Hierarchical Porous Fluorinated Graphene Oxide@MetalâOrganic Gel

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Biological and Medical Applications of Materials and Interfaces

Hierarchical-Porous Fluorinated Graphene Oxide@MetalOrganic Gel Composite: Label Free Electrochemical Aptasensor for Selective Detection of Thrombin Veronika Urbanova, Kolleboyina Jayaramulu, Andreas Schneemann, Stepan Kment, Roland A. Fischer, and Radek Zbo#il ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14344 • Publication Date (Web): 09 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

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ACS Applied Materials & Interfaces

Hierarchical-Porous Fluorinated Graphene Oxide@Metal-Organic Gel Composite: Label Free Electrochemical Aptasensor for Selective Detection of Thrombin Veronika Urbanová,†,# Kolleboyina Jayaramulu,†,‡,# Andreas Schneemann,‡ Štěpán Kment,† Roland A. Fischer,*,‡ Radek Zbořil*,†

†Regional

Centre of Advanced Technologies and Materials, Faculty of Science, Palacký

University Olomouc, Šlechtitelů 27, 783 71, Olomouc, Czech Republic

‡Department

of Chemistry and Catalysis Research Centre, Technical University of

Munich, Ernst-Otto-Fischer-Straße 1, 85748 Garching, Germany

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KEYWORDS: fluorinated graphene oxide (FGO), metal-organic gel (MOG), hierarchical pores, electrochemical aptasensor, thrombin


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ABSTRACT: Current research effort aims at developing and designing new sensing platform architectures for effective assaying biological targets that are significantly important for human health care and medical diagnosis. Here, we proposed a novel nanostructured sensor based on the combination of fluorinated graphene oxide and ironbased metal organic gel (FGO@Fe-MOG). The unique properties including hierarchical porosity along with excellent electron transfer behavior, make it an ideal candidate for electrochemical sensing of thrombin with superior detection limits compared to other (electrochemical, fluorescence, colorimetric) strategies. Specifically, thrombin-binding aptamer (TBA) was immobilized onto FGO@Fe-MOG through strong electrostatic interaction without any special modification or labeling, and the electrochemical impedance spectroscopy was used as the analyzing tool. The introduced aptasensor revealed high selectivity and reproducibility toward thrombin with the detection limit of 58 pM. The effectiveness, reliability, and real applicability of the proposed FGO@Fe-MOG nanohybrid were also confirmed by the determination of thrombin in a complex biological matrix represented by human serum. Taking into account the superior detection limit; high 3 ACS Paragon Plus Environment


selectivity; reproducibility; and precision, the developed scalable and label-free aptasensor meets the essential requirements for clinical diagnosis of thrombin.


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1. Introduction

Increased health care requirements have resulted in the need to develop sensitive, reliable, and cost effective tools for the quantitative detection of biomolecules relevant in medical diagnosis, pathogen detection, and biomedical research.1–5 At the same time, recent advances in nanotechnology have generated a variety of nanomaterials with unique intrinsic properties that can be leveraged for the improvement of biomedical application.6,7 One general advantage of nanomaterials is their high surface to volume ratio, which enables the immobilization of an enhanced amount of bioreceptor units useful for binding and sensing biomolecules.8 Moreover, nanomaterials can be relatively easily synthetically modified with appropriate functional groups to tailor their physico-chemical as well as their structural and dimensional properties. In turn, such nano-engineered platforms offer reproducible immobilization of bioreceptor units, enhanced sensitivity for targets, and increased biocompability.8–10 Aptamers are single-stranded nucleic acid (ssDNA) molecules known as promising alternative to antibodies with potential applicability in immunoassays and biotechnology.11



