Review—Recent Trend on Two-Dimensional Metal-Organic Frameworks for Electrochemical Biosensor Application

Monometallic 2D MOFs generally consist of single metal and organic ligands. Their structures and characteristics depend on the selection of metal and ligand in the desired formation. Liu et al. prepared two types of 2D MOF monometallics using ultrasonic assisted precipitation methods. The MOF comprised of Nickel (Ni) or Cobalt (Co) metal-ion and H₂BDC (1,4-benzenedicarboxylic acid) ligands. The abbreviation of these MOFs are NiBDC and CoBDC. Both MOFs had similar structure of nanosheets with the thickness of 3 nm that was resulted from three or four coordination layers. Their morphological structure is shown in Figs. 2a and 2b.³⁴ Zhao et al. had synthesized another type of 2D monometallic MOF with a Cu metal center. The MOF had a nanosheet structure in the shape of a square lamellar, as shown in Fig. 2c, with the thickness in the range of 5–25 nm and a lateral size of 0.5–4 mm. This structure is related to the nucleation and crystal growth by the slow diffusion process in the intermediate layer. Continuous diffusion of Cu²⁺ and BDCA linker to the middle layer produces more Cu-BDC nanosheets.³⁵ Co-BDC has similar coordination with its Ni counterpart. The metal is octahedrally bonded with six oxygens from BDC ligands and each metal is connected to each other in the certain plane direction forming metal-oxygen layers.^36,37 The layer then bridged to other layers by BDC ligands in another direction. In the case of Cu-BDC, the MOF consisted of two 5-coordinate copper cations in which they covalently bond to each other.³⁸ Each pair of copper bridged with four BDC ligands to other pairs forming a 2D framework.

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Bimetallic 2D MOFs are produced by combining two metals at a certain ratio or substituting a few metal atoms with another metal atom. The resulting structure of 2D MOFs highly depends on the ratio of the two metals. The incorporation of second metal aims to modify the structure and electronic properties of 2D MOFs.^39,40 Ma et al.⁴¹ found that the enhancement of electrochemical properties of NiCo–MOF was caused by the partial electron transfer from Ni²⁺ to Co²⁺ through oxygen in the BDC ligand. Moreover, the synergetic effect of NiCo bimetal MOF also contributed to its high electrochemical activities. The corresponding structure and SEM images of Ni–Co MOF are shown at Fig. 3. However, Li et al. reported a slightly different result. In their NiCo–MOF, the electron transfer was from Co to Ni as observed from the X-ray Photoelectron Spectroscopy (XPS) measurement.⁴² Another study by Hao et al. reported that adding Fe atom to Ni–MOF system nanosheets, in the case of NiFe–MOF, could significantly improve the kinetics of the charge transfer reaction.⁴³

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Furthermore, Zhou et al. had successfully synthesized 2D bimetallic ZnZr–MOF with a new strategy that is MOF-on-MOF method. The structure displayed the bonding between Zn–MOF nanosheet and Zr–MOF layer. The report also found that the order of metal precursors and organic ligands addition would affect chemical structure, crystalline properties, and surface functionality of 2D bimetallic ZnZr–MOFs. The order of adding metal precursors is the difference in the process of forming Zn-MOF-on-Zr-MOF or Zr-MOF-on-Zn-MOF. This significantly affected different structure and characteristics of MOFs. In detail, Zn-MOF-on -Zr-MOF will have better sensing properties than Zr-MOF-on-Zn-MOF-, Zn-MOF-, and Zr-based sensors. This is because Zn-MOF-on-Zr-MOF has a stronger bioaffinity and higher stability. This can be explained by the Zr–MOF substrate that assists the penetration of Zn (II) ions into the Zr–MOF node, which resulting in complete integration of Zn–MOF and Zr–MOF. Meanwhile, Zr (IV) ions cannot easily penetrate the Zn–MOF interior because of the small pore size, so that Zr–MOF will cover the surface of Zn–MOF. Zn-MOF-on-Zr-MOF has a unique flower-like structure with a size around 10 ± 2 μm and has no clear lattice spacing, whereas Zr-MOF-on-Zn-MOF has a similar structure to pure Zr–MOF with identical lattice fringes are widely distributed.⁴⁴ Another study showed that the addition of Zn to the NiZn–MOF nanosheet could adjust the porosity and electronic structure of the nanosheet. The NiZn–MOF had a flower- like structure assembled from nanosheets providing a high specific surface area and a fast electron/mass transfer channel. This fast charge transfer might be resulted from the synergistic interaction of two metal ions in this material.⁴⁵

