Electrochemical Aptasensors Based on Hybrid Metal-Organic Frameworks

Metal-organic frameworks (MOFs) offer a unique variety of properties and morphology of the structure that make it possible to extend the performance of existing and design new electrochemical biosensors. High porosity, variable size and morphology, compatibility with common components of electrochemical sensors, and easy combination with bioreceptors make MOFs very attractive for application in the assembly of electrochemical aptasensors. In this review, the progress in the synthesis and application of the MOFs in electrochemical aptasensors are considered with an emphasis on the role of the MOF materials in aptamer immobilization and signal generation. The literature information of the use of MOFs in electrochemical aptasensors is classified in accordance with the nature and role of MOFs and a signal mode. In conclusion, future trends in the application of MOFs in electrochemical aptasensors are briefly discussed.


Introduction
Aptasensors are biosensors containing aptamers as biorecognition elements [1,2]. Their progress started since 1990, where several papers reported the method of aptamer selection and emphasized advantages of these new molecules over antibodies [3][4][5]. The term aptamer was derived from Latin aptus (to fit) and Greek meros (part) [6]. The aptamers are synthesized in vitro from a random library of nucleotides followed by their selection by means of affine chromatography containing the conjugates of the analyte molecules. The protocol called SELES (Systematic Evolution of Ligands by EXponential Enrichment) [7] consists of several cycles of selection and amplification of the number of copies that allows specification of the sequences with maximal affinity toward the target [8]. To some extent, the reaction of an aptamer with an analyte is similar to the antigen-antibody interactions and results in reversible formation of a molecular complex. In some cases, such complexes can exert enzyme-like catalytic activity. Thus, the aptamer-hemin complex mimics peroxidase activity [9]. Such complexes have found analytical applications in the assembly of appropriate biosensors [10].
Aptamers are more stable in comparison with proteins in extreme conditions, including the interference with proteases and microbial digestion [11]. They can be easily modified by some tags and functional groups intended for their implementation in the assembly of biosensors by covalent binding [12,13] or to the attachment of labels and linkers [14]. RNA aptamers have found predominant In this review, main attention is paid to the aptasensors with the MOF particles in their assemblies intended to improve performance in the determination of various analytes demanded in medicine, pharmacology, and environmental monitoring.