Aptamers display high binding affinity toward their targets, which is derived from their capability of folding in response to binding with their target molecules.12,13 These properties make aptamers ideal tools for diagnostics, therapeutics, and sensing.14,15 To date, a wide range of aptamers with prominent selectivity and high reproducibility have been designed and synthetized along with the target range from small organic molecules and ions, through small peptides, to whole cells and viruses.16,17 As a consequence, their biomolecular recognition ability makes them attractive for the fabrication of highly efficient biosensors. Thrombin, a serine protease, is a key element in the coagulation process, which converts circulating fibrinogen into a fibrin, the building matrix of blood clots.18 Notably, thrombin can also serve as a biomarker for diagnosis of some diseases, including arterial thrombosis, pulmonary metastases, and diseases associated with coagulation abnormalities.19 In virtue of its biological importance, the development of biosensors toward thrombin detection with high sensitivity and selectivity represents a great challenge for fundamental research, and, therefore, assays for its detection are attracting increasing attention. The 6 ACS Paragon Plus Environment

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15-mer thrombin-binding aptamer (TBA) was first described in 1992 and its structure has been well characterized and studied since then.20 Upon folding into an antiparallel chair like G-quadruplex structure with two G-tetrads surrounded by a TGT loop on one side and two TT loops on the opposite side, this DNA oligonucleotide can strongly and selectively recognize the fibrinogen-binding exosite I of thrombin.21 Therefore, the TBA has been extensively explored, coupled to biosensors for thrombin detection, and combined with different methods for the signal read-out, colorimetry,22,23 fluorescence quenching,24 or surface-enhanced Raman scattering (SERS).25 However, these methods are limited by the complexity of the detection process repeatidly requiring multiple binding steps and using specific labels. However, electrochemical detection26–29 using tailored nanostructure sensing platform could overcome these limitation. Indeed, the employment of nanomaterials with hierarchical porosity will increase the electrode surface, leading to a higher number of immobilized aptamers. Thus, the sensitivity of such sensor will be consequently enhanced without any special amplification strategy. Recently in the field of porous materials science, metal-organic frameworks (MOFs) have been recognized to exhibit elevated specific surface areas and higher crystallinity.30–34 7 ACS Paragon Plus Environment


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MOFs are unique in terms of their extraordinary abundant, tunable pores with various functional site, compared to other traditional carbon and inorganic zeolite materials.35-36 The MOFs have been synthesized by various methodologies; however, the synthesis of meso/macroporous MOFs preserving large surface area still remains a big challenge.31 Nevertheless, coordination polymer gels should enable an adaptable approach to the synthesis of porous materials of the desired shape. Such materials combine both a significant

surface

area

and

surface

accessibility

due

to

their

hierarchical

(micro/meso/macroporous) structure, and, therefore, they can be used as catalyst supports or catalysts as such.37–41 MOGs have been synthesized in analogy to MOFs by combining organic linkers and inorganic metal ions, retaining the MOF backbone and keeping its inherent structure after removal of the synthesis solvent.38 Besides their previously mentioned improved mass transfer due to their micro/mesoporous nature, MOGs can also respond to its environment including the change in the pH, light, or temperature, making them interesting candidates for sensing applications.42 Due to these desirable and unique properties, MOGs have


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already showed the potential for a wide range of applications including adsorption, sensing, catalysis, or chiral separation.39–41 In this work, we report a facile and scalable synthesis of a conductive hierarchically porous redox active hybrid based on the combination of fluorinated graphene oxide and iron-based metal organic gel (FGO@Fe-MOG). In this case, fluorinated graphene oxide (FGO) acts as a structure directing agent for the stabilization and shape control of ironbased metal organic gel (Fe-MOG) nanoparticles. The FGO controls the nucleation of the Fe-MOG via coordination modulation through surface oxygen groups. In order to support the relevance of this original nanostructured material, label free electrochemical detection of thrombin was also performed. This approach links the unique properties of the FGO@Fe-MOG hybrid material concerning its hierarchical porosity and electrostatic attraction toward the target molecule. Our nanostructured sensing platform enabled fast, simple, and reproducible detection of unlabeled thrombin with high specificity and detection limit at picomolar concentration level. 2. Materials and Methods