Generally, the stability of MOFs depend on the metal nodes chemical stability that is strongly influenced by the properties and the environment of metal nodes itself, such as rigidity, size of the organic ligands that are connected to metal nodes, and the number or type of surrounding metal nodes interconnected via organic ligands. As an example, in bimetallic NiCo–MOF (NiCo(-BTB)4(bipy)3), not only the Ni-based, but also the Co-based paddle wheel SBUs (secondary building units) interconnects via the organic ligands. This coordination have a more stable framework as compared to the pure Ni–MOF.⁴⁶ The stronger interaction of the metal-ligand bond will protect the MOFs against hydrolysis. Moreover, in the mixed metal or bimetal MOFs, replacing or combining metal nodes with a less valent metal ion will destabilize the final MOFs product, which resulting in a charge deficit that needs to be compensated by exchangeable cations.⁴⁷

The morphological structure of the bimetallic 2D MOFs also depends on the ratio of the two metals themselves. Y. Hao et al. synthesized Ni–BDC, Fe–BDC, and Ni_0.75Fe_0.25-BDC, then observed the morphology transformation after Fe addition. The Ni_0.75Fe_0.25-BDC exhibited 2D structure that was adapted from Fe–BDC. This transformation caused more efficient catalytic performance of 2D MOF and also improved its stability and electrochemical properties. The Other Ni:Fe ratios such as Ni_0.5Fe_0.5-BDC and Ni_0.25Fe_0.75-BDC, had a similar structure to the raw ratio.⁴³ In addition, Lim et al. also found that the addition of Zn could modify the structure of Co–MOF and significantly reduced the thickness of nanoflakes, as shown in Fig. 4. This reduction in thickness allowed a reduction in resistance and provided a larger surface area helped improving the electrochemical performance.⁴⁰

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Alongside the number of metal ions, the types of ligands also influence 2D MOFs structures. Ligands in 2D MOFs can be in the form of single-type ligands or mixed-type ligands which are further explained in the following sections.

2D MOFs can be synthesized using one type of ligand which has two or more binding groups and positioned at the appropriate coordination. The types of ligand commonly used in 2D MOFs are aliphatic organic ligands including hydroxy acetic acid, diacetic acid, and adipic acid.^17,48 Ligands with high symmetry (e.g. C3, C4, D3h and D4h) are commonly used to design 2D MOFs. The C3 and D3h symmetrical ligands are used to form 2D MOF that extend in the equilateral triangular direction to design 2D MOF formation. Those symmetrical ligands have a lot of attention because of their ability to form 2D MOF with a good conductivity. Different results will be obtained if 2D MOF preparation employs D4h symmetry ligands. Coordination with metal will cause the ligand to extend in quadrilateral direction. It has been reported that the D4h symmetry ligand is popular to be used to prepare 2D MOF due to its predictable structure and viability to afford metal doping, especially for D4h ligand that contain porphyrin.¹⁷ In addition, there are also aromatic ligands with carboxylic groups, such as 1,4-benzene dicarboxylic acid and 2,2'-dithiodibenzoic,⁴⁹ and N-heterocyclic rings, such as pyridine. Both are preferred for the preparation of 2D MOF because of their rigid structure and fixed directional angle that make the 2D MOF structures become simpler.^50–53

B. Liu et al. successfully synthesized 2D Co-MOF using 1,4-benzene dicarboxylic acid (BDC) ligand with bottom up method resulting a uniform two-dimensional thickness and exposed metal atoms. This structure had an impact on a faster and more stable electron transfer. As well-known, the attractive characteristics of the 2D MOFs are their interconnected porous structure and high surface area, which can increase electron transfer and mass diffusion. In this case, the Co ion is effectively coordinated with 1,4-BDC ligand,³⁴ In most cases, BDC ligands are used to configure 2D MOF because of the ability to form 2D structures very well.^54,55 Moreover, the modification of BDC ligand with –NO₂ group may affect the electron density of the dicarboxylic ligands. Therefore, different physical and chemical properties of MOF can be obtained.⁵⁶

2D MOF with single-type ligands has limited possible coordinate angles due to the limited number of ligands suitable to form 2D MOFs. This limitation results in a reduced option to tune the characteristic of the 2D MOFs, knowing that the various features of MOF are due to their numerous combination of metal-ion and ligands.^17,57 These drawbacks can be solved by adding more ligands or commonly called as the mixed-types ligand. Lei Wang et al. reported a combination of 4,4'-bipyridine and 1,2,4-benzene tricarboxylate ligand in coordination with Cu metal.⁵⁸ In their case, 2D structure was constructed by Cu₂(CO₂)₄(4,4'-bpy)₂ paddle-wheel like as a secondary building unit (SBU) and 1,2,4-BTC as a linker. X.F. Yang et al. also reported the synthesis of 2D MOF nanosheets, [Cu(Hcpbda)(bpp)(H₂O)]_n, by using two different ligands, H₃cpbda and 1,3-bis(pyridin-4-yl)propane (bpp), with the illustration of the ligand structures are shown in Fig. 5 and the comparison of the crystalline structure at single and mixed type ligand is shown in Fig. 6. Deprotonated Hcpbda²⁻ ligand coordinated with Cu cations forming 1D chains. The bpp ligands acted as a bridge that connect each Cu cation in another direction. They also reported some possible coordination of multi carboxylic acid with different N-donor ligands to produce a two-dimensional MOF.⁵⁹ They concluded that the coordination angles, the ligand length and the size of the rigid ligand affected the 2D structural construction. N-donor ligands, such as ligands that contain pyridine and imidazole, are more developed because of their ability to be the excellent bridge to produce attractive MOF structures.⁶⁰ O-donor ligands, such as polycarboxylate and naphthalene disulfonate, offer stable coordination in MOF formation due to their ability to strongly bind with the metal center.^60,61

