Design of Metal-Organic Frameworks-Based Nanoprobes for

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Design of Metal-Organic Frameworks-Based Nanoprobes for Multicolor Detection of DNA Targets with Improved Sensitivity Shuai Wu, Chao Li, Hai Shi, Yue Huang, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02127 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Analytical Chemistry

Design of Metal-Organic Frameworks-Based Nanoprobes for Multicolor Detection of DNA Targets with Improved Sensitivity Shuai Wu,† Chao Li,† Hai Shi,† Yue Huang,‡ Genxi Li*,†,ǁ †

State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life

Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China ‡

College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China

ǁ

Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R.

China ABSTRACT: Metal-organic frameworks (MOFs) receive more and more interests in the field of analytical chemistry for their diverse structures and multifunctionalities. In this study, we have designed and fabricated nanoscale MOFs-based nanoprobes for multicolor detection of DNA targets with improved sensitivity. To do so, MOFs-based nanoprobes, constructed by using porous MOFs as scaffold to load signal dyes and DNA hairpin structure as capping shell, have been prepared. Once introduction of target, competitive displacement reaction triggers the release of fluorophores from the MOFs’ pores. Consequently, a significantly enhanced fluorescence signal can be observed owing to the high loading capacity of MOFs. Therefore, the stimuli-responsive nanoprobes can enable sensitive detection of DNA targets with a low detection limit of 20 fM and selective identification to discriminate single-base mismatch. Moreover, the MOFs can encapsulate different fluorophores with different DNA gatekeepers designed according to the sequence of target DNA, resulting in more kinds of stimuli-responsive nanoprobes for multiplexed DNA analysis in the same solution. Furthermore, these smart nanoprobes reported in this paper may provide a unique MOF-based tool for detection of various targets via stimuli-responsive systems in the future to widen the applications of MOFs.

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Metal-organic frameworks (MOFs), an emerging class of hybrid porous materials self-assembled from metal-containing nodes and organic linkers by strong bonds, have attracted tremendous attention in the past two decades.1 Ordered nanoporous systems with thermal stability and adaptable surface chemistries have enabled their implementation to many applications, including gas storage and separations,2 catalysis,3,4 chemical sensing,5 and an increasing amount of potential biomedical applications.6 At the initial stage, MOFs related to biomedical applications are focused on their use as drug nanocarriers7 or molecular imaging probes8 due to their highly tunable nature, good biocompatibility and extraordinary capacity for hosting drugs. Since MOFs possess organic components with various chemical moieties (e.g. amino or azide group), great interest has been paid for precisely covalent grafting biomolecules such as nucleotides,9 peptides,10 antibodies,11 and enzymes12 to generate a new breed of biofunctional materials. Therefore, the tailored bio-related MOFs may exhibit great potential to achieve molecular recognition and signal transduction for biosensing applications in recent years.13 The currently reported MOFs-based biosensing primarily relies on the optical properties14,15 or catalytic properties16-18 of MOFs. However, these MOFs-based biosensors also meet some challenges for further applications. For instance, a limitation is their relatively poor sensitivity. The specificity and mimic enzymatic activity of MOFs are not as strong as expected with existing natural enzymes, leading to weak detecting signals.19 Likewise, when used as fluorescent quenching materials, a signal normally labeled with one biomolecule only responds to one recognition event.20 In addition, the noncovalent interaction between biomolecules and the MOFs is not stable enough, affecting the performance of detection. Therefore, it is desirable to design more sensitive and stable MOFs-based biosensors for bioanalysis. The unique advantage of the MOFs is in possession of very large specific surface area with well-defined porosity, which means that MOFs can hold a large number of guest molecules.21 What’s more, external stimuli can well control the release of internal molecules.22-25 Taking full advantage of these superiorities, we speculate that porous MOFs hold great promise to perform as novel alternative nanocarriers for the fabrication of biosensors to make up for these deficiencies. Herein, we present a MOFs-based strategy that allows DNA analysis in a homogeneous solution with significantly improved sensitivity. More importantly, since a variety of probes can be formed when the MOFs encapsulate different signal molecules and functionalized with different DNA structures, our method is able to achieve the increasing demand for multiple DNA detection.26-28 Besides, the strategy proposed in this work may offer an excellent signal transduction platform for the multiple detection of DNA, it can also be integrated with other recognition elements as gatekeepers to broaden its applications in disease diagnosis. EXPERIMENTAL SECTION Materials and Instruments. All DNA sequences were ordered and HPLC purified from Sangon Biotechnology Co., Ltd (Shanghai, China) and are shown in Supporting Information Table S1 and S2 respectively. ZrCl4, aminoterephthalic acid (NH2-BDC), dimethyl sulfoxide