General Characterizations of MOFs
In accordance with the IUPAC definition [50], MOF is "a coordination network with organic ligands containing potential voids". In turn, a coordination network is described as "a coordination compound extending, through repeating coordination entities, in 1 dimension, but with cross-links between two or more individual chains, loops, or spiro-links, or a coordination compound extending through repeating coordination entities in 2 or 3 dimensions". The term previously used for such structures (hybrid organo-inorganic materials) is considered as imprecise because about all the coordination compounds can be ascribed with this wording. Although the above definition does not assume a crystalline character of the MOF structure, it seems important to distinguish them from the more general term "coordination polymer". In addition to the above terms, some other reticular materials with the properties similar to those of the MOFs are known from the literature. They involve zeolitic imidazolate frameworks [51] and covalent organic frameworks (COFs) [52]. In the latter case, organic building blocks create 3-D networks on themselves, whereas MOFs always include bridging metals.
From the point of view of assembling from appropriate precursors [53], the MOF structures consist of the metal atoms or metal clusters (secondary building blocks) linked with polytopic ligands like terephtalic acid or imidazole fragments. The structures of the most common linkers applied are presented in Figure 1. Metal clusters commonly have a polyhedral structure. Depending on the linkers, they can have various coordination numbers. Thus, Zr MOFs with coordination numbers of 6, 8, 10, and 12 have been reported [54]. This determines the network topology and formation of unusual particle forms like cubic, hexagonal particles, etc. [55].
Most common MOFs described in the assembly of electrochemical sensors and especially biosensors are constructed from carboxylate-based linkers. Their stability toward hydrolysis depends on the strength of the bonds between the carboxylate groups and the metal-based nodes. From the hard/soft acid/base principle, hard carboxylates form more robust structures with the metal ions in a high valence state (Ce(III/IV), Zr(IV), Hf(IV), etc.) [56][57][58][59]. MOFs assembled from Ni-, Cu-, and Co-based nodes and carboxylate-bearing linkers degrade to appropriate oxides in basic media [59]. For the same reason, such MOFs can be used for the synthesis of derived materials with electrocatalytic properties. Among the MOFs based on other metals, many reported examples refer Metal clusters commonly have a polyhedral structure. Depending on the linkers, they can have various coordination numbers. Thus, Zr MOFs with coordination numbers of 6, 8, 10, and 12 have been reported [54]. This determines the network topology and formation of unusual particle forms like cubic, hexagonal particles, etc. [55].
Most common MOFs described in the assembly of electrochemical sensors and especially biosensors are constructed from carboxylate-based linkers. Their stability toward hydrolysis depends on the strength of the bonds between the carboxylate groups and the metal-based nodes. From the hard/soft acid/base principle, hard carboxylates form more robust structures with the metal ions in a high valence state (Ce(III/IV), Zr(IV), Hf(IV), etc.) [56][57][58][59]. MOFs assembled from Ni-, Cu-, and Co-based nodes and carboxylate-bearing linkers degrade to appropriate oxides in basic media [59]. For the same reason, such MOFs can be used for the synthesis of derived materials with electrocatalytic properties. Among the MOFs based on other metals, many reported examples refer to UiO-66 consisting of hexa-zirconium nodes and linear dicarboxylate linkers [60,61]. They exert high chemical stability both in acidic and neutral media but are redox inactive and hence should be first labeled Sensors 2020, 20, 6963 4 of 33 with some appropriate groups like porphyrin fragments [62]. In aptasensors, the high affinity of Zr oxides toward phosphate groups in the DNA sequence make such materials attractive in the immobilization of aptamers with no respect of the protocol of signal measurement. MOFs based on nitrogen-containing linkers are considered as an alternative to the described carboxylate-based MOFs. The ZIF (zeolitic imidazolate framework) family is most popular in electrochemical detection systems. They are assembled with Zn 2+ and Co 2+ cations and are quite stable even in basic media due to the formation of rather narrow and hydrophobic pores [63].
In some cases, metal ions form with appropriate linkers the 2-D structures (linear supramolecular polymers), which are then assembled in the 3-D networks with participation of another metal ion. Such bimetallic MOFs show a uniform regular structure. Their properties, e.g., redox activity, porosity, internal volume, and hydrolytic stability, depend on both implemented metals. Ni cations are often applied as bridging ions. They provide assembly of the hierarchical structure, whereas Zn can be used for primary complexation with organic linkers [64]. In such materials, the integer ratio of the metal atoms (1:1, 1:2, 1:3) is mostly observed and the properties of the products highly depend on the ratio of the reagents mixed in the solvothermal synthesis.
Alternatively, bimetallic heterostructured MOFs can consist of two separated materials (MOFs-on-MOFs) or result from consecutive assembling of appropriate layers (core-shell, or MOFs@MOFs structures) [65]. Such heterostructured materials attract attention due to the higher variety of the properties of the materials and the synergetic effect of both components in the reactions of electrocatalysis and analyte recognition. Combination of two metals offers higher hydrolytic stability of the products against mechanical mixture of appropriated MOFs taken separately [66].
The morphology of common MOF materials is outlined in Figure 2. The 2-D films consisting of linear supramolecular polymer chains (Figure 2A) are mostly assembled on solid supports, e.g., electrode surface or crystalline carrier with regular alternation of the binding sites. Figure 2B represents the MOF crystal based on a single metal and polydentant ligand participating in formation of the chain linking bonds. Figure 2C shows a bimetallic MOF particle, where a second binding metal atom forms the 3-D structure of the product. Finally, Figure 2D,E outline heterogeneous MOF-on-MOF and MOF@MOF (core-shell [67]) structures, respectively.  [60,61]. They exert high chemical stability both in acidic and neutral media but are redox inactive and hence should be first labeled with some appropriate groups like porphyrin fragments [62]. In aptasensors, the high affinity of Zr oxides toward phosphate groups in the DNA sequence make such materials attractive in the immobilization of aptamers with no respect of the protocol of signal measurement. MOFs based on nitrogen-containing linkers are considered as an alternative to the described carboxylate-based MOFs. The ZIF (zeolitic imidazolate framework) family is most popular in electrochemical detection systems. They are assembled with Zn 2+ and Co 2+ cations and are quite stable even in basic media due to the formation of rather narrow and hydrophobic pores [63]. In some cases, metal ions form with appropriate linkers the 2-D structures (linear supramolecular polymers), which are then assembled in the 3-D networks with participation of another metal ion. Such bimetallic MOFs show a uniform regular structure. Their properties, e.g., redox activity, porosity, internal volume, and hydrolytic stability, depend on both implemented metals. Ni cations are often applied as bridging ions. They provide assembly of the hierarchical structure, whereas Zn can be used for primary complexation with organic linkers [64]. In such materials, the integer ratio of the metal atoms (1:1, 1:2, 1:3) is mostly observed and the properties of the products highly depend on the ratio of the reagents mixed in the solvothermal synthesis.