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2.1. Chemicals. Iron(III) nitrate nonahydrate, trimesic acid (BTC), sulphuric acid, phosphoric acid, hydrochloric acid, potassium permanganate, hydrogen peroxide, ethanol, methanol, dimethylformamide, fluorinated graphite, potassium ferricyanide (K3[Fe(CN)6]),

potassium

ferrocyanide

(K4[Fe(CN)6]),

sodium

chloride,

tris(hydroxymethyl)aminomethane (TRIS), sodium phosphate dibasic, sodium phosphate monobasic, thrombin from human plasma, bovine serum albumin (BSA), immunoglobulin G from rabbit serum (IgG), and streptavidin were purchased from Sigma-Aldrich. Thrombin-binding aptamer (TBA, 5′-GGTTGGTGTGGTTGG-3′) HPLC purified and lyophilized was provided by Generi Biotech (Czech Republic). 2.2. Synthesis of FGO@Fe-MOG. Typically, 1 mmol of Fe(NO3)3∙9H2O was dissolved in methanol (5 mL), then mixed with 5 mL methanolic solution of trimesic acid (H3BTC, 0.75 mmol) and the obtained mixture was stirred for 15 minutes at room temperature. Afterward, the transparent liquid was sealed into a glass container heated to 100 °C and after ten minutes an orange gel was formed. Subsequently, the wet gel was dried at 60 °C overnight.


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2.3. Preparation of Fluorinated Graphene Oxide (FGO). Fluorinated graphene oxide (FGO) was prepared by chemical oxidation and exfoliation of fluorinated graphite (FG) under acidic condition according to a slightly modified version of Hummers’ method.43 1 g of FG was dissolved in a mixture of H2SO4 (90 mL) and H3PO4 (10 mL). After vigorous stirring at 60 °C for two hours, 4.5 g of KMnO4 were added slowly to the reaction mixture and the mixture was stirred continuously at 90 °C overnight. Subsequently, ice was added to the mixture followed by the addition of 30% H2O2 (2.5 mL). The resulting FGO was filtered off, dispersed in tetrahydrofuran, centrifuged (6000 rpm), and finally washed with HCl and ethanol. The resulting solid was dried in the oven at 60 °C. 2.4 Preparation of FGO@Fe-MOG. The FGO@Fe-MOG composite was prepared and adopting from literature.39100 mg of FGO was exfoliated in dimethylformamide (5 mL) under sonication for one hour. Then, methanolic solution of Fe(NO3)3∙9H2O (1 mmol) and trimesic acid (0.75 mmol) was added into a suspension of FGO and stirred at room temperature for ten minutes. The resulting transparent liquid was then sealed into a glass container and heated to 100 °C.



Subsequently, after ten minutes a black gel formed, and was dried at 60 °C overnight to obtain powder used for further analysis. 2.5. Electrochemical Measurements. All electrochemical experiments were performed using a PGSTAT128N potentiostat (Metrohm Autolab B.V.) monitored by NOVA software. Disposable electrically printed electrodes (DEP-chips, EP-PP model) consisting of a three-electrode system including a carbon-based working electrode, Ag/AgCl reference electrode, and a carbon-based counter electrode were used in the electrochemical setup, and were obtained from Biodevice Technology (Nomi, Japan). For some measurements, glassy carbon electrodes (GCEs) were used as the working electrodes. All electrochemical impedance spectroscopy (EIS) measurements were performed in TRIS-buffered saline (TBS, pH 7.4) containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1 molar ratio) as a redox probe. Impedance measurements were recorded between 0.1 MHz and 0.1 Hz at 10 mV at sinusoidal voltage amplitude and at room temperature. 2.6. Modification of electrode surface (i.e., DEP-chips or glassy carbon electrodes) The DEP chips (or glassy carbon electrodes, GCEs) were modified by drop-coating of water/ethanol (1:1) dispersion of the respective materials (FGO, Fe-MOG, or FGO@Fe12 ACS Paragon Plus Environment