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(DMSO), Tris (2-carboxyethyl) phosphinehydrochloride (TCEP) and N, N-dimethylformamide were purchased from Sigma Aldrich. Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) were purchased from Thermo Fisher. Fluorescein (Flu) and rhodamine 6G (Rho 6G) were provided by Sangon Biotechnology Co., Ltd (Shanghai, China). The reagent of Cyanine5 carboxylic acid (Cy5) was purchased from Little-PA Sciences Co., Ltd. (Wu Han, China). All other chemicals used were of analytical grade without any further purification. Deionized water obtained from a Millipore water purification system (Milli-Q, ≥18.2 MΩ) was used throughout the experiments. The transmission electron microscope (TEM) images of MOFs were recorded on a Tecnai G2 F20 S-TWIN with an accelerating voltage of 200 kV. Powder X-ray diffraction (PXRD) data was measured by a D8 Advance diffractometer (Bruker, Germany). The nitrogen isotherms of MOFs were obtained on a Micropore & Chemisorption Analyzer (Micromeritics ASAP2020, USA) at 77 K. FT-IR spectroscopy was measured by a NEXUS640 infrared spectrometer system (NICOLET). Zeta potentials were carried out on a NanoBrook 90Plus instrument (Brookhaven, USA). Fluorescence emission measurements were performed using F-7000 spectrometer (Hitachi, Japan). The UV-vis spectra were recorded with a UV-1800 spectrophotometer (Shimadzu, Japan). Synthesis of MOFs (UiO-66-NH2). The MOFs were prepared according to a previously reported procedure with some modifications.29 Briefly, 240 mg of ZrCl4, 220 mg of aminoterephthalic acid and 3.8 g benzoic acid were dissolved in 20 mL of DMF with ultrasound for about 3 min. Then the mixture was transferred to a Teflon-liner and was kept in an oven at 120 °C under static conditions. After 20 h, the mother liquor was cooled to room temperature and the precipitates were isolated by centrifugation for 10 min at 10500 rpm. The resulting solid was respectively washed with fresh DMF and absolute ethanol for 10 min to remove the residual reaction precursors, repeat for 3 times. Finally, the solids were dried at 100 °C under reduced pressure overnight for later use. Preparation of DNA-Functionalized MOFs. To prepare monodispersed MOFs-based nanoprobes. Firstly, 1 mg of MOFs was placed into 450 µL HEPES buffer (20 mM, pH 7.2) and sonicated for 5 min. Then 50 µL of Sulfo-SMCC solution (10 mM) was added and mixed for 2 h at room temperature, MOFs-SMCC conjugates were formed, and an excess amount of free Sulfo-SMCC was removed by centrifugation (8000 rpm, 5 min). The resulting products were redissolved in 450 µL HEPES buffer (20 mM, pH 7.2), the maleimide portion of the crosslinker is available for reaction with freshly reduced and purified thiolated Arm-DNA (0.6 µM) under gentle shaking. After 4 h, the MOFs were washed by centrifugation (6000 rpm, 3 min) for three times to remove unbound DNA and dispersed with 950 µL aqueous solution. Loading the Cargo and Capping the Pores. To load the cargo, 50 µL aqueous 10 mM Flu (dissolved with a handful of DMSO before using) solution was added to the above-mentioned mixture that was allowed to 12 hours of incubation while continuous stirring. Afterwards, 0.8 µM hairpin DNA solution was added to the resulting reaction mixture containing 100 mM NaCl to form the duplex DNA structures as the gates

