Fabrication of 3D Amino-Functionalized Metal-Organic Framework on Porous Nickel Foam Skeleton to Combinate Follicle Stimulating Hormone Antibody for Specific Recognition of Follicle-Stimulating Hormone.

In this study, a superficial and highly efficient hydrothermal synthesis method was developed for the in situ growth of amine-functionalized iron containing metal-organic frameworks (H2N-Fe-MIL-101 MOFs) on porous nickel foam (NicF) skeletons (H2N-Fe-MIL-101/NicF). The uniform decoration of the H2N-Fe-MIL-101 nanosheets thus generated on NicF was immobilized with follicle-stimulating hormone (FSH) antibody (Ab-FSH) to detect FSH antigen. In the present work, the Ab-FSH tagged H2N-Fe-MIL-101/NicF electrode was first applied as an immunosensor for the recognition of FSH, electrochemically. With all of the special characteristics, this material demonstrated superior specific recognition and sensitivity for FSH with an estimated detection limit (LOD) of 11.6 and 11.5 fg/mL for buffered and serum solutions, respectively. The availability of specific functional groups on MOFs makes them an interesting choice for exploring molecular sensing applications utilizing Ab-FSH tagged biomolecules.

Introduction

Metal–organic frameworks are rapidly growing hybrid materials that are being incorporated into to the materials science research area because of their versatile characteristics.[1] In general, MOFs possess a structurally diverse platform for a wide range of applications. It includes a large surface area, highly ordered crystalline structure with multidimensional networks, adjustable porosity, specific symmetry, high loading efficacy, tunable surface chemistry, abundant compositions, availability of multiple Lewis acids sites, and a variety of organic linkers.[2−4] These hybrid materials are composed of metal ions and organic ligand moieties in regular symmetry associated via coordination bonding or intermolecular forces.[5] Highly porous MOFs lead to a high exposure active surface area for mass transport thus leads to high capacitance.[6−9] With MOFs as emerging probe materials, it shows a guarantee for sensing in light of their versatile properties.[10−12] The host–guest interaction capability of MOFs enables specific recognition of target molecules.[13,14] However, no MOF-based sensor for FSH detection has been discovered.

FSH is a glycoprotein comprised of monomeric protein subunits attached with a sugar unit. The dimer is incorporated with two polypeptides, α- and β-subunits. The β-subunit assigns the hormone its distinct biological role and is accountable for interaction with FSH receptors. The glycoproteins play a role in clinical areas as a crucial protein-based cancer biomarker.[15,16] The most commonly available FSH detection methods are based on noncompetitive immunoassays using capillary electrophoresis with chemiluminescence, enzyme-linked immunosorbent assay (ELISA), and amperometric assay. The direct identification of glycoprotein is difficult due to the scarcity, heterogeneity, and complex structure of glycans. The availability of carboxylic, amino, and sulfonic functional groups makes MOFs a hydrophilic material.[17] They can form hydrogen bonds with a distinct affinity that facilitates glycopeptide interactions with MOF materials.[18] Additional improvements in the hydrophilicity can be achieved by postsynthetic modification.[19] The functionalized MOFs are useful in enzyme immobilization in which the isoelectric points of enzymes can be tuned and further helps to improve the electrostatic interactions.[20]

Functionalized MOF materials are considered promising candidates for electrochemical sensing applications.[21−24] Because of the insulating nature of MOFs, minimal electrochemical applications were explored. Incorporating MOFs with other functional materials such as biomolecules, polymers, and nanomaterials can furnish functionalized bulk materials with improved characteristics, which is an efficient way to expand their sensing applications.[25,26] A systematic approach to improving the conductivity of MOF is to incorporate additives or coordinating polymers, but in this study we attempted to utilize the nickel foam support that can stock charges and involves the rapid charge–discharge process due to its good conductivity.[27] The sensitivity greatly relies on the electroactive area and mass transfer ability of electrodes and analytes, respectively. Hierarchical porous features of MOFs enhance the accessibility of the active area and promote analytes diffusion, thus benefiting mass transport.[28]

The creation of simple, rapid, low-cost, specific, and sensitive recognitions without mediators or labeling enzymes is an important goal for many researchers to devise an impedimetric electrochemical sensor.[29] This methodology is fruitful to reveal the complex formation of biological molecules that are immobilized on the electrode surface by examining the electrode and electrolyte interface. A typical electrochemical biosensor setup mainly possesses an electrode substrate, recognizing element, transducer, and an electronic detection system. Different types of nanomaterials and organic substrates act as support materials that can tie up the substrate and probe. However, these materials possess a small surface area and low sensitivity and necessitate challenging material preparation. To this end, constructing advanced materials that can function as electrochemical sensors to enhance their detection performance is a great challenge.[30]

