were prepared by mixing adenosine monophosphate (AMP) or guanosine monophosphate (GMP) with various lanthanides (Ln 3+ ) ( Figure 1A). All samples spontaneously formed particles as judged from increased turbidity ( Figure  1B), and the Tb 3+ /GMP sample also showed strong green luminescence, also indicating the coordination reaction. 28 Previous work showed that both the nucleobase and the phosphate of these nucleotides are required for forming such CPs, 28,40,41 and a proposed structure of Ln 3+ /GMP is also shown in Figure 1A.
To gain a comprehensive understanding of these CPs, we first prepared samples with different ratios between Ln 3+ (La 3+ , Eu 3+ , Gd 3+ ,Tb 3+ , and Lu 3+ ) and nucleotides (AMP and GMP). We chose these five Ln 3+ ions to represent the beginning, middle, and end of the series. Because DNA is a polyanion, electrostatic interaction is likely to be important. Therefore, we measured the ζ-potential of the products ( Figure 1C,D). To have a full understanding, we also varied the ratio between Ln 3+ and the nucleotides. All of them were negatively charged regardless of the ratio, except for La 3+ /GMP showing a charge close to zero. The negative charges on these materials are attributable to the phosphate group on the nucleotides. The final product was not very much affected by the initial ratio of the reactants, suggesting well-defined and stable complexes were formed. Considering this, a fixed 1:2 ratio of Ln 3+ / nucleotide was used for preparing the CPs for the subsequent experiments.
The morphology of the Tb 3+ /AMP and Tb 3+ /GMP samples was examined by transmission electron microscopy (TEM, Figure 1E,F) and scanning electron microscopy (SEM, Figure  1G,H). They formed nanoparticle aggregates with individual particles being around 40 nm, which was consistent with the literature report. 22 These samples were freshly prepared and used, and we have recently noticed that for heavy lanthanides mixed with AMP, their CP nanoparticles would transform into hydrogels after overnight storage. 32 DNA Adsorption by CP Nanoparticles. As Ln 3+ ions can strongly bind DNA, and nucleotides may also base-pair with DNA, we were interested in studying their DNA adsorption properties. To achieve a systematic understanding, we varied the sequence of DNA and also the composition of CPs. FAM-A 15 DNA (a 15-mer polyadenine with a carboxyfluorescein label) was used as an example to illustrate our experiment. We fixed the concentration of FAM-A 15 at 20 nM and monitored its background fluorescence for 10 min. Each CP material was then added at various concentrations (Figure 2A−F). In general, a higher CP concentration induced more fluorescence quenching, which indicated more DNA adsorption. As the emission of FAM did not overlap with the absorption spectra of the CPs (they mainly absorbed in the UV region), the quenching here was attributed to photo-induced electron transfer instead of energy transfer.
With 25 μg/mL of La 3+ /GMP, full DNA desorption was achieved, whereas more Tb 3+ /GMP and even more Lu 3+ /GMP were required to achieve the same quenching efficiency. A similar trend was also observed with the AMP-containing CPs. Therefore, with the same nucleotide, lighter lanthanides were more effective for DNA adsorption. For the same lanthanide, the AMP samples adsorbed the DNA more slowly than the GMP ones. For a quantitative understanding, we plotted the relative fluorescence quenching as a function of CP concentration ( Figure 2G). At low CP concentrations, a linear fluorescence quenching was observed. The slopes of these curves represent DNA adsorption capacity. The slope of La 3+ / GMP was ∼2.2-fold higher than that of Tb 3+ /GMP and ∼2.4fold higher than that of Lu 3+ /GMP. For AMP, the slope of La 3+ /AMP was similar to that of Tb 3+ /AMP and ∼1.9-fold higher than that of Lu 3+ /AMP. Therefore, the La 3+ -containing CPs had the highest DNA adsorption capacity, whereas the Lu 3+ ones were the lowest. When AMP and GMP were compared (using the same metal), GMP had a higher adsorption capacity. Therefore, among these combinations, La 3+ /GMP adsorbed the DNA most efficiently. The reason might be its lack of surface charge ( Figure 1D).
To confirm the importance of electrostatic repulsion, we then studied the effect of salt concentration using Tb 3+ /GMP ( Figure 2H). Without salt, no fluorescence quenching was observed, indicating a lack of DNA adsorption. This can be explained by the long-ranged charge repulsion between the CP and DNA. With 50 mM NaCl, fluorescence quenching occurred. Further addition of 100 mM Na + and 1 mM Mg 2+ resulted in even faster quenching. Our results indicated that a certain amount of salt was required to overcome the electrostatic repulsion for DNA adsorption.