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Analytical Chemistry Scheme 1. Schematic Diagram for A) the Fabrication Process of MOFs-Based Nanoprobes and B) Multicolor Detection of DNA Targets

to lock the dyes inside the pores. Finally, the MOFs were washed at least five times with HEPES buffer until negligible background fluorescence was observed. Preparation of other dyes-entrapped stimuli-responsive nanoprobes was similar to above method. Dyes Release Experiments and Multicolor Detection of DNA. To investigate the release process of the dyes from the DNA-capped MOFs, 0.1 mg of the solid were suspended in 900 µL HEPES buffer (10 mM, 100 mM NaCl, pH 7.2). Additionally, 100 µL buffer containing different concentrations of DNA targets were respectively added to the samples, stirring in each case the final mixtures at room temperature. Aliquots of 150 µL were taken at several times and centrifuged 3 min at 6000 rpm (in order to remove the solid) and the fluorescence of the dyes released measured at appropriate excitation wavelengths. As for the multicolor detection of DNA sequences, three fluorescent dyes (Flu, Rho 6G and Cy5) were severally encapsulated into the pores to form different MOFs-probes capped with various hairpin DNA (P1, P2 and P3) and then were mixed in a solution (final concentration 30 µg/mL of each). Subsequently, DNA samples of different DNA triggers (T1, T2 and T3) were added to the mixture and reacted for 30 min. Subsequently, the fluorescence intensities were recorded after centrifuge operation. The Flu fluorescence was excited at 494 nm and recorded at 513 nm; the Rho 6G fluorescence was excited at 525 nm and recorded at 553 nm; and the Cy5 fluorescence was excited at 646 nm and recorded at 664 nm. RESULTS AND DISCUSSION Nanoprobes Design. The strategy of fabricating the nanoprobes based on stimuli-responsive MOFs is depicted in Scheme 1. As for MOFs, we selected UiO-66-NH2, a

representative type of zirconium (Zr) -based MOFs, basically consisting of Zr (IV) and 2-amino-1,4-benzenedicarboxylate (BDC-NH2), expecting its outstanding biocompatibility and successful surface modification.30,31 Initially, the scaffold UiO-66-NH2 was firstly synthesized, then Sulfo-SMCC was incubated with MOFs as covalent crosslinker oriented amine-to-sulfhydryl for further post-synthetic modification.32 After this step, short thiolated single DNA strands named Arm-DNA (A-DNA) were anchored on to the SMCC-functionalized MOFs. Dyes loading was accomplished by soaking MOF nanoparticles in a solution of fluorescein to allow the guest molecules to diffuse into the pores of the MOFs. In most cases, the switches should satisfy two requirements: possessing a certain conformation to serve as lock components and changing their shapes rapidly in response to external stimuli.33 So a single strand hairpin-DNA, the 3’-end of which is partially complementary with A-DNA was selected to be response elements for targets. Without targets, the stem-loop structure of hairpin DNA hindered the release of fluorescent dyes by reason of steric-hindrance effect. However, once introduction of the targets, they would hybridize with the hairpin-DNA, as a result, the heteroduplex separated from the surface of MOFs by binding-induced DNA strand displacement. Consequently, the targets-stimulated disassembly resulted in existence of only the short A-DNA, thus unlocking the MOFs and allowing the release of the signaling molecules. By observing the change of the signal, the DNA targets could be determined. At last, three different fluorescent dyes (Flu, Rho 6G and Cy5) accompanied with three tailored hairpin DNA were introduced to multiple DNA analyses.