In this study, we aim to contrive a high-performance immunosensor for specific recognition of an FSH glycoprotein. In this connection, in situ hydrothermal methods were used to generate the electrode materials with the aid of NicF solid supports. The as-decorated materials were used as working electrodes, which is followed by conjugation with Ab-FSH to detect FSH electrochemically. Amino-functionalized MOFs (H2N–Cr-MIL-101) are highly known for their specificity toward glycopeptide enrichment. This study prefers to use environmentally friendly, nontoxic iron incorporated amino-functionalized H2N–Fe-MIL-101 MOFs for the fabrication of immunosensors to detect FSH analytes. On the basis of the electrochemical analysis, it is found that the as-fabricated electrodes H2N–Fe-MIL-101/NicF possessed fast and excellent selectivity for the FSH target. The uniformly arranged stable MOFs on porous NicF surfaces may also help in achieving more stable results when compared to the other platforms. This study design is an effective way to create a rapid, onsite, and sensitive electrode system for FSH detection.

Results and Discussion

The success of the inorganic–organic hybrids relies on the variety of metal-oxide clusters that are connected with multifarious functionalized organic ligands. The use of a linear terephthalic acid-based organic linker is particularly important since it can generate several framework structures with fascinating features.[31,32] MOFs can show a size-exclusion effect due to their tunable porosity which is uncommon in other porous materials.[33] Moreover, the surface of MOFs possesses affinity-based functionalities that enable any biological molecules entrapped into the pores in terms of shadow effect.[34] Among the variety of MOFs, the exceptionally large breathing effect of MIL-88 have been first sought in 2007. The context “breathing” implies the ability of the unit cell framework to reversibly swell and shrink without affecting the topology under the influence of external stimulus. Thus, it is well suitable for host–guest interactions.

Considering H2N–Fe-MIL-101 materials as an example, the presence of adsorption effect and strong hydrophilicity toward polarity materials were proven to achieve the great potential for glycopeptide enrichment. Easily synthesized amino-functionalized MIL MOF was first used in glycoprotein enrichment because of its miraculous ability in trapping peptides as well as high enrichment function.[35] It is proved that a potential enrichment of proteins and peptides could be achieved by MOFs. Specifically, amino-functionalized MOFs were utilized for glycopeptide enrichment actions via hydrophilic interactions.[36] From these experimental supports, a new strategy was developed involving amino-functionalized MOF for the specific recognition of FSH.

Iron-based MOFs or derived materials exhibit excellent sensing performances because of their potential redox ability and stability when used as electrode materials.[37,38] Construction of electrode materials with a microstructure and uniform morphology is an effectual strategy to strengthen their electrochemical characteristics.[39] The nickel/nickel-based electrodes find great applications in electrochemical devices, supercapacitors, and fuel cells as electrode materials. As NicFs have open-pore structure, and they can participate in active mass supports as well as a charge carrier. These porous materials are more important as they provide substantial use of material mass while maintaining its 3D expanded structure and high surface area. The fine-tuned 3D structural assembly of NicFs builds them as unique materials for multiple applications.[40] The electrochemical applications of direct MOFs are limited due to their inherent drawbacks such as unsteadiness in aqueous environments and poor electron transfer properties.[41,42] Hence MOFs were grown on highly conducting nickel foam substrate. This is the reason for specific utilization of iron and nickel for the creation of immunosensor when compared to other metal ions.

Schematic Illustration of the Stepwise Fabrication Protocol of Immunosensing Biosensing System

The synthesis method for the development of 3D H2N–Fe-MIL-101 nanosheets MOFs on porous nickel foam (labeled as H2N–Fe-MIL-101/NicF) for FSH detection is shown in Scheme . The electrode materials were synthesized from a one-step hydrothermal method using iron salt and H2Bdc-NH2, as starting precursors. The successful formation of H2N–Fe-MIL-101/NicF was checked from the color change of nickel foam. The basic properties of as-synthesized materials were evaluated by performing several characterizations including HRFE-SEM, TEM, PXRD, FT-IR, XPS, BET, and UV–vis and fluorescence techniques. The H2N–Fe-MIL-101/NicF electrodes were generated through multiple steps according to Scheme . To construct the immunosensor matrix, initially, the Ab-FSH were oxidized in the presence of sodium metaperiodate that causes mild oxidation of carbohydrate units to convert it into formyl groups. Then the oxidized anti-FSH antibody was then allowed to react with H2N–Fe-MIL-101/NicF electrode in which the oxidized glycoprotein (formyl moieties) reacts with amines MOFs and forms Schiff base intermediates. The electrodes were subjected to undergo a reductive amination process. Finally, Ab-FSH tagged H2N–Fe-MIL-101/NicF bioelectrodes are readily available for further electrochemical analysis.