Stronger DNA Adsorption by AMP CPs. After confirming DNA adsorption, we further explored the mechanism of adsorption by inducing DNA desorption. DNA is made of nucleosides and a phosphate backbone, which are responsible for interacting with various materials. For example, DNA bases stack and hydrogen bond with graphene oxide, 14 and DNA bases coordinate with a gold surface, 42 whereas the DNA phosphate backbone is mainly responsible for its adsorption on metal oxides. 19  We first successively adsorbed a FAM-labeled DNA on Tb 3+ /AMP and Tb 3+ /GMP and then washed away the free DNA. To test whether the phosphate backbone of DNA was involved in adsorption, we challenged the samples with 10 mM free inorganic phosphate ions ( Figure 3A). An immediate increase in fluorescence was observed in both samples, indicating a quick release of the DNA. The system reached a new equilibrium in a few seconds, after which the signal became stable again. Therefore, the phosphate backbone of DNA was important for its adsorption onto both CPs. This is understandable because the phosphate backbone of DNA can interact with Ln 3+ . 27,40 We calculated the percentage of released DNA, and the Tb 3+ /GMP sample released around 4-fold more DNA than Tb 3+ /AMP, suggesting that the DNA adsorption affinity on Tb 3+ /AMP was higher.
Hydrogen bonding plays an important role in DNA adsorption by materials such as graphene oxide. 14,20 Urea can break hydrogen bonds, and it has been commonly used to probe such interactions. Following this, we exposed our CP nanoparticles with adsorbed DNA to 4 M urea ( Figure 3B). Interestingly, over 60% of the DNA was released from Tb 3+ / GMP, whereas Tb 3+ /AMP barely released any. Therefore, hydrogen bonding is also an important force for DNA adsorption by Tb 3+ /GMP. It is unclear whether hydrogen bonding is important for Tb 3+ /AMP. If not, another type of even stronger interaction must exist, such as Tb 3+ binding with the DNA phosphate backbone, as indicated by the above phosphate washing experiment.
To further understand DNA adsorption, we then examined DNA displacement by proteins and surfactants, which may also compete with DNA for surface adsorption sites. Bovine serum albumin (BSA) had no effect on Tb 3+ /AMP, whereas ∼30% DNA desorbed from Tb 3+ /GMP ( Figure 3C). Similarly, SDS, a small molecule anionic surfactant, had no effect on Tb 3+ / AMP, whereas ∼20% DNA desorbed from Tb 3+ /GMP ( Figure  3D). For cationic CTAB ( Figure 3E) and a higher molecular weight surfactant, Tween 80 ( Figure 3F), Tb 3+ /AMP also released less DNA. Therefore, under all of these washing conditions, the DNA was adsorbed more tightly on the Tb 3+ / AMP sample.
DNA-Induced DNA Desorption. After understanding DNA desorption by various denaturing and competing agents, we then studied DNA-induced DNA desorption, which might be useful for DNA detection. We first adsorbed FAM-A 15 or FAM-T 15 on Tb 3+ /AMP or Tb 3+ /GMP. Then, the four types of nonlabeled 15-mer homo-DNAs were sequentially added to hybridize with or to displace the adsorbed DNA probes as shown in Figure 4A. A few interesting observations were made. First, no DNA desorbed from Tb 3+ /AMP, regardless of the DNA added ( Figure 4B,C). On the other hand, when the probe was adsorbed on Tb 3+ /GMP, it can be desorbed by other DNA sequences, further confirming the much stronger DNA adsorption affinity of the AMP CPs ( Figure 4D,E).
For Tb 3+ /GMP, adding C 15 induced the highest adsorption for both probes ( Figure 4D,E), whereas T 15 and A 15 ranked the next. We have recently observed that poly-C DNA adsorbed on many nanomaterials (e.g., graphene oxide, MoS 2 , and various metal oxides) more tightly than other homo-DNA sequences. 19,22,44 At the same time, C can base-pair with G, which may also contribute to its stronger affinity on Tb 3+ /GMP. At this moment, it is unclear which mechanism played a more important role.