Preparation of DNA-Gatekeeper Mechanized MOFs and Characterization. Amine-functionalized terephthalic acid was reacted with ZrCl4 to yield the porous MOFs. As shown in

Figure 1. A) PXRD patterns of simulated from the crystal structure of (a) UiO-66-NH2, (b) as-synthesized UiO-66-NH2, and (c) Flu-loaded, DNA-capped UiO-66-NH2. B) TEM image of MOFs. C) Nitrogen adsorption−desorption isotherm of UiO-66-NH2 and Flu-loaded, DNA-capped UiO-66-NH2. D) Zeta-potentials of (a) MOFs, (b) MOFs-SMCC and (c) MOFs-SMCC-DNA. Error bars were derived from three parallel experiments. Figure 1A, the powder x-ray diffraction (PXRD) patterns of the synthesized MOF nanoparticles with sharp diffraction peaks were closely consistent with those simulated from the single-crystal structure, which revealed the purity and high crystallinity of the MOFs. Transmission electron microscopy (TEM) analysis (Figure 1B) displayed that the as-synthesized MOFs showed a typical octahedral crystal structure with a particle diameter of 130-150 nm. The nitrogen adsorption-desorption isotherm (Figure 1C) of activated MOFs

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at 77 K was performed to confirm its permanently porous nature, with a specific surface area of 980 m2 /g and a pore volume of 0.59 cm3/g. The pore size distribution of MOFs calculated by DFT model showed two main sharp peaks at about 0.6 nm and 1.2 nm (Figure S1). After MOFs were modified with Sulfo-SMCC to present maleimide groups on the surface, thiolated DNA could be covalently linked to the surface for subsequent formation of caps. The process of surface functionalization was monitored by FTIR spectroscopy (Figure S2). Meanwhile, zeta-potential analysis clearly indicated charge variation of MOFs after post-synthetic modification. As shown in Figure 1D, initially, the MOFs with amine groups showed a positive potential of +25.8 mV, and covalent linkage of Sulfo-SMCC increased the positive charge. Finally, the MOFs reacted to thiolated A-DNA/hairpin-DNA hybrid to form negatively charged probes displaying a potential of -38.5 mV. Feasibility of Sensing Method. To investigate the feasibility of the “on-command” sensing system, the dyes of fluorescein were firstly used as model substrates for guest molecules. The molecule size of Flu is calculated to be 9.5 * 8.0 Å, which facilitates the process to diffuse into the pores of UiO-66-NH2 (Figure S3). After the hybridization step of A-DNA and hairpin-DNA, the excess DNA was removed by washing and centrifugation. The surface area and pore volume of MOFs were decreased after Flu loading. As shown in Figure 1C, Flu-loaded MOFs exhibit a much lower BET surface area (286 m2 /g) and the pore volume is down to 0.14 cm3/g, which reveals the successful adsorption of dyes in the pores. The amount of Flu in the certified material was calculated to be 51.36 nmol/mg MOFs via appropriate calibration curve (Figure S4) according to the difference in concentrations of the initial and left dyes, demonstrating the high loading ability of MOFs. And as shown in Figure 2A,

Figure 2. Release profiles of fluorescein dyes from MOFs probes A) capped with hairpin-DNA (blue dot-line), linear DNA without stem-and-loop structure (red dot-line) and only modified with short arm-DNA (black dot-line). B) in the presence of targets after 30 min utilizing hairpin-DNA gated MOFs probes. Error bars were derived from three parallel experiments.