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Layered MOF can be achieved by combination of two or more ligands to get entanglement phenomena. Entanglement is an interesting phenomenon in MOF that are constructed by longer organic ligands, such as 1,4-bis(benzimidazole-1-yl)butane (bimb) and the rigid 1,6-naphthalene disulfonate (1,6-NDS).⁶⁰ It produces large voids in its network. Some examples of entanglement systems are interpenetration, polycatenane, polyrotaxane, polythread, interdigitation and polyknotting. Zhao et al. constructed the [Cd(Seb)(1,4-mbix)] entanglement MOFs with the aliphatic dicarboxylic acid (Seb) as the host ligand and two isomeric bis(2-methyl-imidazole) ligands (1,4-mbix) as the auxiliary ligands and Cd as a metal ion. The 1,4-mbix ligand and the aliphatic dicarboxylate anion ligand were in parallel binding to form a 2D-2D interpenetration network. The Cd (II) ion had the same six-coordination that bonds with two nitrogen atoms of the two 1,4-mbix ligands and four oxygen atoms of the two anionic Seb²⁻ ligands established an octahedral geometry. These [Cd₂ (1,4-mbix)₂] rings were linked by Seb²⁻ (O1 and O2) along a direction as a linear linker without penetrated into the ring [Cd₂ (1,4-mbix)₂] and another Seb²⁻ (O3 and O4) along c direction as a rod that penetrated into the ring [Cd2 (1,4-mbix)2] of the other layers. These produced two identical 2D single network that would be linked to each other in parallel, resulting in a 2D + 2D → 2D interpenetration layer by polyrotaxane feature.⁶³ Figure 7 shows the SEM images of single ligand and mixed-type ligand 2D MOFs.

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A top-down method is a synthesis approach that is performed as an attempt to exfoliate the bulk layered-materials.⁶⁷ This exfoliation process is the disintegration process of MOF crystals into single or multilayer structures which can easily occur due to the existence of noncovalent interactions that involve hydrogen bonding, π-π interactions, and van der Waals forces.²³ The exfoliating of bulk layered material can be advantageous for synthesizing 2D materials because it can produce an exfoliated 2D material with a simple and low-cost procedure. Moreover, many layered-material types can not be synthesized using other methods except exfoliation. The exfoliated 2D material can also be processed in solution, which may facilitate their fabrication into desired structures and applications.⁶⁷ There are several methods to do exfoliation, such as sonication exfoliation, shaking treatment, ball-milling, mechanical exfoliation, and intercalation or chemical exfoliation.⁶⁸ Mechanical exfoliation is the commonly used synthesis method for the top-down method since it can produce 2D MOFs with good crystallinity and purity.⁶⁹ However, it should be noted that exfoliation requires multistep processes, which may cause the layer interactions to be weakened.⁷⁰

Sonication exfoliation is a simple and effective method to synthesize ultrathin single-layer 2D nanomaterials. This exfoliation utilizes sound waves to agitate particles in solution. In this context, sonic waves are used to break the bonds between layers of bulk MOF, so that 2D MOF can be obtained. Currently, this approach has been developed by many researchers. For instance, in the earlier 2015, Alavi et al. had successfully used the sonication approach to exfoliate {[Cu₂(BDC-NH₂)₂(dabco)]DMF.3H₂O}. They found that sonication time dramatically influenced the quality and the distribution of the nanostructures. They claimed that 60 min is the optimum time of the sonication process because it gave the materials' best structure. It was confirmed that 30 min of sonication duration was not enough that the structures of 2D MOF did not grow. When the duration of sonication was longer than 60 min, 90 min and 120 min, the 2D MOF structures had agglomerated.⁷¹

Two groups of researchers also successfully applied sonication exfoliation in their research. They claimed that a suitable solvent in the sonication process is water. Firstly, In earlier 2019, Wu et al. synthesized 2D Cu–MOF nanosheets using a sonication exfoliation approach for 20 min in water. In their work, they confirmed that the successful exfoliation result using TEM and atomic force microscopy (AFM). Based on the TEM analysis, the MOF nanosheet showed its ultrathin nature. Meanwhile, the AFM image showed that the MOF nanosheet had a uniform thickness of 3.4 nm.⁷² Moreover, in early 2020, Pang et al. used this approach to obtain 2D MOF nanosheets. ([M₂(bdc)(dabco)] (M = Zn, Co, Ni, H₂bdc = 1,4-benzene dicarboxylic acid, dabco = 1,4-diazabicyclo-[2.2.2]octane)) was subjected to sonication in water. The exfoliation process provided an ultrathin layer of 2D nanomaterial (0.9 nm).⁷³ It was confirmed that the MOF nanosheet had a good stability in the water at the sonication process.