In general, FAM-T 15 was adsorbed more tightly on Tb 3+ / GMP than FAM-A 15 , as its signal was much lower. This suggests that in addition to DNA phosphate interactions, DNA bases may also contribute. For example, G/T can form a wobble base pair. At the same time, T 15 induced more FAM-A 15 desorption than A 15 ( Figure 4D), and A 15 induced more FAM-T 15 desorption than T 15 ( Figure 4E). This suggests that DNA hybridization also played a role in DNA desorption.
The effect of DNA length was then studied using poly-C DNA ( Figure 4F,G). C 15 and C 30 induced the highest desorption, followed by C 10 , whereas no desorption was observed with C 5 . Note that the amount of DNA desorbed by C 30 remained the same as that by C 15 . Considering that C 30 has double the number of nucleotides (at the same DNA molar concentration) compared to C 15 , it is likely that C 15 was already in a favorable configuration for DNA adsorbing on Tb 3+ /GMP, and even longer poly-C DNA could not further improve the affinity.
Fine Tuning Affinity by Mixing AMP/GMP. It seemed that changing from GMP to AMP had a huge influence on the DNA adsorption affinity of the CPs. We suspected that we might be able to fine-tune the interactions by mixing AMP and GMP at different ratios. To test this idea, we prepared a series of CPs by using GMP/AMP mixtures. We adsorbed FAM-A 15 on these CPs, and nonlabeled C 15 or T 15 was added to desorb the FAM-A 15 DNA ( Figure 5A,B). In both cases, the more the GMP added, the more the DNA desorbed, and a roughly linear relationship was observed ( Figure 5C). This confirmed that we could indeed tune DNA adsorption affinity by using their mixtures.  DNA Sensing. After the aforementioned fundamental studies, we then tested these CP nanoparticles as a sensing platform for DNA detection. As pre-adsorption of probe DNA did not seem to offer sufficient selectivity (e.g., all DNA sequences can induce probe desorption), we used the prehybridization strategy as shown in Figure 6A. 45−47 Here, the probe was a FAM-labeled 24-mer DNA with a random sequence. We first successively hybridized various concentrations of the target complementary DNA (cDNA) with the probe for 10 min, to which Tb 3+ /AMP or Tb 3+ /GMP was added. The kinetics of signaling on Tb 3+ /GMP ( Figure 6B) and Tb 3+ /AMP ( Figure S1A) were followed. The final fluorescence was higher with a higher concentration of the target DNA, suggesting that adsorption of duplex DNA was less favorable. The kinetics of adsorption was quite fast and the system reached a stable signal in just a few minutes. This cDNA concentration-dependent fluorescence indicates potential sensing applications.
We quantified the relative fluorescence enhancement at 10 min after adding the Tb 3+ /GMP CPs (Figure 6D), and a linear relationship was observed up to 30 nM DNA. We also measured the detection limits of these sensors based on the 3σ/slope calculation (σ = background variation in the absence of cDNA) to be 0.9 nM DNA for Tb 3+ /GMP. This is comparable to most nanomaterial based DNA sensors. 13,48−50 The selectivity was further tested (Figure 6C), where 100 nM nonlabeled 24-mer DNA did not inhibit probe adsorption and the fluorescence still dropped, suggesting that DNA hybridization forming a duplex was required for the inhibited probe adsorption. We also tested the same sensing method with Tb 3+ /AMP ( Figure S1B,C). As it has stronger DNA adsorption affinity, the difference between single-stranded and double-stranded DNA was smaller, and the sensitivity was lower. Finally, we also tested the Tb 3+ /GMP based assay in a lake water sample ( Figure S1D), also confirming its selective recognition of the target DNA.

■ CONCLUSIONS
In summary, we systematically studied DNA adsorption and desorption from various Ln 3+ /nucleotide CP nanoparticles using AMP and GMP as metal ligands. We discovered that DNA was adsorbed more tightly by Ln 3+ /AMP. The phosphate backbone binding was very important for DNA adsorption onto these CPs, although DNA bases also contributed to adsorption. Urea, proteins, and surfactants were more effective in displacing DNA from Tb 3+ /GMP than from Tb 3+ /AMP. By mixing AMP and GMP at various ratios, we continuously tuned the affinity of DNA adsorption. Using FAM-A 15 as a pre-adsorbed probe, C 15 induced more probe desorption than T 15 from Tb 3+ /GMP, suggesting that C 15 adsorption by Tb 3+ /GMP might be more favorable than its hybridization between T 15 and A 15 . Tb 3+ /GMP exhibited higher probe DNA detection sensitivity. Considering the modular nature of such CP materials, it is likely that one could also mix other components including metal ions and different lengths of phosphate (e.g., AMP and ATP) to further fine-tune material properties. These fundamental understandings are valuable for the designing and optimization of materials and devices based on these CP nanoparticles.