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Figure 3. A) Fluorescent emission spectra of released fluorescein from MOFs upon addition of the target DNA with varying concentrations: (a) control, (b) 0.05 pM, (c) 0.2 pM, (d) 1 pM, (e) 5 pM, (f) 20 pM, (g) 100 pM, (h) 500 pM and (i) 1000 pM and (j) 2000 pM. B) Calibration curve based on variation of fluorescent signal at maximum emission wavelength versus DNA concentration; inset shows the linear relation of fluorescent response versus logarithm of DNA concentration. Error bars were derived from three parallel experiments.

negligible fluorescence was measured from MOFs in the absence of external stimuli (blue dot-line), while the fluorescence intensity of the MOFs only modified by A-DNA displayed an increasing trend over time (black dot-line). It could be interpreted that short single oligonucleotides were unable to be gatekeepers in solution and led to the leakage of dye molecules from the pores. In addition, in order to further explore the impact of the DNA structure on capping efficiency. Another ssDNA that complementary to A-DNA without stem-and-loop structure were designed to form the “bio-gates” of MOFs as comparison, but the effect was inferior to that of the hairpin-DNA (red dot-line). We attributed it to that the loops of the hairpin structure were close to each other generating the steric-hindrance effect to form a capping shell. Accordingly, the results forcefully confirmed that hairpin-DNA hybridization could act as an efficient cap for retention of guest molecules. Additionally, the responsive release of fluorescein was investigated by addition of target DNA (T-DNA) to the system of MOFs. Figure 2B showed the time-dependent fluorescence changes upon unlocking the caps. Within the first 30 minutes without T-DNA addition, the curve kept a very small change. However, in the presence of the targets, considerable amount of fluorescein leaked from the pores, generating a good response with an amplified fluorescence signal and the fluorescence intensity began to level off after 90 min, which is due to the liberation of the formed DNA hybrid from the nanocontainers by competitive displacement reaction. Therefore, based on the signal amplication by the controllable-release MOFs, the ultrasensitive detection of DNA could be achieved. Optimization of Experimental Conditions. To achieve the best performance of the assay, various experimental conditions were then optimized. The amount of signal molecules and the concentration of gatekeeper DNA would indirectly determine the analytical system. First, we employed different concentrations of the dyes to incubate with 1 mg/mL MOF nanoparticles. As shown in Figure S5, it was found that the dyes solution of 500 µM achieved the highest signal-to-background ratio, excess signal dyes may cause more physical absorption on surface that would reduce the sensitivity of this assay. Second, the concentrations of anchor

strands and hairpin strands were optimized, as shown in Figure S6, with the increased concentrations, the signal intensity increased and reached a plateau after 600 nM and 800 nM respectively, providing the best signal response. In addition, the concentration of crosslinker Sulfo-SMCC was optimized to be 1 mM. Besides, 90 min was chosen as the detection time point according to the aforementioned time dependent fluorescence change curve. Assay Performance. The above optimized method was applied to the detection of DNA targets. As shown in Figure 3A, the signal response could increase proportionally with the raised concentrations of target DNA. Figure 3B exhibits a good linear correlation of the fluorescent intensity changes at maximum emission wavelength to the logarithm of the concentrations ranging from 50 fM to 2 nM with a linear equation of Y = 982.9 + 501.0 lg C (where Y is the change of fluorescence intensity and C is the concentration of DNA target) and a squared correlation coefficient of 0.998. The limit of detection (LOD) in this work was determined to be 20 fM. This excellent sensitivity may be derived from the unique porous structure of MOFs to lock abundant of dye molecules, and only a trace of target molecules could be the “keys”

Figure 4. The selectivity of MOFs-based probes for the discrimination of target DNA (I), one-base mismatched DNA (II) and noncomplementary random DNA (III), curve IV displays the control group without DNA addition. The concentration of DNA is 1 nM.