Alternatively, bimetallic heterostructured MOFs can consist of two separated materials (MOFs-on-MOFs) or result from consecutive assembling of appropriate layers (core-shell, or MOFs@MOFs structures) [65]. Such heterostructured materials attract attention due to the higher variety of the properties of the materials and the synergetic effect of both components in the reactions of electrocatalysis and analyte recognition. Combination of two metals offers higher hydrolytic stability of the products against mechanical mixture of appropriated MOFs taken separately [66].
The morphology of common MOF materials is outlined in Figure 2. The 2-D films consisting of linear supramolecular polymer chains ( Figure 2A) are mostly assembled on solid supports, e.g., electrode surface or crystalline carrier with regular alternation of the binding sites. Figure 2B represents the MOF crystal based on a single metal and polydentant ligand participating in formation of the chain linking bonds. Figure 2C shows a bimetallic MOF particle, where a second binding metal atom forms the 3-D structure of the product. Finally, Figures 2D and 2E outline heterogeneous MOF-on-MOF and MOF@MOF (core-shell [67]) structures, respectively. In general, about 70,000 crystalline structures, which exert unique topology, structural backbone, and appended functional groups, have been reported in the Cambridge Structural Database to 2017 [68]. In general, about 70,000 crystalline structures, which exert unique topology, structural backbone, and appended functional groups, have been reported in the Cambridge Structural Database to 2017 [68].
The rather simple protocol of assembly anfogurd variety of the building blocks and linkers make it possible to customize the structures with a wide range of pore size distribution. As a result, the internal surface area of the MOF structures can reach 8000 m 2 /g against 1000 m 2 /g for zeolites and 3500 m 2 /g for activated carbon [69]. For this reason, MOFs have found increasing application in gas storage and Sensors 2020, 20, 6963 5 of 33 separation [70][71][72], drug delivery [73], and as supporting matrix for catalysts [74]. Recently, similar structures have become popular in sensors and biosensors as carriers of recognition elements and signal-generating agents. It should be noted that the high porosity of the MOF crystals is not the only requirement for their successful application in the analyte accumulation and recognition. In many cases, release of the molecules from the internal volume of the MOF pores results in disturbance of their regular structure followed by decay of the adsorption capacity and specificity toward the species determined. This prevents sensor/biosensor recovery after the contact with an analyte and their multiple use. The problem can be solved by formation of the hierarchical structures described above. Thus, Ru bipyridine complexes suitable for electrochemiluminescence measurements and able to form open frameworks are mostly used as modifiers in more rigid structures based on carboxylate-bearing linkers [75,76]. Another approach suggests the combination of the Ru complexes with bridging metal centers [77].
Pore tuning is considered as one of the principal advantages of the MOF structures in chemical analysis and sensor assembling. Thus, the MOF-5 structure assembled from Zn acetate and terephtalic acid possesses a 3-D cubic network with 12 Å pores. Substitution of the linker with 2-metyhlimidazole changes the topology of the network to that typical for zeolites with large open pores of 11.6 Å in diameter interconnected with small ones (3.4 Å). Similar MOF with Cr 3 O clusters has a cubic structure with extra-large pores (30 to 34 Å) [78]. Thus, variation of the precursors allows the assembly of various structures that are uniform on the atomic level and tunable in pore size and its variation.
Another way to modify the 3-D structure of the MOFs is to specify synthesis conditions, namely, to use surfactants and stabilizers like polyvinylpyrrolidone (PVP) [79] or poly(ethylene imine) (PEI) [80] added on the stage of the precursors' mixing and dispersion of low-soluble components in water or aqueous solvents. Thus, the synthesis of Ni MOF with 2-aminoterephtalic acid as a linker has been described in dimethylformamide (DMF), its mixture with water, and that containing PVP [81]. In pure organic solvent, spindle-shaped particles of 1−1.5 µm were obtained, and in 50% DMF, the size of the particles decreased to 700−800 nm and their shape changed to irregular spheres. The addition of the surfactant decreased the size of spherical particles to 350−400 nm. Their shape became more regular. The voltammetric response to methylene blue as a redox indicator changed with the size of the MOF particles deposited on the electrode so that smaller particles produced a higher current. The influence of the solvent was attributed to the deprotonation of carboxylate groups of the linker promoted by water addition and to the PVP influence on the rate of the MOF nuclei growth. Together with the temperature of solvothermal synthesis, the choice of solvent or their mixtures makes it possible to affect the morphology of the particles obtained and their electrochemical properties. However, changes in the currents recorded can be related to the difference in the specific surface area of the particles or in the surface-to-volume ratio if intrinsic redox properties of the MOFs are measured. The influence of the shape itself is less mentioned and discussed.
Three types of the MOFs in the assembly of the aptasensors are described: • "Pure MOFs" obtained from the metal salt and organic linker providing assembly of the 3-D structure. They are mostly synthesized in rather mild conditions (at 80−140 • C) in an autoclave or even at atmospheric pressure during 40−240 min [82]. The product is precipitated, separated, and washed from residuals of reactants and the solvents. Then, the particles are dried and used as received without further mechanical disintegration. Such MOFs mostly contain transient metals (their clusters), which can be involved in redox reactions or act as adsorbents for accumulation of the redox indicators and aptamers. Bimetallic MOFs of the regular structure (MOF-on-MOF and MOF@MOF) described above can also be included in this group though their synthesis is complicated and involves separate steps of the reagent's addition.