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MOG). First, the dispersion (1 mg mL–1) was sonicated for 10 min and then 3 μL was drop-coated onto the working electrode surface and allowed to dry at room temperature. The excess of the material was removed from the electrode surface by gentle rinsing with Milli-Q water. 2.7. Immobilization of TBA (5′-GGTTGGTGTGGTTGG-3′) and Thrombin Detection. The thrombin-binding aptamer (TBA) was immobilized onto the surface of either nonmodified or modified DEP chip by dry physical adsorption. 3 µL of the TBA in PBS buffer solution (pH 7.0) at the optimum concentration of 10 µM was drop-coated onto the electrode surface and allowed to dry at 60 °C for ten minutes. Subsequently, the electrode was washed three times in PBS buffer solution at room temperature and afterward gently stirred in order to remove the excess of the non-adsorbed aptamer. The DEP chips modified with TBA were afterward incubated with the desired concentration of thrombin (THR) in TRIS buffered saline for 1 h at 37 °C under gentle stirring. Three washing steps were then performed in the PBS buffer solution. Blank experiments were performed under the same protocol mentioned above using IgG, BSA, or streptavidin instead of thrombin. 13 ACS Paragon Plus Environment


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3. Results and Discussion 3.1. Synthesis and Structural Characterization of FGO@Fe-MOG. Recently, we focused on the synthesis of metal-organic frameworks (MOFs) and graphene oxide composites for energy related applications and environmental technologies because the integration of these two fascinating classes of materials yields new and unexpected synergistic properties.39,44,45 The Fe-MOG is synthesized by simple mixture of methanolic solution of trimesic acid ( BTC) and Fe(NO3)3∙9H2O in 20 ml glass vial and heated at 100 oC (see supporting information for complete details and optical photographs of gels as shown in Figure S1). Fe-MOG is built up by interconnected nanoparticles of the metalorganic

framework

MIL-100

(Fe)

(MIL

=

Material

Institute

de

Lavoisier,

Fe3X(H2O)2O(BTC)2, X = F-, OH-, BTC3– = 1,3,5-benzenetricarboxylate Figure S2).46 A hypothetical Fe-MOG gelation mechanism is shown in Scheme 1. In its initial step, the formation of Fe-BTC occurs, which is composed of chains of the trinuclear iron oxo cluster building unit connected with the BTC by metal-ligand coordination.38 These Fe-BTC chains agglomerate to form MOF nanoparticles (Fe-BTC NPs) in the second step. In the 14 ACS Paragon Plus Environment

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final step, these Fe-BTC NPs self-assemble, which results in porous MOGs through coordination perturbation.38 Typical powder XRD of the Fe-MOG shows several broad reflections with low crystallinity. The observed reflections are shown in Figure 1A and match with MIL-100 (Fe) simulated PXRD pattern.46

Scheme 1. Schematic illustration of the formation of iron-based metal-organic gel (FeMOG) and its hybrid with fluorinated graphene oxide (FGO@Fe-MOG).



Figure 1. (A) X-ray powder diffraction patterns simulated pattern of MIL-100 (Fe)(black curve), Fe-MOG (red curve), and FGO@Fe-MOG (blue curve). (B) FT-IR spectra of corresponding FGO, Fe-MOG, and FGO@Fe-MOG samples. Nitrogen adsorptiondesorption isotherm measured at 77 K (C) Fe-MOG; (E) FGO@Fe-MOG. Pore size distribution of (D) Fe-MOG; (F) FGO@Fe-MOG.


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Herein, a simple preparation to prepare novel hybrid nanocomposites of fluorinated graphene oxide and iron-based metal-organic gel Fe-MOG is presented (FGO@FeMOG). The powder XRD pattern of hybrid FGO@Fe-MOG shows broad reflections and that can be indexed to different planes of pristine MIL-100(Fe) ( Figure 1A). The Fourier transform infrared (FT-IR) spectrum of the Fe-MOG exhibits stretching frequencies of Fe-O and carboxylate at 758 cm–1 and 1523 cm–1 respectively, where iron metal centres are coordinated with BTC organic linkers (Figure 1B). It should be noted the band at 950 cm–1

is accredited to bridging µ-OH groups and clearly supports the MIL-100(Fe)

coordination environment.