Scheme 1

Stepwise Illustration

Illustration of the synthesis of iron containing 3D H2N–Fe-MIL-101 nanosheets MOFs on porous NicF substrate by in situ hydrothermal methods derived from FeCl3·6H2O salt and H2Bdc-NH2 ligand precursors and NicF solid support producing uniformly decorated H2N–Fe-MIL-101/NicF electrodes followed by bioconjugation of FSH antibody for FSH detection.

Characterization of Surface Morphology of the as-Prepared H2N–Fe-MIL-101/NicF Electrodes and Scratched off H2N–Fe-MIL-101 Particles by SEM and TEM

The surface morphology and particle size of the H2N–Fe-MIL-101/NicF were analyzed by HRFE-SEM and TEM techniques. The results point out the uniform assembly of nanosheets on NicF that holds an average size of 25 nm as shown in Figure . The elemental distribution on the H2N–Fe-MIL-101/NicF was known from the EDX results. The composition measurement by EDX analysis displays the presence and uniform distributions of major elements such as carbon (C), iron (Fe), oxygen (O), and nitrogen (N) on the porous NicF substrate. The SEM-EDX area elemental mappings of H2N–Fe-MIL-101/NicF are displayed in Figures S1 and S2. To further prove the surface morphology of the materials grown on the solid support, some of the H2N–Fe-MIL-101 were scratched off from the NicF via ultrasonication in ethanol and then analyzed for SEM. The SEM image of the powdered material yields a hexagonal spindle pattern which is a characteristic of typical H2N–Fe-MIL-101 reported earlier.[43] This confirms that the material grown on the NicF skeleton is H2N–Fe-MIL-101. It concluded the uniform distribution of H2N–Fe-MIL-101 on the NicF surface. The analyzed results demonstrated that H2N–Fe-MIL-101 shares a similar elemental composition as that of their molecular formula. It is found that the experimental atomic % values and the theoretically calculated values share a similar value. The elemental contents of the materials calculated from the EDX analyses are consistent with the reported values of H2N–Fe-MIL-101.[44] The SEM EDX area elemental mapping images of H2N–Fe-MIL-101 are displayed in Figures S3–S5. The particle size and morphology of H2N–Fe-MIL-101 were further confirmed by TEM observation.

Figure 1

(a–f) SEM images of H2N–Fe-MIL-101 grown on porous NicF substrate after in situ hydrothermal treatment using FeCl3·6H2O precursor, H2Bdc-NH2 ligand, and NicF solid support precursors. The inset in (d–f) shows the markings of each nanosheet that was grown on the porous NicF skeleton.

As shown in Figure , the results revealed that the hexagonal microspindle H2N–Fe-MIL-101 has been decorated on the NicF surface on a large scale. The fabricated material consists of solid hexagonal microspindles of 200 nm size. It is composed of a hexagonal microspindle with a solid structure.[45] The TEM images showed a regular hexagon outline which is consistent with the SEM patterns.

Figure 2

(a–f) TEM images of the iron-containing amino-functionalized H2N–Fe-MIL-101 MOFs.

Interpretation on XRD Patterns and FT-IR Spectra of H2N–Fe-MIL-101 MOFs

Figure a illustrates the powder XRD patterns obtained for H2N–Fe-MIL-101 MOFs. The as-prepared materials showed main diffraction peaks at 9.39° and 17.95° which are similar to that of the reported diffraction patterns of the MIL 101 series.[46,47] The XRD patterns of the synthesized H2N–Fe-MIL-101 samples exhibited strong peaks fairly at high intensity and flat background, indicating high crystallinity of the materials. In addition, no other peaks were recorded for the as-obtained sample evidencing the high phase purity of H2N–Fe-MIL-101. To further understand the molecular structure and to identify the changes in the functional groups of H2N–Fe-MIL-101. The FT-IR spectrum was recorded in the frequency range of 400–4000 cm–1. The resulting spectrum is displayed as Figure b. The strong intensity bands obtained in the 1600–1400 cm–1 indicate the presence of symmetrical and asymmetrical stretching modes of O–C–O MIL-101 frameworks. The absorption peaks at 3464 and 3375 cm–1 were ascribed due to the symmetrical and asymmetrical stretching vibrations of amine groups, respectively.[48] The strong apparent peak located at 541 cm–1 was attributed to Fe–O vibration. In addition, the bands at 1624 and 1336 cm–1 are responsible for σ(N–H) and υ(C–N), respectively.

Figure 3

(a) Images displaying the powder XRD patterns of H2N–Fe-MIL-101 with a simulated pattern report. (b) The FT-IR spectrum of H2N–Fe-MIL-101 MOF. (c) Comparison of UV–vis absorption spectra of free linker H2Bdc-NH2 and H2N–Fe-MIL-101 MOF. (d) Emission spectra (λex = 360 nm) of H2N–Fe-MIL-101 MOF and free linker H2Bdc-NH2.