•
Hybrid materials consisting of the MOF as a platform for deposition of other components accumulated in their internal volume (low molecular species like redox-active dyes [83]) or on the surface. From inorganic species, hybrid particles can contain metal complexes [84] and oxides [85]. They can vary by the degree of surface loading, conditions of modification, and the properties Sensors 2020, 20, 6963 6 of 33 of the products obtained. Organic modifiers can also be covalently attached to the functional groups of organic linkers (amino group of 2-aminoterephtalic acid as an example). Modification can simplify the following attachment of the MOF-based particles in the biosensor assembly or exert amplified redox properties attributed to the modifier.

•
Nanoparticles obtained by calcination of the MOFs prepared in advance by conventional methods of low-temperature solvothermal synthesis. Actually, the product of calcination does not belong to the MOF family because high temperatures result in carbonization of organic matter and seriously influence the initial 3-D structure of the particles. Nevertheless, the MOF-derived materials show attractive electroconductive and electrocatalytic properties due to the high concentration of mesoporous carbon and oxides of transient metals able to complete redox reactions [86]. Fe 3 O 4 -modified particles derived from Fe containing MOFs in calcination are also used in magnetic separation as a part of the measurement protocol [87].
MOFs are commonly characterized prior to use in the biosensor assembly using conventional tools of nanotechnologies and nanomaterials investigation. Thus, the structure and elemental content of the particles are confirmed by attenuated total reflectance FT-IR spectra, X-ray diffraction analysis and energy-dispersive X-ray spectroscopy (EDX elemental mapping), the morphology and size of the particles by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), surface charge, and hydrodynamic diameter by dynamic light scattering (DLS) [66].
In many cases, the characterization of the MOF particles is performed for a certain content of the reactant mixture used for the synthesis together with the results of molecular modelling and crystallographic presentation of the MOF unit structure (see Figure 3 as an example). This might be useful to follow the relationships between the chemical content and structure of the MOF particles. However, it seems insufficient to assess the contribution of the MOFs to the sensor/biosensor performance. Moreover, such characterization is mostly related to idealized conditions and does not ascribe the same MOFs in contact with the liquid phase, especially on the electrode-solution interface. For the same reason, an unusual shape of the MOF particles quite different from that of more common metals sometimes looks amazing but does not influence the electrochemical properties of the MOF material, mostly independent of the particular crystallographic description.  In addition, electrochemical impedance spectroscopy (EIS) provides valuable information on the conditions of electron exchange on the electrode interface and offers broad opportunities for monitoring various steps of the assembly of the surface layer [89]. In the EIS experiments, the ferri/ferrocyanide redox pair [Fe(CN)6] 3−/4− is used at its equilibrium potential. Changes in the electron transfer resistance and to a lesser extent the interface capacitance make it possible to  Adsorption capacity, internal pore volume, and other porosity characteristics are described by nitrogen adsorption isotherm and differential scanning calorimetry-thermoporosimetry [66]. It should be noticed that the characteristics determined by these methods have a limited relation to the performance of electrochemical sensors. Hydration and specific interactions with polar water molecules and their adsorption on internal walls of the pores significantly affect the quantities of the reagents that can be adsorbed in the MOF particles implemented in the sensor. For this reason, electrochemical aptasensors are mostly characterized by the signals of redox species adsorbed.
In addition, electrochemical impedance spectroscopy (EIS) provides valuable information on the conditions of electron exchange on the electrode interface and offers broad opportunities for monitoring various steps of the assembly of the surface layer [89]. In the EIS experiments, the ferri/ferrocyanide redox pair [Fe(CN) 6 ] 3−/4− is used at its equilibrium potential. Changes in the electron transfer resistance and to a lesser extent the interface capacitance make it possible to conclude about the charge of the layer and its permeability for small ions. It is important that the redox indicator has the negative charge like phosphate residuals of the aptamer chain. This excludes interferences caused by non-specific adsorption or electrostatic accumulation of the redox indicator.

General Assessment and Bibliography Statistics
Biosensor assembling is a protocol of consecutive attachment of biochemical and auxiliary components on the transducer interface to reach optimal conditions for target biochemical interactions and signal generation. Regarding electrochemical biosensors, heterogeneous mediators of electron transfer are mostly required to compensate for decreased conductivity of the surface layer caused by implementation of biopolymers and to amplify the signal of redox active labels and indicators. Immobilization of biomolecules should maintain their microenvironment to be most comfortable for biochemical functioning and protect them from reactive species and undesired working conditions (extreme pH, influence of reactive species on the steps of biosensor assembly and signal measurement, etc.). Besides, immobilization should take into account steric limitations of the analyte binding related to the partial shielding of biochemical receptors on the electrode interface. In the history of biosensors, many approaches have been elaborated for biosensor assembly that offer stable signal and long-term operation of biosensors in different and changing conditions. Below, some of them are briefly considered in relation to the use of the MOFs in aptasensors. It should be clearly noted that the protocols described below do not exhaust all the methods and are limited with those most frequently used within the subject of the review.
Although MOFs belong to rather new materials that appeared in the scope of chemists in the past few decades, they have found intensive application in electrochemical sensors and biosensors from the very beginning. Figure 4 represents the results of the bibliography statistics performed with Web of Science to follow annual changes in the number of publications and general principles of the MOF application in the aptasensor assembly.
Starting from 2012, an exponential growth of the articles devoted to the use of electrochemistry tools in MOF synthesis and application was observed. Meanwhile, a significant part of such works utilized redox activity of the metals in the assembly of the MOF particles as a way to assess the structural specificity of the materials and to monitor the assembling of reticular structures from precursors, especially in 2-D nets on solid supports. Then, the electrocatalytic properties of the metal clusters in the knots of the frameworks were successfully applied in the protocols of mediated oxidation of many organic species attractive from the point of view of medical diagnostics or food safety control. Application of the MOFs in aptasensors covers about 10−12% of the publications and among them about half are devoted to electrochemical aptasensors. As could be seen from the histograms, enormous growth of the interest in the MOF-based aptasensors is expected in the nearest future. It is related to the remarkable variety of their properties and obvious advantages briefly mentioned above in the introduction.
Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors structural specificity of the materials and to monitor the assembling of reticular structures from precursors, especially in 2-D nets on solid supports. Then, the electrocatalytic properties of the metal clusters in the knots of the frameworks were successfully applied in the protocols of mediated oxidation of many organic species attractive from the point of view of medical diagnostics or food safety control. Application of the MOFs in aptasensors covers about 10−12% of the publications and among them about half are devoted to electrochemical aptasensors. As could be seen from the histograms, enormous growth of the interest in the MOF-based aptasensors is expected in the nearest future. It is related to the remarkable variety of their properties and obvious advantages briefly mentioned above in the introduction. The role of the MOFs in the aptasensors described ( Figure 4D) varies from a rather traditional application as a surfaced enhancer and to their use as a source of redox mediators collected or released in the target binding event. MOFs are also compatible with biochemical strategies of the signal amplification assuming the use of enzymatic and DNAzyme amplification or exonuclease-assisted DNA amplification protocols. To some extent, the sandwich assay with two aptamer/DNA sequences utilized for immobilization of the bioreceptor and its labeling can be considered as a biochemical approach previously elaborated for immunoassay. All of the assemblies combining MOFs and aptamers with electrochemical transducer are considered in more detail below in the review. The role of the MOFs in the aptasensors described ( Figure 4D) varies from a rather traditional application as a surfaced enhancer and to their use as a source of redox mediators collected or released in the target binding event. MOFs are also compatible with biochemical strategies of the signal amplification assuming the use of enzymatic and DNAzyme amplification or exonuclease-assisted DNA amplification protocols. To some extent, the sandwich assay with two aptamer/DNA sequences utilized for immobilization of the bioreceptor and its labeling can be considered as a biochemical approach previously elaborated for immunoassay. All of the assemblies combining MOFs and aptamers with electrochemical transducer are considered in more detail below in the review.