In some special cases, bulk MOFs have been delaminated using a shaking treatment. Araki et al. successfully did this approach. in 2013 they synthesized La(BTP) (BTP = 1,3,5-benzene triphosphonic acid) using a shaking treatment. They produced nanosheets with approximately 1.3 nm of thickness.⁷⁴ A similar result was also reported by Kondo et al. in 2013. They synthesized the ultrathin 2D [Cu(bpy)₂(OTf)₂] (named CuMOF) (bpy = 4,4'-bipyridine; OTf = trifluoromethanesulfonate). In their research, MOF nanosheets were obtained by shaking treatment in acetone. Similar to the sonication approach, shaking treatment also depends on the duration of the treatment. Kondo et al. had investigated the effect of treatment duration on the 2D structure. The duration was varied to 1 h, 3 h, 6 h, and 12 h. They found that 1 h and 3 h of the treatment resulted in thick MOF sheets. Meanwhile, the 6 h shaking treatment resulted 2D CuMOF nanosheets to have a thickness of 4–5 nm based on the AFM characterization. Lastly, the 12 h shaking treatment could break 2D CuMOF into small pieces with a size of less than 500 nm. They concluded that the best duration treatment is 6 h because it gave a smooth surface with a sufficient thickness.⁷⁵ Based on these results, we know that the duration time has an important role in shaking treatment.

This approach is relatively new for synthesizing 2D material and still has limited in the heterogeneities of the product, moderate porosity, and limited crystallinity. Several researchers have successfully performed this approach. In 2014, Peng et al. synthesized Zn₂(bim)₄ (bim = benzimidazole) using a ball milling approach. They found that the final MOF crystals had interlayer distance of approximately 0.988 nm.⁷⁶ In 2019, another study about ball-milling had been reported by Yoon et al. who synthesized the bimetallic conductive 2D metal-organic framework (Co_0.23Ni_0.77-CAT). Ball-milling processes produced a relatively thick lateral layer with approximately 1.91–2.21 nm that was confirmed by HRTEM characterization.⁷⁷

Mechanical exfoliation is one of exfoliation technique that gives a high possibility to produce a good quality of a few-layer nanocrystals.⁷⁸ Currently, 2D material samples which synthesized by mechanical exfoliation have an unsurpassed quality in terms of purity.⁷⁹ Zhu et al. (2019) stated that mechanical exfoliation can exfoliate the bulk crystal using plastic tape and can be used in almost all 2D materials, including 2D MOFs.⁸⁰ In 2018, López-Cabrelles et al. reported that they had successfully exfoliated [Fe(bimCl)₂] (HbimCl = 5-chlorobenzimidazole) using this method. Their research declared that the material had a layered structure with the packing thickness of approximately 400 μm.⁸¹

Chemical exfoliation from MOFs potentially allows efficient and controllable formation of 2D MOF nanosheets. This approach was reported by Ding et al. in 2017. They demonstrated the chemical exfoliation approach to obtain MOF nanosheets from intrinsically layered MOF crystals. They claimed that the chemical exfoliation process could proceed at room temperature. The thickness of the produced 2D MOF nanosheets was approximately 1 nm, with an overall yield of 57%.⁸² In another case, the delamination procedure was applied to Cu–BDC bulk to obtain 2D Cu–BDC. Generally, the coordination of Cu–BDC was formed by the coordination of Cu ion and H₂BDC in planar geometry. Each layer was connected by DMF molecules in the a-axis direction, forming stacked layers.⁸³ Post-treatment of Cu–BDC by immersing it in the ethanol solution containing amino compounds was reported could delaminate the stacked layers. This post-treatment procedure did not affect the crystal structure of Cu–BDC.