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Figure 5. Fluorescence spectra for multiplexed DNA detection. Three probes of MOFs were mixed in a solution (P1, P2 and P3, final concentrations 30 µg/mL of each), probe mixture in the presence of different targets T1 (green, 0.5 nM), T2 (blue, 2 nM), and T3 (red,1 nM), with the excitation/emission wavelengths of (a) 494 nm/513 nm, (b) 525 nm/553 nm and (c) 646 nm/664 nm.DNA gates were designed according to the selected three kinds of target tumor-suppressor genes T1, T2, and T3 (exon segments of p16, p21, and p53 genes).

to trigger the releasing behavior. To further evaluate the specificity of our method, control experiments were conducted by using mismatched DNA and random DNA with same concentration of 1 nM. As shown in Figure 4, in comparison to the full complementary target, much smaller increase in fluorescence intensity was measured for single-base mismatched DNA and the signal readouts of non-complementary was largely close to that of blank control. All these results manifest that this controlled-release sensing system could be used for detecting DNA targets with good sensitivity and selectivity. Multiple DNA Detection. Simultaneous analysis of multiple targets is of great significance for molecular diagnosis. For instance, cancers are generally associated with mutations in a variety of tumor-suppressor genes that have been confirmed that testing of them plays a significant role in the early diagnosis of cancer.34 Given the excellent ability of the MOFs to wrap the signal molecules, multiple targets detection may be realized by using this sensing system. Considering the effect of dye-to-dye energy transfer, we selected three fluorescent reagents, Flu, Rho 6G, and Cy5 which each emits at 513, 553 and 664 nm respectively. All of them were able to locked into the interior of MOFs (Figure S7) by different DNA gates producing three stable MOF probes (P1, P2 and P3). The loading capacities of the other two signal dyes were calculated to 58.32 nmol/mg and 37.8 nmol/mg according to the standard curves (Figure S8 and S9). Intriguingly, as shown in Figure 5, the MOF probes responded only to the specific target and generated characteristic fluorescence at the corresponding wavelength. The addition of T1 (0.5 nM) only led to the specific emission at 513 nm, with minimal emission of P2 and P3 (Figure 5a). Analogously, the presence of T2 (Figure 5b) or T3 (Figure 5c) brought about the emission at 553 nm and 664 nm, respectively. All the data imply that this detection system can not only recognize target DNA with excellent selectivity, but also exhibits the power for the multicolor detection of DNA targets. To be noted, the MOFs-based multiple assay also improved sensitivity greatly compared to those of previously reported multiple detection strategies (listed in Table 1).

Table 1. Comparison of Assay Methods for Multiple DNA detection Materials used

LOD

Analytical methods

Ref

Quantum dots

100 pM

Fluorescence

35

Encoding metal ions

33 pM

Electrochemistry

36

Gold nanoparticles, silver nanoparticles, and gold nanorods

0.4 nM

Colorimetric

37

Transition metal dichalcogenide nanosheets

50 pM

Fluorescence

38

MOFs

20 fM

Fluorescence

This work

CONCLUSIONS In summary, we have designed new stimuli-responsive nanoprobes based on nucleic acid-functionalized MOFs to achieve direct, sensitive and multiple detection of DNA targets. In this study, molecular beacons with hairpin structure hybridized with the Arm-DNA were demonstrated to be able to effectively cap signal molecules within the pores of porous MOFs due to the effect of steric hindrance. We employ competitively DNA strand displacement to trigger the release of a great deal of dye cargo, achieving a large signal amplification and sensitive detections are ensured, the detection limit of the assay can reach 20 fM. Selectivity experiments showed excellent ability of this sensing system to discriminate mismatched DNA. More importantly, the unique properties of MOFs make it possible to integrate the two processes of multiple detection and signal amplification into one system, providing great versatility to develop biosensors. Moreover, the concept proposed in this work could be further extended to recognize variety of targets like proteins through replacing response elements into relative aptamers. With the merits, we believe that the stimulus-release process based on MOFs shows great promise for more applications in biomedical research in the future.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Oligonucleotide sequences, pore size distribution of the MOFs, FT-IR analysis of the MOFs, UV absorption calibration curves of the dyes, optimization of experimental conditions and nitrogen adsorption−desorption isotherm of the MOFs after loading dyes (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail address: [email protected], Fax: +86 25 83592510.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 81772593, 21235003).

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