Assembling of the Surface Layer
As was briefly mentioned above, MOF particles are mostly deposited on the surface of the transducer by drop casting. In some cases, the electrode can serve as a solid support for their synthesis if it does not assume necessity in high temperatures and pressure. In such cases, the nature of the electrode material is important because it determines the structure of the modifier and characteristics of the electron transfer. Contrary to most modern biosensors, aptasensors with the MOF particles are mostly designed on gold instead of glassy carbon electrodes (GCEs) or screen-printed carbon electrodes frequently applied in enzyme or immunosensors. This might be due to the compatibility of gold with the protocol of the aptamer immobilization via formation of the Au-S bonds between the electrode and terminal thiol group of the aptamer molecule. In addition, Au nanoparticles can be chemically or electrochemically deposited onto the electrodes or on the MOF particles to increase their specific surface area.
Deposition of the MOFs can be accompanied with the introduction of other nanomaterials. Besides the Au nanoparticles mentioned, carbonaceous nanomaterials play the same role of surface enhancement and increase the quantities of aptamer molecules attached to the surface. Carbon nanotubes, reduced graphene oxide, and g-C 3 N 4 nanoparticles also increase the rate of electron Sensors 2020, 20, 6963 9 of 33 transfer and serve as carriers for aptamer immobilization. Polymer binders like chitosan can extend the operation period of aptasensors due to the prevention of leakage of the surface components and better mechanical durability of the surface layer.
Aptamers are immobilized on the electrode interface by physical or covalent immobilization. In the first case, physical adsorption is mostly directed by electrostatic interactions [90]. Although pore volume and size are often discussed in the step of the MOF synthesis optimization, their importance for aptamer immobilization is questionable because of the rather large volume of bioreceptor molecules and necessity to provide accessibility of the analyte molecules, which are often rather big to fill in the internal volume of the MOF crystals. Electrostatic immobilization is addressed to the attraction of phosphate groups of the aptamer chain to the positively charged metal cations of the MOF units. In case of Zr MOFs, the covalent interaction of phosphate residues with ZrO clusters is also discussed as a driving force of the aptamer attachment [91].
Physical adsorption can be additionally amplified by polycationic species added on the stage of the MOF synthesis. The same approach is used for suspending MOF particles prior to their casting on the electrode surface [92]. Hydrophobic interactions of the aromatic π-electrons of the MOF linkers and of the nucleobases in the DNA main sequence are also mentioned as a driving force of physical adsorption [93].
In chemical (covalent) immobilization, terminal groups of aptamers form covalent bonds with certain functional groups of the electrode material or of the additives previously deposited on its surface. The previously mentioned interaction between thiolated aptamer and Au nanoparticles is also related to the covalent immobilization. In comparison with physical adsorption, covalent immobilization is less sensitive to the pH and reaction media content due to the higher stability of the covalent bonds toward hydrolysis and oxidation against coulomb interactions. Then, the formation of the bonds always involves a particular site of the electrode/carrier and of the aptamer molecules. This makes the structure of the product obtained predictable (so-called site-specific immobilization). In comparable conditions, covalently attached aptamer is more accessible for bulky analyte than that electrostatically immobilized. Sufficiently lower steric hindrance of target interactions of the aptamer-analyte is achieved by the introduction of long chain fragments (spacers) in the aptamer molecule. These spacers consist of methylene groups or homonucleobases poly(A)-poly(T) lift the aptamer over the electrode surface and simplify its interaction with the target molecule [94]. Spacer introduction increases the flexibility of the aptamer chain in its linear configuration and simplifies the conformational changes of the aptamer structure in the analyte recognition event. Spacers affect the hydrophobicity of the aptasensor interface and hence can influence the transfer of the organic analyte molecules from aqueous solution to the electrode surface for the following binding with the biorecognition element [95].
Covalent immobilization is mostly performed by bifunctional reagents that form bonds with the functional group in the assembly of the carrier or electrode material and with another one in the structure of an aptamer molecule. Aminated aptamers with terminal primary amino groups are commercially available and can interact with two such reagents, i.e., carbodiimides and glutaraldehyde. The reaction scheme is briefly outlined in Figure 5. Covalent immobilization is mostly performed by bifunctional reagents that form bonds with the functional group in the assembly of the carrier or electrode material and with another one in the structure of an aptamer molecule. Aminated aptamers with terminal primary amino groups are commercially available and can interact with two such reagents, i.e., carbodiimides and glutaraldehyde. The reaction scheme is briefly outlined in Figure 5. Glutaraldehyde reacts with many terminal functions but most efficiently with primary amino groups. In the latter case, the Schiff bases are formed. The reaction of the >C = N-group formation is reversible and sensitive to hydrolysis [96]. Additional treatment with a chemical reductant like NaBH4 results in the formation of a more stable amide bond. In the MOF particles, 2-aminoterephltalic acid can be involved in this reaction together with aminated aptamer. Glutaraldehyde reacts with many terminal functions but most efficiently with primary amino groups. In the latter case, the Schiff bases are formed. The reaction of the >C = N-group formation is reversible and sensitive to hydrolysis [96]. Additional treatment with a chemical reductant like NaBH 4 results in the formation of a more stable amide bond. In the MOF particles, 2-aminoterephltalic acid can be involved in this reaction together with aminated aptamer.
Carbodiimide binding requires aminated and carboxylated reactants that interact with formation of the amido group. Carboxylic groups are present on the surface of carbonaceous materials like carbon nanotubes. Their number can be increased by preliminary oxidation of the carbon materials with strong oxidants. Carboxylic groups of the MOF linker in the structure of terephtalic acids etc. are hardly involved in such a reaction due to their participation in formation of the 3-D MOF structure. Among the many reagents described for carbodiimide binding, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the cyclohexyl derivative are most often used for the aptasensor design. The reaction is accelerated by the addition of N-hydroxysuccinimide (NHS) [97]. Carbodiimide binding is fulfilled in mild conditions at ambient temperature or even at 4 • C. Similar processes can be used for the implementation of labels in the structure of aptamers and auxiliary DNA sequences used for the signal detection.
Affinity immobilization is based on the use of specific interactions with natural or artificial receptors that utilize or mimic natural biospecific interactions [98]. Avidin-biotin interaction is one of the most frequently mentioned examples. A biotin fragment is introduced in the aptamer structure ( Figure 6) while a protein part (avidin or streptavidin) is placed on the electrode. For immobilization on gold, the synthetic derivative of avidin, neutravidin, which is from thiolated groups, is commercially available [99]. Each avidin molecule can bind up to four biotin fragments so that their interaction allows the formation of multilayered structures with participation of various biotinylated counterparts.
Glutaraldehyde reacts with many terminal functions but most efficiently with primary amino groups. In the latter case, the Schiff bases are formed. The reaction of the >C = N-group formation is reversible and sensitive to hydrolysis [96]. Additional treatment with a chemical reductant like NaBH4 results in the formation of a more stable amide bond. In the MOF particles, 2-aminoterephltalic acid can be involved in this reaction together with aminated aptamer.
Carbodiimide binding requires aminated and carboxylated reactants that interact with formation of the amido group. Carboxylic groups are present on the surface of carbonaceous materials like carbon nanotubes. Their number can be increased by preliminary oxidation of the carbon materials with strong oxidants. Carboxylic groups of the MOF linker in the structure of terephtalic acids etc. are hardly involved in such a reaction due to their participation in formation of the 3-D MOF structure. Among the many reagents described for carbodiimide binding, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the cyclohexyl derivative are most often used for the aptasensor design. The reaction is accelerated by the addition of N-hydroxysuccinimide (NHS) [97]. Carbodiimide binding is fulfilled in mild conditions at ambient temperature or even at 4 °C. Similar processes can be used for the implementation of labels in the structure of aptamers and auxiliary DNA sequences used for the signal detection.
Affinity immobilization is based on the use of specific interactions with natural or artificial receptors that utilize or mimic natural biospecific interactions [98]. Avidin-biotin interaction is one of the most frequently mentioned examples. A biotin fragment is introduced in the aptamer structure ( Figure 6) while a protein part (avidin or streptavidin) is placed on the electrode. For immobilization on gold, the synthetic derivative of avidin, neutravidin, which is from thiolated groups, is commercially available [99]. Each avidin molecule can bind up to four biotin fragments so that their interaction allows the formation of multilayered structures with participation of various biotinylated counterparts.  High affinity of avidin-biotin complexation (K D~1 0 −14 -10 −15 M [100]) makes this protocol competitive with covalent attachment of aptamer by carbodiimide binding. Affinity interactions involve other natural receptors specific to a certain fragment in immobilized molecules. Thus, concanavalin A is applied for protein binding via the glycan domain [101]. However, they are rarely used in aptasensor assembling.
Being structurally relative to conventional DNA probes, aptamers can be involved in hybridization with complementary DNA sequences immobilized on the electrode surface. Such an interaction can be also considered as affinity immobilization though double stranded helix formed can then dissociate in the presence of the analyte molecules and leave aptamer sequence to go away form the electrode interface (Figure 7).
Hybridization involves a rather short sequence of the aptamer and solves both the problems of immobilization and accomplishment of a proper orientation of the aptamer against the electrode surface. Interaction with bulky analyte is more effective with orthogonally standing DNA probe-aptamer duplexes. Auxiliary DNA applied for an aptamer collection is called capturing DNA contrary to the signaling DNA that makes the label detectable with an appropriate transducer. It is mostly immobilized to the electrode via Au-SH interactions [102,103]. Application of the capturing and signaling DNA sequences in the sandwich assay is described in more detail below.
Being structurally relative to conventional DNA probes, aptamers can be involved in hybridization with complementary DNA sequences immobilized on the electrode surface. Such an interaction can be also considered as affinity immobilization though double stranded helix formed can then dissociate in the presence of the analyte molecules and leave aptamer sequence to go away form the electrode interface (Figure 7). Hybridization involves a rather short sequence of the aptamer and solves both the problems of immobilization and accomplishment of a proper orientation of the aptamer against the electrode surface. Interaction with bulky analyte is more effective with orthogonally standing DNA probe-aptamer duplexes. Auxiliary DNA applied for an aptamer collection is called capturing DNA contrary to the signaling DNA that makes the label detectable with an appropriate transducer. It is mostly immobilized to the electrode via Au-SH interactions [102,103]. Application of the capturing and signaling DNA sequences in the sandwich assay is described in more detail below.