Further, The FT-IR spectroscopy analysis of hybrid of

FGO@Fe-MOG clearly shows stretching frequency at 1224 cm-1 corresponding to the covalent C-F bond along with significant bands of pristine Fe-MOG. 39 Figure 1C shows nitrogen adsorption-desorption isotherms of Fe-MOG have typical type-IV isotherm. The steep uptake of nitrogen at low pressure region (up to P/P0~ 0.4) suggesting the Fe-MOG contains high degree of micro pores. The BET and Langmuir surface area of the Fe-MOG area about 2109 m2/g and 2900 m2/g respectively. The N2 adsorption isotherm exhibites wide pore size distribution centered at 0.5 to 20 nm as 17 ACS Paragon Plus Environment


calculated using Non-Localized Density Functional Theory (NLDFT) method (Figure 1D and denoted as A, these pores originated from within the MOG nanoparticle as shown Scheme 1). The resultant FGO@Fe-MOG hybrid material preserved the type-IV isotherm of the meso porous materials. The BET and Langmuir surface areas of 672 and 1100 m2/g (Figure 1E), respectively. The pore size distribution reveals the pores are in the range from 2 to 80 nm (Figure 1F). The textural parameters suggest the presence of three types of pores: (a) the inherent micro/meso pores of the MOF NPS, (b) the mesopores formed by the aggregation of MIL-100(Fe) NPs (0.5–20 nm corresponding to Fe-MOG pores type A, and (c) macropores formed at the interface between the Fe-MOG and the FGO 20–80 nm type B as shown in Scheme 1. Therefore, the introduction of the fluorinated graphene oxide disrupts the MIL-100(Fe) crystal growth, which enhances mismatch growth over oriented crystallization, leading to these resultant macro pores in the FGO@Fe-MOG. The morphology and the chemical arrangement of FGO@Fe-MOG nanohybrid were characterized various microscopy methods and elemental mapping by energy dispersive spectroscopy (EDS). The SEM micrographs of pure Fe-MOG clearly show the existence 18 ACS Paragon Plus Environment

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of a sponge like morphology (Figure 2A). In contrast the SEM micrographs of FGO@FeMOG exhibit a bundle of uniform octahedral crystals with the size ranging from 500 nm to 2 µm covered by FGO layers (Figure 2B,C). The TEM measurements clearly confirm that some of the Fe-MOG nanoparticles are well intercalated between the FGO layers (Figure 2D–F). The typical HAADF-TEM images of the hybrid revealed a uniform distribution of Fe, C, F, and O over the whole nanocomposite (Figure 2G–J). The shape and the morphology of the composites drastically differ from the pristine Fe-MOG and FGO, strongly suggesting that the FGO acts as a selective nucleation center conducting the precise growth of the MOF nanocrystals, controlling the morphology, and, more importantly, enhancing the MOF crystallinity.



Figure 2. Scanning electron micrographs of (A) Fe-MOG and (B,C) FGO@Fe-MOG. HRTEM images of (D–F) FGO@Fe-MOG along with EDS elemental maps for (G–J) Fe, C, F, and O.


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The X-ray photoelectron spectroscopic (XPS) measurements of FGO@Fe-MOG show sharp signals for Carbon, Oxygen, Fluorine, and Iron atoms (Figure 3). and an atomic content of 22% of fluorine was determined. The characteristic Fe peaks at binding energies of 530.14 eV were observed corresponding to the Fe 2p3/2 signal of an ironoxide (Fe-O) bond (Figure S3). The F1S deconvoluted spectra shows covalent behavior of C-F bond in the composite, which was not changed upon the synthesis procedure (see Figure 3C). Therefore, in the FGO@Fe-MOG hybrid, Fe-BTC nanoparticles undergo controlled growth and are selectively chelated with oxygen functionalities of FGO sheets which provide free C-F pendant groups.



Figure 3. XPS spectra of FGO@Fe-MOG: (A) Survey, (B) high resolution C1s XPS spectrum, (C) high resolution F1s XPS spectrum, and (D) high resolution O1s XPS spectrum.