Analysis of UV–vis and Emission Properties

The UV–vis and emission spectroscopic methods were used to demonstrate the optical properties of H2N–Fe-MIL-101 and free linker H2Bdc-NH2. A sharp band that appeared at 350 nm (Figure c) for the H2Bdc-NH2 may be due to π–π* and n−π* transitions whereas H2N–Fe-MIL-101 displayed more broadband which is well deviated from the H2Bdc-NH2. The broadening effect was assigned to the electron transfer between the metal-oxo (Fe–O) cluster and H2Bdc-NH2. In addition, the strong interactions between Fe–O clusters and H2Bdc-NH2 might cause ligand-core charge transfer (LCCT) transitions. This strongly suggests the H2N–Fe-MIL-101 MOF formation.[49] Similarly, room-temperature emission spectra were recorded for H2N–Fe-MIL-101 and H2Bdc-NH2 at an excitation of λex = 360 nm. The emission peaks were obtained at 437 and 448 nm (Figure d) for H2Bdc-NH2 and H2N- Fe-MIL-101, respectively.[43] A slight red shift in the emission peak was noticed when compared with free H2Bdc-NH2, supporting the strong coordination of H2Bdc-NH2 and Fe–O clusters.

Details XPS Evaluation of H2N–Fe-MIL-101 MOFs

To evaluate the nature of chemical states and the surface components of the samples, the XPS analysis was carried out, and their results are displayed in Figure . As can be seen from the whole range survey spectrum (Figure a), it is obvious that the sample contains the Fe, C, O, and N elements that are consistent with EDX data. The Fe 2p spectrum results in two peaks appeared at a binding energy of 709.2 and 722.1 eV for Fe 2p3/2 and Fe 2p1/2 (Figure b). The satellite peak with a binding energy of 714.8 eV indicates the presence of Fe in a +3 oxidation state.[43,50] A strong peak at about 529.7 eV corresponds to O 1s (Figure c). Figure d represents the deconvolution peak of C 1s. This exhibits three peaks at binding energies of about 282.8, 284.3, and 286.7 eV indicates the existence of C–C, C–O, and C=O, in the frameworks, respectively.

Figure 4

(a) Full range XPS survey of H2N–Fe-MIL-101 MOFs showing the presence of major elements. (b–d) High-resolution deconvoluted XPS spectra of Fe 2p, O, and C1, respectively.

BET Analysis of H2N–Fe-MIL-101 MOFs

The nitrogen (N2) adsorption and desorption isotherms of H2N–Fe-MIL-101 are displayed in Figure . The isotherm results indicate the isotherms are compatible with type IV isotherm which is the characteristic of mesoporous materials. On the basis of this knowledge, it can be evident that the as-prepared H2N–Fe-MIL-101 MOFs are mesoporous materials. The porosity and surface properties of H2N–Fe-MIL-101 were calculated using the BET–Langmuir method. The BET surface area was found to be 1755 m2/g and the BJH adsorption pore size was measured to be 2.3 nm.

Figure 5

N2 adsorption and desorption isotherm curves of H2N–Fe-MIL-101 MOFs.

Important Parameters of the Electrochemical Measurements of the Constructed Bioelectrode System

On account of the successful Ab-FSH-tagged H2N–Fe-MIL-101/NicF electrode fabrication and FSH detection, EIS and CV were selected as investigation tools to analyze the electrochemical process happening between the constructed electrode and electrolytic solution interface.[51] For a better understanding of the electrochemical performance of Ab-FSH/H2N–Fe-MIL-101/NicF electrodes for FSH detection, the EIS spectra were displayed as Nyquist plots. The charge transfer resistance (Rct) and the other related parameters were extracted by fitting the measured EIS data with a suitable Randle’s model. Figure S6 represents Randle’s equivalent circuit model, which has four different elements: (i) the solution resistance (Rs), (ii) the charge transfer resistance (Rct), (iii) constant phase element (CPE), and (iv) the double layer capacitance (Cdl).[47,52] The changes in the morphology of the bioframed electrodes after Ab-FSH immobilization and FSH treatment were investigated by SEM, CLSM, and CV analysis.