Similar to the top-down method, the bottom-up method also has several types of synthesis processes such as interfacial synthesis, three-layer synthesis, surfactant-assisted synthesis, and modulated synthesis.⁶⁸ Bottom-up method is considered as the most promising method that can produce more applicative and potential material. However, 2D MOF nanosheets that are synthesized using this method are sometimes still relatively thick, around 5–50 nm, and the production process is limited. Therefore, the large scale synthesis process using this method remains a challenge.⁸⁴ Although the bottom-up method allows a direct growth-ensuring, the morphological structure and thickness of 2D MOF can be entirely controlled. However, the addition of foreign substances will inevitably lead to an inadequate utilization of active sites on the surface.⁸⁵ The bottom-up method's critical point is to limit the vertical growth direction of the 2D MOF layer due to the strong interaction between each layers to achieve the lowest surface energy.⁸⁶ Hence, some of the researchers also used the bottom-up method to synthesize 2D MOFs, especially the interfacial synthesis approach.⁸⁷ Interfacial synthesis is generally a liquid-liquid or liquid-air interfacial reaction.⁸⁸

This approach depends on the reactant's dispersive behaviors in two separate phases, which could control the diffusion of the reactants from one phase to the other phase.⁸⁹ This method is one of the most widely used of the bottom-up methods to synthesize MOF nanosheets. This method is often used with several approaches, such as liquid/liquid interface or liquid/air interface in controlling the growth of MOF nanosheets. For instance, the synthesis of nickel bis(dithiolene) nanosheets was successfully reported by Kambe et al. in 2013. They achieved the synthesis by reaction of nickel(II) acetate and benzenehexathiol at liquid-air interface. Their research was done by spreading a thin layer of ethyl acetate solution that contained benzenehexathiol to the surface of aqueous solution that contained nickel(II) acetate. The evaporation of ethyl acetate in the liquid-air interface formed the nanosheets. Then, the nanosheets were investigated using scanning tunneling microscopy and it was observed that the hexagonal pattern of single-layer nanosheets had a height of 0.6 nm.⁹⁰

This approach was conducted by Rodenas et al. (2015) in order to synthesize 2D MOF Cu–BDC. Rodenas et al. synthesized the Cu–BDC in a glass test tube. This method uses three layers which are differentiated by density. The top layer is the metal precursor solution and the lowest layer is the H₂BDC ligand solution with the middle layer is the intermediate solvent layer. Under static conditions, the diffusion of metal cations and BDC ligands into the intermediate layer causes the growth of MOF crystals to occur locally in a highly diluted medium. This method requires no immiscible liquid phase, as opposed to the interfacial method, in which the interface rate determines the MOF growth. Their research produced a 2D MOF CuBDC with a lateral dimension of 0.5–4 μm and the thicknesses in the range of 5–25 nm.⁵⁴

This approach not only controls the MOF growth in the vertical direction, but also improves the dispersion of MOF nanosheets in the liquid phase.⁶⁸ This approach had been successfully reported by Wang et al. in 2020. Their research reported a sodium dodecyl sulfate (SDS)-assisted solvothermal to produce 2D c-MOF nanosheet including HHB-Cu (HHB = hexahydroxytriphenylene), HHB–Ni and HHTP–Cu (HHTP = 2,3,6,7,10,11- hexahydroxytriphenylene). The SDS surfactant was used to direct the MOFs' anisotropic growth in the 2D direction and resulting in the formation of ultrathin and large-sized nanosheets. SDS had hydrophobic chains that could weaken the interactions of interlayers and disassembled it to form ultrathin nanosheets during the sonication treatment. Moreover, it prevented further agglomeration by helping to stabilize the as-synthesized HHB–Cu MOF NSs in solution. The AFM measurement revealed a thickness of 4.5 ± 1.4 nm.⁹¹

Apart from surfactants, several small molecules (e.g. acetic acid, pyridine) have also been used in the synthesis of the MOF nanosheet. These molecules, also known as modulators, have the same functional groups as organic bridges, which competitively coordinate with metal nodes to regulate the growth kinetics of MOFs. More importantly, the modulator's selective coordination in a particular crystal plane would inhibit the growth of MOFs along that direction, leading to the anisotropic growth of MOF with different morphologies. Two groups of researchers have reported this approach. Firstly, in 2017 Hu et al. successfully applied this approach to obtain 2D layered MOF nanosheets, NUS-8(Zr/Hf). These 2D layered MOFs were composed of Zr₆O₄(OH)₄ or Hf₆O₄(OH)₄ clusters and 1,3,5-benzene tribenzoate (BTB⁻³). Examination by field-emission scanning electron microscopy (FE-SEM) strongly indicated that the 2D layered nanosheets had been formed. It exhibited the nanosheets morphology with a thickness of 10–20 nm and the lateral size of approximately 500–100 nm.⁹² Secondly, in 2019 Nian et al., also synthesized 2D cobalt-based MOF CO₂(bim)₄ (bim = benzimidazole) nanosheets. Their research found that the lateral size of the 2D MOF nanosheets was up to 800 nm–3 μm. The SEM characterization revealed that the layered had a thickness of approximately 30 nm.⁸⁶

As we know, both bottom-up and top-down synthesis methods can produce MOFs in two-dimensional form. However, according to several studies top down methods based on exfoliation have a chance of restacking 2D layered MOFs to the bulk form after volatilizing the solvent. This will be a limitation of the application in electrochemical biosensors.⁶² The obtained 2D MOFs from the exfoliation synthesis approach also display low stability against aggregation that will reduce the catalytic performance in electrochemical sensing. Thus, we suggest that it is better to use a bottom-up strategy to obtain 2D MOFs for electrochemical applications. Moreover, bottom-up approaches such as layer by layer synthesis or surfactant-assisted synthesis allowed a controllable MOF structure and properties such as vertical orientation that facilitates more active surface area and interlayer spacing that provide additional channels for ion transport, leading to the increased sensing performance.