■ MATERIALS AND METHODS
Chemicals. All DNA samples were from Integrated DNA Technologies (IDT, Coralville, IA). The DNA sequences were Preparation of Nucleotide/Lanthanide CPs. The CPs made of Ln 3+ /AMP and Ln 3+ /GMP were synthesized according to a previously reported method. 28 Typically, 1 mL of LnCl 3 aqueous solution (5 mM) was added to 1 mL of AMP or GMP disodium salt solution (10 mM) dissolved in HEPES buffer (100 mM, pH 7.6) under stirring, and white precipitates were formed immediately. After stirring for 2 h at room temperature, the CP products were collected by centrifugation at 14 000 rpm for 10 min followed by washing three times with water and centrifugation. Finally, the CPs were dispersed in 1 mL of water and stored at 4°C for use. Tb 3+ /(AMP + GMP) CPs with different ratios of AMP/GMP were prepared using the same method.
TEM, SEM, and Dynamic Light Scattering (DLS). TEM was performed on a Philips CM10 microscope. An aqueous droplet containing 100 μg/mL of Tb 3+ /AMP or Tb 3+ /GMP dispersion was placed on a 230 mesh holey carbon copper grid. After 30 s, the excess solution was removed by a piece of filter paper. SEM was performed on a JSM-6330F (acceleration voltage, 15 kV). After drying the CP deposited filter membranes in vacuum, they were coated with Pt using a Hitachi E-1030 ion sputter before imaging. The ζ-potential of the CP nanoparticles was measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS90 system with a He−Ne laser (633 nm) at 90°collecting optics at 25°C. In a typical experiment, CPs were dispersed in 10 mM HEPES buffer (pH 7.6).
DNA Adsorption. The kinetics of DNA adsorption were investigated by adding different concentrations of Ln 3+ /AMP or Ln 3+ /GMP with a molar ratio of 2:1 to 100 μL of solution containing 20 nM FAM-labeled DNA in buffer A (10 mM HEPES, pH 7.6, 100 mM NaCl, and 1 mM MgCl 2 ) at 25°C. Different salt concentrations were also tested. The fluorescence was measured using a microplate reader (Infinite F200 Pro, Tecan) with 490 nm excitation and 520 nm emission. To test DNA desorption, a 500 μL solution containing Ln 3+ /AMP or Ln 3+ /GMP (2 mg/mL) was respectively incubated with 200 nM FAM-A 15 or FAM-T 15 in buffer A in the dark at room temperature for 1 h. Then these solutions were washed with buffer A by centrifugation at 15 000 rpm for 10 min and they were finally dispersed in 500 μL of buffer A and stored at 4°C (called sensor solution).
DNA Desorption. To study DNA desorption, 10 μL aliquots of the above sensor solutions were separately centrifuged, and the supernatant was removed. The pellets were then dispersed in 100 μL of 4 M urea, or 10 mM phosphate, and the fluorescence intensity was measured. To study DNA desorption induced by proteins and surfactants, each well contained 10 μL of the above sensor solution and 90 μL of buffer A (10 mM HEPES, pH 7.6, 100 mM NaCl, and 1 mM MgCl 2 ). Then different concentrations of BSA or surfactant (SDS, Tween 80, and CTAB) were added to induce desorption. To displace the adsorbed DNA by DNA, 10 μL of the above DNA sensor solutions was added to each well containing 90 μL of buffer A. Then a final concentration of 0.4 μM nonlabeled DNA homopolymers (A 15 /T 15 /C 15 /G 15 ) was added. To study the effect of DNA length, a final concentration of 0.4 μM C 5 , C 10 , C 15 , and C 30 was added using the same method.
DNA Sensing. In a typical DNA assay, 50 μL of the fluorescent probe FAM-24-mer DNA (20 nM) was hybridized with different concentrations of target c-24-mer DNA in buffer A for 10 min, to which 2 μL of Tb 3+ /AMP or Tb 3+ /GMP (150 μg/mL) dispersion was added. The kinetics of fluorescence change was then measured using the microplate reader.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01217.
DNA sensing using Tb 3+ /AMP as the probe adsorbing material (PDF)