Morphological Variations on Ab-FSH/H2N–Fe-MIL-101/NicF Electrodes after the FSH Biorecognition

In this study, the SEM analyses were recorded to confirm the antibody conjugation on the working H2N–Fe-MIL-101/NicF electrodes after Ab-FSH immobilization. The morphological changes were monitored during each stage of electrode construction as given in Figure . Figure a represents the SEM images for bare NicF and the surface is smooth, plain, and pure. The uniform nanosheets were decorated on the NicFs after being subjected to in situ hydrothermal synthesis along with starting precursors (Figure b). The morphological variations are noted when the antibodies were attached to the H2N–Fe-MIL-101/NicF surface (Figure c). As a result of Ab-FSH attachment, clear globular structures are visible on the surface of H2N–Fe-MIL-101/NicF, yielding an effective area for the biomolecule attachment. The SEM image of Figure d illustrated that the FSH biomolecules were immobilized directly on the Ab-FSH tagged H2N–Fe-MIL-101/NicF electrode. The morphological changes were caused due to specific immune-interaction between Ab-FSH and FSH. All of these morphological variations on the electrode surface were evident in the successful fabrication of H2N–Fe-MIL-101/NicF bioelectrode.

Figure 6

SEM images of (a) bare porous NicF substrate. (b) NicF decorated with H2N–Fe-MIL-101 MOF nanosheets after in situ hydrothermal treatment. (c) H2N–Fe-MIL-101/NicF electrodes after bioconjugation with Ab-FSH. (d) FSH treated Ab-FSH/H2N–Fe-MIL-101/NicF bioelectrodes.

Characterization of H2N–Fe-MIL-101/NicF Electrodes after Ab-FSH Bioconjugation by Confocal Laser Scanning Microscopy (CLSM)

To visualize Ab-FSH conjugation on H2N–Fe-MIL-101/NicF electrodes, the electrodes were incubated with Rhodamine B dye (Rhod B) alone, Ab-FSH alone, and Rhod B dye combined Ab-FSH for 30 min. Then the electrodes were rinsed with PBS buffer to remove excess Rhod B and Ab-FSH and then observed under CLSM. The Rhod B doped H2N–Fe-MIL-101/NicF electrodes (Figure S7a) shows that fluorescence indicates the presence of Rhod B dye. The Ab-FSH conjugated H2N–Fe-MIL-101/NicF electrodes (Figure S7b) that did not display any fluorescence shows the conjugation of Ab-FSH. The merged images of Rhod B dye/Ab-FSH combined with H2N–Fe-MIL-101/NicF electrodes (Figure S7c) clearly displayed the presence of both Rhod B and Ab-FSH, confirming the Ab-FSH conjugation on the H2N–Fe-MIL-101/NicF electrodes.

Changes in the Cyclic Voltammetric Response during the Fabrication of the Proposed Biosensing System and FSH Detection

To further demonstrate the successful bioelectrode H2N–Fe-MIL-101/NicF fabrication for FSH detection, the CV profiles were recorded. Here, the CV analysis was recorded at each step to confirm the Ab-FSH conjugation with H2N–Fe-MIL-101/NicF electrodes as displayed in Figure . When the electrodes were uniformly decorated with nanosheet MOFs, the peak current was distinctly reduced because these nanosheet MOFs tend to catalyze the charge transfer process of the redox K3[Fe(CN)6]/K4[Fe(CN)6] probe.[53]

Figure 7

Measurement of CV response during the H2N–Fe-MIL-101/NicF biosensing fabrication process for FSH detection in 10 mM K3[Fe(CN)6]/K4[Fe(CN)6] electrolyte at a scan rate of 100 mV s–1.

The Ab-FSH tagged H2N–Fe-MIL-101/NicF electrodes displayed a reduction in the larger peak current for the redox probe. It is worth mentioning that a decrease in peak current reflects the strong interaction of antibodies on the H2N–Fe-MIL-101/NicF electrode surface that may be caused by a dramatic steric hindrance on the redox probe charge transfer via electrode surface. The significant changes in CV peak currents were noticed as the scan rate increases, revealing the successful immobilization of Ab-FSH on H2N–Fe-MIL-101/NicF for FSH detection. The Ab-FSH functionalized electrodes Ab-FSH/H2N–Fe-MIL-101/NicF were incubated with FSH, and changes in the peak current were significantly reduced, indicating that the FSH was successfully combined on the electrode surface. Additionally, the interaction between the Ab-FSH and FSH generates an antibody–antigen immunocomplex protein layer that can perturb the electron transfer between the constructed bioelectrodes and electrolyte interface. Thus, a reduction in peak current was observed.[51] Finally, the fabricated electrodes Ab-FSH/H2N–Fe-MIL-101/NicF are ready to treat with different concentrations of FSH. It is exciting to find that the nanosheets grown on NicF displayed wonderful CV behaviors of decreasing peak currents. The superior electrochemical performance was achieved due to uniformly grown nanosheets on NicF that have breathing nature and high surface area.