3.2. Electrochemical Performance of FGO@Fe-MOG. Herein, we applied FGO@Fe-MOG as a platform with unique morphology for electrochemical aptasensing of thrombin. The interfacial features of the surface modified glassy carbon electrodes (GCEs) were investigated by means of cyclic voltammetry and


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EIS measurements. The CVs of GCEs modified with FGO, Fe-MOG, and FGO@Fe-MOG were recorded in 0.1 mol L-1 KCl containing 5 mmol L-1 [Fe(CN)6]3–/4– as a redox probe (see Figure S4A). A couple of quasi-reversible and well-defined redox peaks were presented in all three cases. It was observed that the redox current response toward [Fe(CN)6]3–/4– ions was higher for the FGO@Fe-MOG modified GCE when compared to the FGO or Fe-MOG modification. This current response increase was attributed to the extended electrode active surface area due to the higher porosity of FGO@Fe-MOG composite. Furthermore, peak-to-peak separation ΔEp was estimated to be 117, 91, 83 mV for the FGO, Fe-MOG, and FGO@Fe-MOG modified GCE, respectively. The nonmodified GCE showed ΔEp of 113 mV. This reveals that the introduction of the FGO@FeMOG onto the electrode surface clearly enhances the electron transfer efficiency at the modified electrode. Figure S4B shows the linear dependence of the anodic and cathodic peak currents on square root of the scan rate for the FGO@Fe-MOG modified GCE. These data points out that the electrochemical reaction appearing at the modified electrode surface is a diffusion controlled.



Thereafter, electrochemical impedance spectroscopic (EIS) measurements were performed by means of Nyquist plots recorded for the various modified electrodes (data not shown). In principle, EIS is used to observe the kinetics of the charge transfer through the impedance in an electrochemical process.47 The electrode interface can be replaced by Randles equivalent circuit that consists of resistors that act as solution resistance (Rs), charge transfer resistance (Rct), Warburg impedance (Zw) substituting diffusion-controlled processes, and common phase element (CPE) mimicking the non-ideal capacitive nature of the interface. Similar values of charge resistant transfer Rct values were reached with GCE modified by FGO@Fe-MOG (635 Ω) or Fe-MOG (695 Ω), whereas the highest value was obtained for the FGO modified GCE (1347 Ω). This indicated that the incorporation of the FGO within the Fe-MOG did not affect its conductivity and electron transfer properties. These findings were in good agreement with the voltammetric observation described above.

3.3. The Detection of Thrombin Using FGO@Fe-MOG as a Sensing Platform.


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The aptasensor for the thrombin detection was prepared by immobilizing the thrombinbinding aptamer (TBA) onto either the bare or the modified DEP chip with subsequent incubation with thrombin. Impedance measurements were carried out after each step of the protocol (i.e., after modification, TBA immobilization, and finally THR incubation) in order to be able to follow the variation of the impedance values and thus clearly describe the obtained data. The impedance spectroscopy measurements were used to monitor the electrochemical responses in the [Fe(CN)6]3–/4– redox probe. Typical Nyquist diagrams related to the modification of FGO@Fe-MOG with TBA and consequently with thrombin are shown in Figure 4A. The charge transfer resistance Rct obtained with FGO@Fe-MOG was 281 Ω (curve a), whereas once the TBA was immobilized onto the surface, the Rct value increased to 552.3 Ω (curve b). This increase in the Rct value might be ascribed to the repulsive electrostatic forces between the phosphate backbone of the DNA aptamer and the [Fe(CN)6]3 −/4 − redox probe that both are negatively charged. This in turn also reflected the successful loading of the TBA onto the surface of the FGO@Fe-MOG platform. Further hindering of the electron transfer is observed since the electron transfer resistance became larger (1396.9 Ω) after incubation 25 ACS Paragon Plus Environment


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with the target analyte thrombin (curve c). Such observation is associated with the formation of a complex upon the folding of the TBA and the thrombin, which hinders the electron transfer of the redox probe [Fe(CN)6]3–/4– at the electrode surface. The slower electron transfer in this case is caused mainly by electrostatic repulsion and steric hindrance presented by the TBA-THR complex. The impact of the other materials, i.e., FGO and Fe-MOG on the sensing ability for thrombin detection was also analyzed to verify that the exceptional sensing activity is exclusively due to the composite (see Figure S5 in Supporting Information). In order to validate the results obtained from different electrodes, the impedance spectra were analyzed in terms of the Rct ratio defined by the following equation48 and represented as a histogram in Figure 4B.