Signal Measurement Protocols
Recently described aptasensors based on the MOF structures offer a great variety of approaches to the surface layer assembling and signal measurement. They influence the characteristics of appropriate analyte determination to a great extent. The performance of the MOF-based aptasensors is summarized in Table 1. The analytes are presented in alphabetical order. In the description of precursors, hydrate water is omitted from the metal salts formulae.
In electrochemical aptasensors, the following strategies of signal generation have been described and successfully used together with the MOFs:

Signal Measurement Protocols
Recently described aptasensors based on the MOF structures offer a great variety of approaches to the surface layer assembling and signal measurement. They influence the characteristics of appropriate analyte determination to a great extent. The performance of the MOF-based aptasensors is summarized in Table 1. The analytes are presented in alphabetical order. In the description of precursors, hydrate water is omitted from the metal salts formulae.
In electrochemical aptasensors, the following strategies of signal generation have been described and successfully used together with the MOFs: • Measurement of permeability of the surface layer for small ions.

•
Redox signals of labels implemented in the aptamer or auxiliary DNA sequences.

•
Redox signals related to the MOF components (mainly transient metals).

Permeability Assessment
These protocols are based on the fact that specific aptamer-analyte interactions result in the implementation of bulky inert molecules preventing the access of redox-active species to the electrode surface and hence decreasing their signal recorded by voltammetry. Alternatively, increased charge transfer resistance can be recorded in the presence of the [Fe (CN) 6 ] 3−/4− ions as a redox probe. The scheme of the reactions is outlined in Figure 8. These protocols are based on the fact that specific aptamer-analyte interactions result in the implementation of bulky inert molecules preventing the access of redox-active species to the electrode surface and hence decreasing their signal recorded by voltammetry. Alternatively, increased charge transfer resistance can be recorded in the presence of the [Fe (CN)6] 3−/4− ions as a redox probe. The scheme of the reactions is outlined in Figure 8. This protocol first elaborated for immunoassay techniques shows good results for bulky analyte molecules like proteins or living cells. In case of small analytes, aptamers with guanine residues change their configuration from a linear one to a G4 quadruplex [150,151]. This results in a denser packing of the surface layer and suppresses the transfer of redox indicators to the electrode.
The MOFs are mostly utilized as aptamer carriers. The MOF particles can be decorated with Au nanoparticles to extend the surface area and simplify immobilization of thiolated aptamers. Non-specific adsorption is main factor influencing the sensitivity and selectivity of the response, especially in the presence of proteins attaching to the bare gold surface by cysteine residuals. For this reason, the electrodes are additionally treated after the aptamer immobilization with inert organic thiols, preventing such adsorption. 6-Mercaptohexanol is used in many protocols due to its hydrophilicity and easy attachment to the naked part of the electrode. In both EIS and DPV measurements, a [Fe(CN)6] 3−/4− redox indicator is applied so that the sensitivity of both methods is expected to be close to each other. However, voltammetry often shows slightly lower limits of detection (LOD) than EIS contrary to the common behavior of similar immune-and DNA sensors. On the one hand, this can be related to a high resolution of the peaks in DPV and the similar method of square wave voltammetry (SWV). On another hand, EIS data are much more affected by surface phenomena like counter ion transfer and high charge capacity of the interface in comparison with the voltammetry measurements. Meanwhile, the EIS parameters offer more information on the mechanism of signal generation. For this purpose, trends in the changes of the electron transfer resistance are compared with those of capacitance. If the diffusion factor dominates and increased resistance is caused by a lower rate of redox indicator transfer, capacitance is insensitive to the surface layer assembling, including the addition of an analyte that changes irregularly. If the charge transfer resistance is affected by the charge separation, the electron transfer resistance and capacitance tend to change in opposite directions (increasing resistance vs. decreasing capacitance). It should be mentioned that the impedimetric determination requires the selection of most suitable equivalent circuit for data fitting and control of non-ideal behavior of the system (correspondence of the constant phase element to the real capacitance of the electrode interface). The latter requirement related to the assessment of the roughness factor is rarely checked in the EIS measurements with the MOF-based aptasensors.
Another drawback of the measurement scheme described is the necessity to quantify the decay of the signal, which is maximal for a blank experiment and decreases with increasing analyte This protocol first elaborated for immunoassay techniques shows good results for bulky analyte molecules like proteins or living cells. In case of small analytes, aptamers with guanine residues change their configuration from a linear one to a G4 quadruplex [150,151]. This results in a denser packing of the surface layer and suppresses the transfer of redox indicators to the electrode.
The MOFs are mostly utilized as aptamer carriers. The MOF particles can be decorated with Au nanoparticles to extend the surface area and simplify immobilization of thiolated aptamers. Non-specific adsorption is main factor influencing the sensitivity and selectivity of the response, especially in the presence of proteins attaching to the bare gold surface by cysteine residuals.
For this reason, the electrodes are additionally treated after the aptamer immobilization with inert organic thiols, preventing such adsorption. 6-Mercaptohexanol is used in many protocols due to its hydrophilicity and easy attachment to the naked part of the electrode. In both EIS and DPV measurements, a [Fe(CN) 6 ] 3−/4− redox indicator is applied so that the sensitivity of both methods is expected to be close to each other. However, voltammetry often shows slightly lower limits of detection (LOD) than EIS contrary to the common behavior of similar immune-and DNA sensors.
On the one hand, this can be related to a high resolution of the peaks in DPV and the similar method of square wave voltammetry (SWV). On another hand, EIS data are much more affected by surface phenomena like counter ion transfer and high charge capacity of the interface in comparison with the voltammetry measurements. Meanwhile, the EIS parameters offer more information on the mechanism of signal generation. For this purpose, trends in the changes of the electron transfer resistance are compared with those of capacitance. If the diffusion factor dominates and increased resistance is caused by a lower rate of redox indicator transfer, capacitance is insensitive to the surface layer assembling, including the addition of an analyte that changes irregularly. If the charge transfer resistance is affected by the charge separation, the electron transfer resistance and capacitance tend to change in opposite directions (increasing resistance vs. decreasing capacitance). It should be mentioned that the impedimetric determination requires the selection of most suitable equivalent circuit for data fitting and control of non-ideal behavior of the system (correspondence of the constant phase element to the real capacitance of the electrode interface). The latter requirement related to the assessment of the roughness factor is rarely checked in the EIS measurements with the MOF-based aptasensors.
Another drawback of the measurement scheme described is the necessity to quantify the decay of the signal, which is maximal for a blank experiment and decreases with increasing analyte concentration. For such systems, not only the current (resistance) deviation but also the uncertainty of the concentrations related to the signal should be assessed, especially near the LOD value. The signal-to-noise ratio commonly used for the LOD calculation does not work properly for the calibration slopes with a relatively slow slope.

Signals of the Redox Labels and Indicators
Although the [Fe(CN) 6 ] 3−/4− indicator remains the most frequently used in aptasensors, other species have found increasing application for signal generation. Some of them denoted as labels are attached to the carrier or electrode surface while other ones (redox indicators) are diffusionally free and can be accumulated in the surface layer due to analyte binding. In case of MOFs, they can serve as specific sorbents for redox-active species. Their activity is then used for the signal measurement. Thionine [121,142,149], fullerene [126], ferrocene [121], and toluidine blue [128] have also been applied for voltammetric signal detection. The signal of labels covalently attached to the electrode surface was the most sensitive for the sandwich assay. In them, analyte is captured by the aptamer attached to the electrode and the complex formed is involved in specific binding with another (signaling) aptamer-bearing label. The scheme of the sandwich assay with aptasensor is schematically presented in Figure 9. concentration. For such systems, not only the current (resistance) deviation but also the uncertainty of the concentrations related to the signal should be assessed, especially near the LOD value. The signal-to-noise ratio commonly used for the LOD calculation does not work properly for the calibration slopes with a relatively slow slope.