Impedimetric Detection of FSH Analytes with the Proposed Immunosensor Matrix

Different concentrations of FSH analytes ranging from 100 ng/mL to 10 fg/mL were prepared to test as-fabricated Ab-FSH/H2N–Fe-MIL-101/NicF electrodes in buffer and 10% serum buffered solutions. Figure displayed the Nyquist plots generated from EIS measurements recorded under buffered and serum conditions. Increasing the FSH concentration results in increasing the charge transfer resistance in both the reaction conditions. This increase in Rct can be assigned due to increased steric hindrance as well as the electrostatic interactions between the FSH and electrolyte. The conductance was found to decrease when the electrodes are exposed to FSH which may be ascribed due to the hindrance in the diffusion of electrolyte toward the electrode surface as a result of FSH layering. Moreover, there is an increased binding of FSH molecules to the immobilized antibodies which is a major kinetic barrier for the electron transfer. It can also be correlated with the fact that that the increasing Ab-FSH/FSH interactions effectively block the free space on the working bioelectrode. The calibration curve indicates that a wide linear detection range is possible. Detailed analysis of the results revealed that the linear range of detection was within 100 ng/mL to 100 pg/mL. The calculated LOD was found to be 11.6 fg/mL for buffered solutions and 11.5 fg/mL for serum solutions. The proposed biodevice achieved low LOD which is attributed to superior antibody immobilization on porous H2N–Fe-MIL-101/NicF electrodes.

Figure 8

EIS analysis after FSH treatment on Ab-FSH/H2N–Fe-MIL-101/NicF electrodes in buffer and 10% serum buffered solutions. (a) Nyquist plot generation from EIS data for the electrodes when treated against various FSH concentrations between 100 ng/mL and 10 fg/mL in the presence of [1:1] [Fe(CN)]3-/4- redox probe, pH 7.4 under buffered conditions. (b) A plot of ΔR (kΩ) versus concentration was obtained for (a). (c) Nyquist plot generation from EIS data for the electrodes when treated against various FSH concentrations between 100 ng/mL and 10 fg/mL in the presence of [1:1] [Fe(CN)]3-/4- redox probe, pH 7.4 under serum conditions. (d) A plot of ΔR (kΩ) versus concentration was obtained for (c).

This excellent electrochemical performance of the nanosheets’ electrodes is credited to their unique structural aspects. First, the 3D macroporous NicFs are highly conductive that enables potential charge transport and attainable electrolyte diffusion. Second, there is an excellent charge transport between nanosheets and current collect, so that the active reservoir can be involved in electrochemical performance. Finally, well-aligned nanosheet construction on NicF provides a high surface area for electrochemical reactions. Moreover, there are electrostatic interactions between amino (−NH2) groups of MOFs and negatively charged carboxylic acid (−COOH) groups of glycan sialic acids. Additionally, the hydrogen bond formation between glycan hydroxyl (−OH) groups and amine (−NH2) groups of MOFs is also involved in the binding process. The material’s thermal stability and pH are added benefits. Most importantly, these solid state electrodes are binder free which allows a fast electrochemical rate. On the basis of these studies, these nanosheets structures are excellent candidates for the specific detection of FSH. Compared with other substrates, nickel foam substrates has an effective surface area, and MOF has a large surface area and significantly improved ligands thereby increasing the number of electrode response sites that further promote current response toward analytes. The as-prepared MOF electrode displayed a low detection limit of 11.6 and 11.5 fg/mL for FSH monitoring when tested under buffered and serum conditions. A comparison table for FSH detection methods and their sensitivities is provided in Table S1. The chemical and mechanical stability of H2N–Fe-MIL-101/NicF and Ab-FSH/H2N–Fe-MIL-101/NicF electrodes was also discussed (Figure S8 and Figure S9)

Analysis of the Specificity and Reproducibility of the Ab-FSH/H2N–Fe-MIL-101/NicF Electrode

The specificity of the proposed Ab-FSH/H2N–Fe-MIL-101/NicF electrode was evaluated against different glycoproteins as shown in Figure a. The obtained selectivity data demonstrated that the conductance of the Ab-FSH/H2N–Fe-MIL-101/NicF sensor toward FSH detection yields insignificant changes when present with other glycoproteins, showing its specific recognition for FSH. The reproducibility of Ab-FSH/H2N–Fe-MIL-101/NicF sensors was analyzed on three different electrodes produced from three different batches under similar experimental conditions. The electrodes were tested against 10 ng/mL and 10 fg/mL FSH. The reproducibility results are presented in Figure b and Rct values obtained indicates that all of the electrodes produced from different batches displayed similar response, highlighting good reproducibility of the fabricated Ab-FSH/H2N–Fe-MIL-101/NicF electrodes.

Figure 9

(a) Selectivity of H2N–Fe-MIL-101/NicF bioelectrodes toward other glycoproteins such as LH (10 μg/mL), HCG (10 μg/mL), TSH (10 μg/mL), and FSH (1 ng/mL). (b) EIS responses of the two devices were prepared from different batches against the selected FSH concentrations (10 ng/mL and 10 fg/mL).