Besides, the ultra-large surface area of 2D MOFs can be obtained from both top-down and bottom-up synthesis process. MOFs in two-dimensional form also promisingly have high mechanical stability and optical transparency that give a lot of benefits in the development of flexible and transparent biosensors devices in the future. Moreover, the ultra-thin thickness layer of 2D MOFs also allows fast electron mobility to the electrode surface and provides a high surface to volume ratio that facilitates easier contact between the biological analyte and the active sites.

As well explained in the previous paragraphs, the top-down and bottom-up methods have their own benefits and also drawbacks. These are summarized in Table I. Additionally, the summary of various 2D MOFs synthesized using the top-down and bottom-up method is provided in Table II.

The ligand design formed in the MOF synthesis also provides the strength in the MOF assembly procedure. Topological predictions with the geometric predictability of the ligands making the constituent materials excellent in the MOF synthesis process. The ligand design aims to enrich the MOF topological types and modify the MOF functionality to be applied in certain applications. For example, some carboxylic ligands have been designed for both synthesis convenience and crystal discomfort purposes. The various ligand designs will also have various functions and also the collection of these ligand designs systematically makes it easier to synthesize according to the desired functionality.⁹⁶

the substitution process has been identified as a new strategy to obtain the desired MOF material. This substitution process is expected to increase the controllability of the MOF material both in terms of structure and properties. The parameters can be controlled for the substitution. For example, the concentration of reactants or reaction time. This process was carried out to obtain the substituted product as desired, either partial or complete substitution. Either partial or complete substitution involves cleavage and regeneration of the coordination bond between the metal ion and the organic ligand. Partially substituted products usually produce unique functionality. On the other hand, the substitution process also can be carried out on metal ions. This process is usually observed in materials that are less chemically stable, namely the coordination bond between the metal and the ligands which are relatively unstable. In this context, the substitution reaction is expected to be an ideal approach in the process of synthesis and modification of material properties including MOF.⁹⁷ For example, in 2D MOF this process was investigated by Chen et al. in 2015. They investigated the effect of substitution of coordinated metal ions, e.g. from Ni to Cu in the MOF, on the structural and electronic properties of the MOF bulks and 2D sheets. With substitution of the metal sites, the band structures of the Cu₃(HITP)₂ sheet near the fermi level became quite different from those of Ni₃(HTIP)₂.⁹⁸

In the MOF deposition process, layer by layer is considered an important approach for depositing films on various types of substrates. In this approach, a solution containing a source of metal ions and organic ligands is mutually supplied to the crystalline growth surface to allow the growth of MOF with a thin thickness and a well-controlled surface. This approach shows the effective modulation technique in controlling crystal morphology. The main advantage of using this approach is having the precision to control the film thickness up to the molecular scale.⁹⁹

Due to the large surface area, good conductivity, fast electron and mass transfer of 2D MOF, this novel material becomes very potential in many electrochemical biosensor applications, specifically for non-enzymatic electrochemical sensors. Compared with the enzymatic electrochemical biosensors that commonly use 3D MOFs, the non-enzymatic electrochemical biosensor is more interesting to be explored because there are many advantages based on their stability in ambient conditions such as thermal and pH, easy to use, high sensitivity, good selectivity, and low cost.¹⁰² In recent years, some studies have reported the development of 2D MOFs as sensing material for non-enzymatic electrochemical biosensors. This mechanism is based on high electrocatalytic activity of 2D MOFs that can catalyze the reduction and oxidation reaction toward the analyte such as glucose, hydrogen peroxides, dopamine, ascorbic acid, and uric acid that can indicate the health condition of human bodies.