Signals of the Redox Labels and Indicators
Although the [Fe(CN)6] 3−/4− indicator remains the most frequently used in aptasensors, other species have found increasing application for signal generation. Some of them denoted as labels are attached to the carrier or electrode surface while other ones (redox indicators) are diffusionally free and can be accumulated in the surface layer due to analyte binding. In case of MOFs, they can serve as specific sorbents for redox-active species. Their activity is then used for the signal measurement. Thionine [121,142,149], fullerene [126], ferrocene [121], and toluidine blue [128] have also been applied for voltammetric signal detection. The signal of labels covalently attached to the electrode surface was the most sensitive for the sandwich assay. In them, analyte is captured by the aptamer attached to the electrode and the complex formed is involved in specific binding with another (signaling) aptamer-bearing label. The scheme of the sandwich assay with aptasensor is schematically presented in Figure 9. The sandwich assay is most effective when the MOF particles are utilized as labels. Thus, in the aptasensor for detection of β-amyloid oligomers as biomarkers of Alzheimer' disease, both capturing and signaling aptamers were immobilized on the Au nanoparticles. However, capturing aptamer was attached to the GCE surface while signaling aptamer was placed on the surface of the Cu MOF. As a result of the sandwich assay, each analyte molecule resulted in the linkage of the MOFs particles to the electrode interface. Their quantities were monitored by the reduction peak current attributed to the partial reduction of Cu 2+ ions in the assembly of the MOF particles [106]. It should be noted that the accessibility of the Cu 2+ ions in the assembly of the MOF particles has not been proved by an appropriate electrochemical tool and the measurement required preliminary deaeration of the working solution to eliminate the interfering effect of dissolved oxygen. The Cu 2+ /Cu + pair was also applied for detection of thrombin [139]. Here, Cu MOF based on 1,4-cyclohexanedicarboxylic acid was applied as a modifier of the GCE covered with the Au nanoparticles and thiolated aptamer. Specific interaction with thrombin decreased the DPV signal of Cu 2+ though the reasons of such a response remain unclear.
The cathodic peak current of the Pb 2+ ions released from appropriate MOFs treated with nitric acid was applied for the determination of carcinoembryonic antigen [109]. The sensitivity of the response was additionally increased by the hybrid chain reaction, which resulted in the formation of a dendritic DNA scaffold. The formation of appropriate products of the DNA amplification was monitored by a Ru(NH3)6 3+ redox indicator. Its oxidation current was proportional to the quantity of the double-stranded DNA scaffolds on the electrode surface.
Zr MOFs synthesized from ZrCl4 and 2-aminoterephtalic acid and saturated with Pb 2+ or Cd 2+ ions were successfully applied for the determination of antibiotics [116,127]. The Zr MOFs particles were modified with appropriate aptamers and saturated with the metal ions. They were then The sandwich assay is most effective when the MOF particles are utilized as labels. Thus, in the aptasensor for detection of β-amyloid oligomers as biomarkers of Alzheimer' disease, both capturing and signaling aptamers were immobilized on the Au nanoparticles. However, capturing aptamer was attached to the GCE surface while signaling aptamer was placed on the surface of the Cu MOF. As a result of the sandwich assay, each analyte molecule resulted in the linkage of the MOFs particles to the electrode interface. Their quantities were monitored by the reduction peak current attributed to the partial reduction of Cu 2+ ions in the assembly of the MOF particles [106]. It should be noted that the accessibility of the Cu 2+ ions in the assembly of the MOF particles has not been proved by an appropriate electrochemical tool and the measurement required preliminary deaeration of the working solution to eliminate the interfering effect of dissolved oxygen. The Cu 2+ /Cu + pair was also applied for detection of thrombin [139]. Here, Cu MOF based on 1,4-cyclohexanedicarboxylic acid was applied as a modifier of the GCE covered with the Au nanoparticles and thiolated aptamer. Specific interaction with thrombin decreased the DPV signal of Cu 2+ though the reasons of such a response remain unclear.
The cathodic peak current of the Pb 2+ ions released from appropriate MOFs treated with nitric acid was applied for the determination of carcinoembryonic antigen [109]. The sensitivity of the response was additionally increased by the hybrid chain reaction, which resulted in the formation of a dendritic DNA scaffold. The formation of appropriate products of the DNA amplification was monitored by a Ru(NH 3 ) 6 3+ redox indicator. Its oxidation current was proportional to the quantity of the double-stranded DNA scaffolds on the electrode surface. Zr MOFs synthesized from ZrCl 4 and 2-aminoterephtalic acid and saturated with Pb 2+ or Cd 2+ ions were successfully applied for the determination of antibiotics [116,127]. The Zr MOFs particles were modified with appropriate aptamers and saturated with the metal ions. They were then immobilized by interaction with antibodies on the surface of magnetic beads. The reaction with target analytes resulted in release of the aptamers and their reaction with the drugs. Then, the products were digested with the RecJ f exonuclease and analytes returned to the aptasensor for recycling. The released MOFs generated cathodic signals of Pb 2+ and Cd 2+ reduction. Their simultaneous determination by SWV made it possible to detect two drugs in one measurement. The reaction scheme was applied for sensitive determination of kanamycin, oxytetracycline, and chloroamphenicol. Similar to the above examples, Cd 2+ /Cd 0 [129], Co 2+ /Co 3+ [143], and Ce 3+ /Ce 4+ [142,146] redox pairs derived from the MOF structure have been applied for the detection of other target aptamer-analyte interactions.
Methylene blue deserves special attention due to its importance and frequency of application in DNA sensors and aptasensors. It can intercalate double-stranded DNA helix and/or interact at minor grooves of the DNA helix. In addition, it can be covalently attached to the terminal tags of the aptamers and auxiliary DNA sequences and changes its redox signal in accordance with accumulation on the electrode interface or partial shielding in the target biomolecular interactions at the proximity of the electrode. In addition, methylene blue can be used for saturation of the MOFs utilized as a label of the aptamer immobilized by hybridization with the complementary DNA sequence (see above in Figure 7) [132]. The reaction with patulin as an analyte resulted in release of the aptamer from the surface so that the cathodic peak current of methylene blue was decreased. In a similar manner, aptamer bearing covalently attached methylene blue was first adsorbed on the black phosphorus particles mixed with Mn MOF on the electrode surface and then removed from the surface in specific binding of stress-induced phosphoprotein 1 [138] ( Figure 10A). The reaction was monitored by a decrease of the DPV current of methylene blue reduction.
Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors target analytes resulted in release of the aptamers and their reaction with the drugs. Then, the products were digested with the RecJf exonuclease and analytes returned to the aptasensor for recycling. The released MOFs generated cathodic signals of Pb 2+ and Cd 2+ reduction. Their simultaneous determination by SWV made it possible to detect two drugs in one measurement. The reaction scheme was applied for sensitive determination of kanamycin, oxytetracycline, and chloroamphenicol. Similar to the above examples, Cd 2+ /Cd 0 [129], Co 2+ /Co 3+ [143], and Ce 3+ /Ce 4+ [142,146] redox pairs derived from the MOF structure have been applied for the detection of other target aptamer-analyte interactions. Methylene blue deserves special attention due to its importance and frequency of application in DNA sensors and aptasensors. It can intercalate double-stranded DNA helix and/or interact at minor grooves of the DNA helix. In addition, it can be covalently attached to the terminal tags of the aptamers and auxiliary DNA sequences and changes its redox signal in accordance with accumulation on the electrode interface or partial shielding in the target biomolecular interactions at the proximity of the electrode. In addition, methylene blue can be used for saturation of the MOFs utilized as a label of the aptamer immobilized by hybridization with the complementary DNA sequence (see above in Figure 7) [132]. The reaction with patulin as an analyte resulted in release of the aptamer from the surface so that the cathodic peak current of methylene blue was decreased. In a similar manner, aptamer bearing covalently attached methylene blue was first adsorbed on the black phosphorus particles mixed with Mn MOF on the electrode surface and then removed from the surface in specific binding of stress-induced phosphoprotein 1 [138] ( Figure 10A). The reaction was monitored by a decrease of the DPV current of methylene blue reduction.
As an example of a more sophisticated approach of methylene blue application, a pinhole aptamer was covalently attached to the electrode and saturated with methylene blue accumulated in the hybridized part of the aptamer molecule ( Figure 10B). Target reaction with E. coli results in opening of the circle of the aptamer and release of the methylene blue molecules so that its DPV signal decreased with the cell number in the sample tested [113].  As an example of a more sophisticated approach of methylene blue application, a pinhole aptamer was covalently attached to the electrode and saturated with methylene blue accumulated in the hybridized part of the aptamer molecule ( Figure 10B). Target reaction with E. coli results in opening of the circle of the aptamer and release of the methylene blue molecules so that its DPV signal decreased with the cell number in the sample tested [113].