Conclusion

In summary, stable, easily synthesized iron-based amino-functionalized nanosheets of H2N–Fe-MIL-101 were successively grown on porous NicF surfaces by in situ hydrothermal methods. These materials were first applied to recognize the analyte FSH. The electrodes were successfully immobilized with Ab-FSH and then examined for FSH detection. The successful fabrication and FSH detection were demonstrated by electrochemical methods. The LOD was calculated to be 11.6 and 11.5 fg/mL for buffered and serum solutions. This methodology may offer the generation of many such new immunosensors in the future. This method can be facily expanded to develop other nanoMOFs on other solid supports for various applications. Enhancing the specificity of the probe toward a specific target is a major challenge and the creation of more suitable glycan binding functional moieties will continue to be a concern for the predicted future.

Experimental Section

Materials

2-Aminoterephthalic acid (H2Bdc-NH2), iron(III) chloride hexahydrate (FeCl3·6H2O), N,N-dimethylformamide (DMF), 2-morpholinoethanesulfonic acid (MES buffer), sodium cyanoborohydride (95%), and sodium metaperiodate (98%) were procured from Sigma-Aldrich (MO, U.S.A.). Human follicle-stimulating hormone (FSH) beta protein, follicle-stimulating hormone beta antibody (Ab-FSH), thyroid stimulating hormone (TSH, 98%), luteinizing hormone (98%, LH), human chorionic gonadotropin (98%, HCG), and bovine serum albumin (BSA) were procured from Gentex and Biotech companies (Hsinchu, Taiwan). The stock solutions of Ab-FSH and FSH were freshly prepared in phosphate-buffered saline solution (PBS), pH 7.4. All other chemicals and solvents were of analytical grade and ordered from Sigma-Aldrich/Merck. All experiments were performed at room temperature unless otherwise mentioned.

Methods

The surface morphology of the synthesized nanosheet MOF materials was investigated by high-resolution thermal field scanning electron microscope (HRFE-SEM, JEOL, JSM-7610F) and transmission electron microscopy (TEM, Hitachi H-7100) with charge coupled device (CCD) camera. The elemental composition of the materials was analyzed with Oxford X max80 energy dispersive spectrometer (EDS). The crystalline phase purity of the MOFs was examined with powder X-ray diffraction method using X-ray powder diffractometer (PXRD, Riagu (Japan)_TTRAX III) using Cu Kα radiation. To reveal the chemical state of the elements, the X-ray photoelectron spectra (XPS) were recorded in JEOL Analyzer (THMFLAb478) with Al Kα excitation source. The pore properties of the synthesized materials were characterized with Brunauer–Emmett–Teller (BET) specific surface area and pore size distribution analyzer (Micrometrics ASAP2020) at 77 K. The presence of the chemical components was analyzed with Fourier-transform infrared spectrometer (FT-IR, PerkinElmer, U.S.A., L1280127) using potassium bromide pelletized materials. The absorbance and emission spectroscopic analyses were carried out in UV–visible and fluorescence spectroscopic techniques (UV–vis, U-3010, Hitachi, Japan) and (F-7000, Hitachi, Japan). A full setup CHI 6116E model CH instruments (U.S.A.) was used to perform the electrochemical measurements such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The confocal imaging studies were carried out using Confocal laser scanning microscopy (LSM780, ZEISS, Germany). The absorbance in the ELISA was measured using the ELISA reader (VersaMAX, Molecular device, U.S.A.).

In Situ Preparation of H2N–Fe-MIL-101/NicF Electrodes by Hydrothermal Method

Before use, the NicFs were sliced into small pieces with a size of 2.7 cm × 2.7 cm. The sliced NicFs were ultrasonicated in 3 M hydrochloric acid for about 10 min to fully remove any oxidized surface. Then the acid-treated NicFs were alternately washed with water and ethanol thrice and dried in an oven at 60 °C. The synthesis of 3D H2N–Fe-MIL-101 nanosheets MOFs were grown on porous nickel foam (NicF) (labeled as H2N–Fe-MIL-101/NicF) surfaces by in situ hydrothermal methods following the reported protocols with modifications.[47,54,55] The detailed procedure is described as follows. Typically, a clear solution of H2Bdc-NH2 (2.5 mmoL, 0.45 g) and FeCl3·4H2O (5 mmoL, 1.35 g) was prepared by dissolving in DMF (15 mL) separately. Then a mixture of the iron solution was added to the H2Bdc-NH2 solution. The solution mixture was allowed to stir for 1 h continuously using a magnetic stirrer at room temperature. Then the resultant solution was carefully passed on to a Teflon-lined autoclave (100 mL) along with a pretreated NicF kept inside at an inclined angle. The autoclave was then placed in a preheated oven for 20 h at 115 °C temperature for hydrothermal reactions to takes place. The autoclave was removed from the oven after natural cooling. The MOF-loaded NicF termed H2N–Fe-MIL-101/NicF was gently removed from the bottom of the autoclave reactor. The as-generated nickel foam and powdered products were collected into separate containers for washings. The products were centrifuged first and then washed several times with alternate solutions of DMF and methanol and finally allowed to dry for 24 h under vacuum to obtain the H2N–Fe-MIL-101/NicF electrode products. The MOF nanosheets materials were subjected to ultrasonication for 1 h in ethanol when structural analyses such as HR-FESEM, TEM, PXRD, FT-IR, XPS, and BET are required.