𝑅𝑐𝑡 𝑟𝑎𝑡𝑖𝑜 =

(𝑅𝑐𝑡 𝑡ℎ𝑟𝑜𝑚𝑏𝑖𝑛 ― 𝑅𝑐𝑡 𝑏𝑙𝑎𝑛𝑘) (𝑅𝑐𝑡 𝑎𝑝𝑡𝑎𝑚𝑒𝑟 ― 𝑅𝑐𝑡 𝑏𝑙𝑎𝑛𝑘)

where Rct thrombin was the electron transfer resistance measured after incubation with the thrombin, Rct

aptamer

after aptamer immobilization and finally Rct

transfer resistance of the modified electrode and buffer solution.


blank

was the electron

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The data presented in Figure 4B clearly demonstrated that the thrombin-binding aptamer (and thus the thrombin) was strongly attracted onto the Fe-MOG and FGO@FeMOG while no interaction between the FGO and the bare electrode was observed. These findings suggested and confirmed strong electrostatic attraction between the Fe-based MOG and the thrombin-binding aptamer (TBA). In addition, one can observe the enhancement of sensing signal with the FGO@Fe-MOG that is arising from both hierarchical porosity and better electron transfer properties of this hybrid material.



Figure 4. (A) The comparison of EIS responses for the FGO@Fe-MOG modified DEP chip (curve a), the FGO@Fe-MOG modified DEP after the aptamer immobilization (curve b) and with consequent incubation with thrombin (curve c). (B) Histogram representing a comparison of Rct ratio value for the DEP chips modified with FGO, Fe-MOG, and FGO@Fe-MOG and for bare DEP chip (blank). (C) Calibration plot corresponding to the increasing concentration of thrombin. Inset - respective EIS spectra. (D) Comparison of impedimetric responses to different proteins recorded with the FGO@Fe-MOG modified DEP chip. The error bars correspond to triplicate experiments. All measurement were performed in TRIS saline buffer (pH 7.4) containing 5 mmol/L [Fe(CN)6]3–/4– redox probe.

3.4. Analytical Performance of Thrombin Aptasensor Based on FGO@Fe-MOG. In order to obtain the best analytical performance of the suggested aptasensor, it was essential to determine the aptamer concentration to be immobilized onto the FGO@Fe-MOG sensing platform. Such optimization is shown in Figure S6. As can be seen, the highest Rct value and thus the biggest amount of immobilized aptamer onto the


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electrode surface was observed to be 10 µmol L-1. This concentration of TBA was used in further experiments. The behavior of the aptasensor with the increasing concentration of thrombin is shown in Figure 4C together with respective EIS spectra (see inset). As can be seen, the increasing concentration of thrombin led to the higher Rct ratio values which means, and is in correlation with previous observations, that the interfacial electron transfer resistance between the modified electrode surface and the solution also increased. The calibration curve thus reveals a linear variation (y= 2.35 + 0.38x; R2 = 0.976) of the Rct ratio with thrombin concentration in the range of 2–14 ng mL-1. The limit of detection (LoD) for thrombin was established from the calibration curve based on the standard deviation (σ) of the response and the slope (S) that can be expressed as LoD = 3.3 × (σ/S). In this way, the LoD was found to be 2.2 ng mL-1 (equals to 58 pM). This value is more than satisfactory regarding the concentration range (nM to low µM level) of thrombin that can actually be found in blood during the coagulation process.49 Table S1 (see Supporting Information) summarizes the comparison of FGO@Fe-MOG based aptasensor with other sensing strategies and platforms described in literature. As can be seen, our proposed 29 ACS Paragon Plus Environment