Besides resulting in a high sensitivity, the applied 2D MOFs as electrochemical non-enzymatic sensors also displayed excellent selectivity even in mixed compound-based detection. The selectivity generally is due to the size exclusion (molecular sieving) mechanism wherein atoms or molecules that are smaller than the MOF's apertures can be adsorbed, but larger molecules cannot.¹⁰³ Designing pore and aperture size for MOFs is an essential consideration to determine the selectivity of the proposed MOFs. Pore modulation can be achieved by removing non-structural ligands or solvent guest molecules from framework nodes or replacing node-coordinated solvent molecules with larger or smaller ligands.¹⁰⁴

One of the analytes that can be detected using a non-enzymatic electrochemical sensor is dopamine. Dopamine, a type of neurotransmitter, is mostly found in the central nervous system and responsible for the hormonal response, cognition, and feelings in the human body.¹⁰⁵ Low amount of dopamine may cause neurological disorders including Parkinson's, Huntington's, and schizophrenia.¹⁰⁶ Qiu et al. proposed an electrochemical non-enzymatic biosensor for the detection of dopamine (DA) using 2D MOF nanosheets doped with Gold nanoparticles (AuNPs) and poly xanthurenic acid (PXA) composite. Their idea is to combine AuNPs with PXA and assemble it on the Cu-tetrakis (4-carboxyphenyl) porphyrin (TCPP) by using an electrodeposition technique to improve the electrocatalytic performance, as shown in Fig. 9. Under cyclic voltammetry measurement, the redox peaks current greatly increased when the (PXA) was incorporated into AuNPs/Cu-TCPP/GCE modified electrodes. This is due to the excellent electron transfer and good synergetic effect between AuNPs/Cu-TCPP/GCE and (PXA). Furthermore, in DPV measurement they used different concentrations of dopamine (DA) as the analyte and Paracetamol (PA) as the interference substance in an electrolyte solution since these analytes have nearly two different redox peaks that can be observed in a single measurement. The linearity and limit of detection (LOD) of the dopamine was 5–125 μM and 1.0 μM, respectively.⁹⁵

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Another substance that is commonly analyzed by the electrochemical method is hydrogen peroxide (H₂O₂). H₂O₂ is naturally produced from an uncompleted metabolite of oxygen in living cells.¹⁰⁷ However, an imbalance of H₂O₂ concentrations in the human body can indicate many diseases, such as cardiovascular disease, degenerative disease, or even cancer.¹⁰⁸ The early detection of H₂O₂ is vital to prevent the malignancies of these diseases. Liu et al. synthesized two-dimensional NiCo-MOF nanosheets as non-enzymatic electrochemical biosensors for H₂O₂ detection. This 2D MOF nanosheets based on Co, Ni was synthesized at room temperature. By using cyclic voltammetry measurement, they obtained the redox peak potential of Co–MOF, Ni–MOF, and NiCo–MOF hydrothermal toward H₂O₂ was at 0.16 and 0.23 V, 0.25 and 0.5 V, 0.2 and 0.36 V, respectively. Besides, having the lowest working potential at oxidation potential of 0.25 V, Co–MOF displayed the best electrochemical response for H₂O₂ compared with other synthesized MOFs. The sensitivity of Co–MOF towards H₂O₂ was 0.69 μM with wide linearity of 0.5–832.5 μM.¹⁰⁹

Similarly, Shu et al. reported non-enzymatic hydrogen peroxide detection using Ni–MOF and hemin. Hemin is a type of heme protein that has similar properties to the peroxidase enzyme. Hemin has several advantages compared to natural enzymes, including chemical and thermal stability, more resistant to denaturation, and low-cost synthesis process. To evaluate the electrochemical behavior of modified electrodes, the CV and DPV measurements were conducted. The combination of MOFs/Hemin/GCE, have the highest redox peak currents in hydrogen peroxide detection as compared to GCE/MOF or GCE/Hemin. This is due to the combination of the large surface area of Ni–MOF and the high peroxidase-like activity of hemin. These fabricated biosensors displayed a low detection limit of 0.2 μM with the linearity of 1 μM–0.4 mM towards hydrogen peroxide.¹¹⁰

Moreover, 2D MOFs which form nanosheets structure show more active sites, increased stability, and also more uniform dispersibility in solution, making this material also often used as an electrochemical biosensor material for glucose detection. The detection amount of glucose is crucial for diabetic patients. The quantitative detection of glucose can be used for monitoring the therapy, the severity of the disease, and determining the effectiveness of the drug delivery system. Li et al. reported the superior non-enzymatic glucose sensor using Co–MOF on a nickel foam array. The synthesis process used a hydrothermal method in aqueous solution. Even though bare nickel foam (NF) only provided the ability for glucose redox reaction due to the presence of nickel as the transition metal that can catalyze redox reaction, they claimed that there were quite weak responses towards glucose. Thus, the Co–MOF with excellent electrocatalytic performance is needed. After combining Co–MOF with NF, the electro-oxidation peak of glucose significantly increased. The measurement was taken in the alkaline condition which is at pH 13, the optimum value that was achieved-as we know that the electrochemical measurement of glucose can be easily conducted under base condition. However, if the measurement is established at pH over 13, this can cause an interference peak of the glucose oxidation since the oxygen evolution reaction phenomena occur at pH 14. Finally, based on their study, Co-MOF/NF exhibited strong activity for glucose detection in alkaline media. The detection of glucose was achieved with a wide linear range of 0.001 mM–3 mM and limit of detection was 1.3 nM.¹¹¹