Immunosensor Design and Fabrication of H2N–Fe-MIL-101/NicF Bioelectrodes for FSH

In this current work, in situ 3D H2N–Fe-MIL-101 MOF nanosheets were grown on NicF skeleton surfaces for the first time using the hydrothermal approach, which is employed as electrode materials for FSH recognition. On account of FSH detection, as-prepared H2N–Fe-MIL-101 MOFs were immobilized with Ab-FSH to specifically detect FSH which is highly associated with regulating the reproductive system of the human body. The development of the entire fabrication of the immunosensor was demonstrated by the considerable changes in electrochemical characteristics of the modified electrodes. The sensing performance was improved by integrating the breathing effect and large surface area of biocompatible H2N–Fe-MIL-101 MOFs and the highly porous nature of NicF substrates that can facilitate the interaction of glycopeptides with MOF materials. The as-created H2N–Fe-MIL-101/NicF electrodes were conjugated with Ab-FSH to execute its electrochemical changes.

Fabrication of FSH Specific Ab-FSH/H2N–Fe-MIL-101/NicF Bioelectrode

To get a stock solution, 50 μL of Ab-FSH (1 mg/mL) was added to the freshly prepared PBS solution (5 mL), and the mixed solution was incubated for 30 min at 4 °C. The metaperiodate reaction causes mild oxidation of carbohydrate units to convert them into formyl groups. The presence of excess sodium metaperiodate was removed by dialysis (500–1000D membrane) using PBS buffer at 4 °C for 2 h. The oxidized Ab-FSH was then allowed to react with H2N–Fe-MIL-101/NicF electrode relatively using carbonate buffer, pH 9.6 at 4 °C for 2 h. This is followed by the addition of sodium cyanoborohydride (5 M) and extended incubation for 30 min at 4 °C. The oxidized glycoprotein (formyl moieties) reacts with amines MOFs and forms Schiff base intermediates. The chemical reduction can stabilize the labile Schiff base interaction. Sodium cyanoborohydride is preferred as a reducing agent which offers a milder reduction in the reductive amination process reducing only the Schiff bases but not the aldehydes. Finally, 25 mL of BSA (0.5 mg/mL) was used to block the nonspecific binding sites without interfering with Ab-FSH binding and incubated for 30 min at 4 °C. The quantification of Ab-FSH bound on the H2N–Fe-MIL-101/NicF electrodes was determined using ELISA and the relevant details are provided in the Supporting Information (Figure S10). Then Ab-FSH treated Fe-MIL-101/NicF electrodes were then tested for FSH detection electrochemically.

Important Parameters of Electrochemical Measurements

To analyze the as-prepared Ab-FSH/H2N–Fe-MIL-101/NicF electrodes for their electrochemical properties, the electrochemical experiments were conducted using three electrode systems and K3[Fe(CN)6]/K4[Fe(CN)6] (1:1, 10 mM) electrolytic bath. The CV and EIS were used to conduct the electrochemical related tests. A common three electrode setup having Ab-FSH/H2N–Fe-MIL-101/NicF electrodes, platinum wire (Pt), and Ag/AgCl (saturated KCl) was used as the working, counter, and reference electrodes, respectively. The impedimetric spectroscopy was measured at an open circuit potential in a frequency range of 1–105 Hz at an amplitude of 5 mV. In this study, EIS data were analyzed and displayed as Nyquist plots. To ensure the accuracy of the experimental data, each measurement was repeated three times at least. The CV profiles were measured in the range of −1 V to +1 V at a scan rate of 100 mVs–1.

Impedimetric Measurement of FSH Analytes

To test the feasibility of the Ab-FSH modified electrodes, a small aliquot (5 μL) of different concentrations of FSH was introduced on the contact area of the Ab-FSH modified H2N–Fe-MIL-101/NicF electrode. The blank analysis was performed for the baseline correction. The LOD was estimated according to the equation: LOD = 3.3 × SD/s, where “SD” represents the standard deviation of the blank and “s” denotes the slope from the calibration curve. The specificity of the developed Ab-FSH/H2N–Fe-MIL-101/NicF electrode was tested against different glycoproteins. The reproducibility of the fabricated electrodes was also tested.