aptasensor offered superior detection limits to other label-free reported aptasensors based on different signal output strategies (e.g., colorimetry, electrochemistry, or fluorescence). The selective detection of the target molecule among the various proteins is an important feature of the sensing system. Hence, the FGO@Fe-MOG modified electrode were after the immobilization of TBA incubated also with different target proteins, i.e., bovine serum albumin (BSA), immunoglobuline G (IgG), or streptavidin. From Figure 4D, it can be seen when BSA, IgG, and streptavidin were used to replace the THR, the changes in the Rct were negligible. These control experiments indicate that the aptasensor offered high selectivity toward thrombin. The reproducibility of aptasensor was investigated at the thrombin concentration of 10 nM, and the relative standard deviation for five times was 5.8%. In addition, five freshly prepared modified electrodes exhibit similar impedimetric response to 10 nM thrombin, with relative standard deviation of 4.9%. This demonstrates high reproducibility and precision of our aptasensor for thrombin detection.


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Finally, the applicability of our aptasensor in a complex matrix was also investigated by detecting thrombin in human serum. Serum samples were prepared by spiking different amounts of thrombin into 10-times diluted human serum. The recoveries varied from 95.4% to 101.2% with RSD around 4.3%, which is satisfactory for analytical purposes (see Table 1). Here in, we have shown that the overall electro-analytical performance of thrombin aptasensor based on FGO@Fe-MOG met essential characteristics of a promising tool for clinical diagnosis of important biomarkers and/or point-of-care analysis.

Table 1. Recovery of thrombin in spiked human serum. Sample

Spiked (pM)

Found* (pM)

Recovery (%)

RSD (%)

1

80

76.3

95.4

4.7

2

132

128.8

97.6

3.8

3

316

319.8

101.2

4.5

*Mean

values of three measurements.

In summary, we have demonstrated a facile and scalable methodology to fabricate hierarchical porous fluorinated graphene oxide (FGO) and a metal-organic gel (MOG) 31 ACS Paragon Plus Environment


composed of BTC organic linkers and Fe(III) metal ions. The resultant gel possesses unique properties including significant chemical stability, hierarchical micro/meso/macro porous behavior, and large surface area. Large hierarchical surface of the nanocomposite enabled immobilization of a substantial amount of thrombin-binding aptamer and thus served as a nano-engineered sensing platform. The suggested thrombin aptasensor possesses superior detection limits compared to the previously developed colorimetric and/or electrochemical thrombin sensors along with high selectivity and reproducibility. Besides, the FGO@Fe-MOG nanostructured platform also showed adequate results when applied in a complex matrix (i.e., human serum), and, thus, it might be interesting for further investigation in clinical practice. We also anticipate extension of the FGO@FeMOG sensing platform for the quantification of other important biomarkers (e.g., myoglobin; BRCA 1,2; CEA, or PSA) with a potential high added value in clinical diagnosis.


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ASSOCIATED CONTENT

Supporting Information. Characterization methods; optical images showing Fe-MOG and FGO@MOG; 3D view of MIL-100 (Fe); XPS spectrum; graphs showing electrochemical properties; graph showing variation of Rct values for different sensing platforms; graph showing optimization of TBA concentration.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] (R.Z.).



Page 34 of 47

*E-mail: [email protected] (R.A.F.).

Author Contributions #These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS V. Urbanová acknowledges support of the Czech Science Foundation (Project GACR No. 1722194Y). K. J. R. is grateful to the Alexander von Humboldt (AvH) foundation for a post-doctoral fellowship. We thank C. Aparicio (Palacký University) for XRD measurements, O. Tomanec (Palacký University) for HRTEM measurements, M. Petr (Palacký University) for measurement of XPS data. The authors also gratefully acknowledge support by the Catalysis Research Centre at TU Munich and the support from the Ministry of Education, Youth and Sports of the Czech Republic (LO1305) and the assistance provided by the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2015073. The work was further supported by the Operational Programme Research, Development

and

Education—European

Regional

Development


Fund,

Project

No.

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CZ.02.1.01/0.0/0.0/15_003/0000416 and CZ.02.1.01/0.0/0.0/16_019/0000754 of the Ministry of Education, Youth and Sports of the Czech Republic.



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