Other Detection Techniques
In addition to the high selectivity of aptamer-analyte interaction, the use of MOFs offers amplification of the signal via implementation of catalytic cycles. For this purpose, horseradish peroxidase [127,137] was used in the framework of the traditional competitive and sandwich assay with hydroquinone substrate and redox detection of benzoquinone formed in the reaction. Besides, catalytic reaction of H 2 O 2 oxidation catalyzed by the MOFs bearing noble metals has been proposed for thrombospondin-1 [146] and led [133] determination. Pd nanoparticles on the Fe-based MOF carrier were involved in catalytic oxidation of tetramethylbenzidine monitored by voltammetry [122]. The measurement systems described can be considered as peroxidase-mimicking systems. The only example of other catalytic systems is the detection of lipopolysaccharides with the Ce-based MOFs bearing Au nanoparticles that catalyze oxidation of ascorbic acid [118]. In all the cases described, the aptasensors show LOD values below picomolar levels and a rather wide range of the analyte concentrations determined. The idea to use MOFs as a catalyst carrier is related to the rather high surface-to-volume ratio and significant increase of the catalyst quantities attached to the reaction site against conventional techniques with the same metal nanoparticles directly deposited on the electrode or introduced as labels in the reactants.
On the other hand, the possible interfering influence of readily oxidized compounds and partial poisoning of the catalytic particles by thiolated organic species can be expected. In most cases, the investigation of matrix effects in a real sample assay is rather limited and leaves space to think about possible underestimation of the analyte content in complex media.

Conclusions
MOFs belong to rather 'new' materials that appeared in the focus of the researchers involved in biosensor development about 10 years ago. Most of the literature devoted to the application of MOFs in electrochemical (bio)sensors covers the period from 2010. The interest in MOFs is mostly related to the diversity of their properties and was inspired to a high extent by great interest in nanotechnologies and nanomaterials in many areas of science and industry. It should be, however, noticed that most of the MOF materials described in the articles related to the design of the electrochemical sensors and biosensors do not belong to nano scale. Their size is mostly about 0.1-1 µm (mesoparticles). Hence, some obvious advantages of nanoparticles (high surface-to-volume ratio, catalytic activity, aggregation-disaggregation abilities, and high chemical and electrochemical activity) cannot be simply extended to the MOF materials and should be proved for each particular case. Application of the MOFs started from their use as reticular materials in gas sensing, storage, and separation. Traditionally, characterization of the new MOF materials is based on crystallography data and porosity assessment though aqueous solutions offer their own requirements and limitations for use of such particles. The idea to apply the MOFs as containers for small molecules (redox indicators) seems in this respect evident but not always effective. Thus, leaching metal cations from the internal volume of the MOF particles was mentioned as one of the reasons of the high background signal [98]. The use of metal atoms positioned in the knots of the 3-D networks requires further investigation because the rigid structure of the MOF can prevent direct contact of such cations with the electrode. To some extent, the last limitation can be excluded by implementation of the MOF particles into the conductive matrix [113,119,131]. In many cases, nanoparticles of noble metals are deposited on the MOF surface. They serve as support for the immobilization of thiolated aptamers and catalysts for voltammetric signal generation. In this sense, MOFs are hardly different from carbonaceous nanomaterials and advantages of their application do not seem obvious.
All of the above mentioned is applicable for electrochemical aptasensors utilizing MOFs to enhance surface square, immobilize aptamers, and generate redox signals. The use of bulky and electrochemically inactive aptamer molecules makes the aptasensor behavior sensitive toward the conditions of electron transfer. Contrary to electroconductive carbonaceous materials, MOF particles offer a shuttle mechanism of the electron exchange with participation of both free and immobilized mediators, including the metals as counterparts of the MOF structure. Such an electron transfer path is easily broken by the inclusion of analyte molecules. For this reason, aptamers based on the MOF structures often show a record sensitivity of detection especially for small analyte molecules that affect the transfer of conventional redox indicators like [Fe(CN) 6 ] 3−/4− to a rather small extent if common approaches of the biosensor assembling are used.
Although most of the articles devoted to the electrochemical aptasensors based on MOF materials do not provide a comprehensive comparison of the characteristics with conventional analogs, it can be concluded that introduction of MOF particles can decrease the LOD values by at least one order of magnitude against the application of similar heterogeneous mediators of electron transfer. The difference might be even higher if MOF particles are used as parts of indicators attached to the electrode interface via target biochemical interactions with analytes. These benefits follow from the size of the particles and multiplication of the signal due to the much higher number of redox sites involved in the electron transfer. Appropriate examples are presented in Table 1. It should also be noted that other figures of merit like a dynamic range of concentration or measurement duration did not demonstrate similar progress against other biosensor materials. Then, application of biochemical approaches to the signal increase, including implementation of auxiliary pinhole aptamers and exonuclease-assisted DNA amplification, the level of influence of other components of the aptasensor assembly.
Summarizing the modern progress in the development of aptasensors based on the MOF materials, one can conclude that further achievements can be expected from the broadened application of hybrid MOF-on-MOF and MOF@MOF particles that combine the advantages of several metals and linkers and can be easily adapted to a particular measurement mode or signal detection requirements. Thus, the use of such hierarchic materials with Ru bipyridine complex shows high efficiency of electrochemilumenscent measurements [124]. Such hybrid metal particles are free from drawbacks related to the leaching metals from the modifier layer and offer better opportunities of the analyte measurements related to very low background signal. The sensitivity of the assay is greatly increased by covalent linking of the MOF particles as labels to the aptamer or auxiliary DNA sequences used.
The variety of hybrid particles can also be extended by variation of the organic linkers and conditions of the synthesis, which can involve chemical and electrochemical steps and variation of the solvents and temperatures applied. A similar conclusion can be made about MOF-derived materials, where the initially synthesized MOF particles are then calcinated at high temperature or treated with chemical reagents. The products do not formally belong to the MOF family but retain the porosity, specific shape, and high adsorption capacity typical for precursors. Although many such derived materials have an alternative path of synthesis, the use of MOF precursors might be beneficial from the point of view of simplicity, and labor and time intensiveness.
The following trends in the further progress of MOF-based aptasensors can be mentioned.

1.
Faster progress is expected in the multiplex assay and simultaneous determination of several analytes based on the number of aptamers introduced in the aptasensor structure.

2.
Growing interest will be directed to the signal-on aptasensors, where an increased concentration of the analyte increases their response. This kind of sensor seems more beneficial both from the point of view of metrological assessment of the results and application of the aptasensors in the real sample assay.

3.
Although the term 'MOF' is mostly related to the use of the linkers with carboxylic binding groups, the variety of the organic part of the MOFs will be increased by the introduction of new structures and precursors derived from imidazole fragments and some others exerting an unusual architecture of the internal space of the particles and their morphology.

4.
The interest in the synthesis of new MOF materials will be shifted to their implementation in supramolecular structures consisting of supramolecular polymers and aptamer molecules.