Xue et al. reported glucose detection using nickel modified copper metal organic framework (Ni@Cu-MOF) nanosheet. The synthesis process was prepared by a simple method at room temperature. Ni@Cu-MOF nanocomposite modified electrodes can be used for glucose detection without any further modification. The same as previously explained work, the electrochemical measurement establishes in alkaline conditions. The doping of Ni can enhance the conductivity and mobility of Cu–MOF modified electrodes. However, too much Ni accumulation can reduce these properties due to the covered surface of the electrode. This strategy displayed a wide linear range of glucose detection (5 μM to 2500 μM) with LOD of 1.67 μM.¹¹²

Furthermore, the promising thing is, if we were able to make 2D material in a vertical orientation. With this orientation, more available active areas can be exposed and consequently can provide a faster mass and electron transfer. Li et al. designed vertical two-dimensional NiCo–MOF nanosheets and demonstrated its application as a non-enzymatic glucose sensor.⁴² Usually, the synthesis of 2D MOF uses bottom-up strategy which needs a large amount concentration of surfactants^113,114 and also required long synthesis process time.⁵⁴ The most challenging thing is to prepare 2D MOF nanosheets with high uniformity without using any surfactant agents-considering that surfactants have several deficiencies. Therefore, establishing 2D MOF on conductive substrates which were proposed by Li et al. to be the solution of this problem. The good interactions between Nickel and Cobalt in the NiCo–MOFNs resulting in an excellent catalytic activity towards glucose. Under amperometric measurement, the linear range of glucose detection was from 0.0010 to 8 mM with a limit of detection of 0.29 μM.⁴²

As we know, the large active surface area of 2D MOF can increase the loading efficiency and available site of biomolecules probing that is very needed in electrochemical biosensor applications, especially for nucleic acid-based biosensor.¹¹⁵ One type of nucleic acid biosensor that is commonly used is the aptamer biosensor (aptasensor). Aptamers is oligonucleotides that can be formed as signaling DNA (sDNA) or capturing DNA (cDNA) which commonly used for various target detection such as cancer makers, protein, or heavy metal ions.¹¹⁶ Aptasensor have many advantages compared with traditional nucleic acid recognition such as high stability, inherent selectivity and excellent bio-affinity. Recently, some studies have been reported for developing aptamers in biosensor application for measuring some tumor markers, which become a significant contribution in cancer monitoring, including evaluation of therapy and predicting cancer recurrence.¹¹⁷ In the biomolecules immobilization strategy to the 2D MOF surfaces, the majority of studies reported the absence of surface treatment for covalent linkages such as cross-linking agents since that 2D MOF nanosheet provide a large active site for π−π stacking, hydrogen bonding and electrostatic interactions for bioconjugation. Nevertheless, a small number of studies also reported the use of EDC/NHS as a cross-linking agent for covalent interactions. Cross-linking agents are chemical reagents that chemically activate a molecule, enabling it to directly couple with another molecule through covalent bonding. Immobilization chemistries using EDC/NHS cross-linker created an activated surface after forming an amide bond between the amine-functionalized substrate and the carboxyl group on the antibody.¹¹⁸ This strategy resulted in some advantages, such as not-requiring antibody modification, oriented antibody, prolonged stability, and commercial availability. However, the requirement of additional process steps and susceptible to hydrolysis still a limitation of this method.¹¹⁹

He et al. proposed 2D zirconium-based metal-organic framework (521-MOF) for the detection of mucin 1.¹¹⁷ Mucin 1 (MUC1) is a type of transmembrane protein that commonly presence at cancer malignancies such as breast cancer, ovarian cancer, prostate cancer, and pancreatic cancer.¹²⁰ They synthesized 521-MOFs using polyvinyl pyrrolidone (PVP) as a capping agent to prevent the agglomeration, then followed by a synthesized processes under mild conditions to create a nanosheet structure. The immobilization of aptamer is achieved by strong bio-affinity between the 521-MOF and the aptamer strands without using any additional material like noble metal nanoparticles for bioconjugation or any surface treatment. The sensitivity of Mucin 1 detection was evaluated by the EIS method with a linear range from 0.001 to 0.5 ng·ml⁻¹ and low detection limit of 0.12 pg·ml⁻¹.¹¹⁷

In recent years, bimetallic MOFs have become an exciting topic for MOFs study due to the unique combination characteristic between two metals in one structure. Some studies showed that bimetallic MOFs have more hydro stability compared with single metal MOFs.⁴⁶ Based on this idea, Zhou et al. synthesized two-dimensional ZnZr bimetallic MOF for cancer marker protein tyrosine kinase-7 (PTK7) detection.⁴⁴ Bimetallic ZnZr–MOF was obtained by changing the synthesis parameters such as the sequence of metal ion precursors and organic ligands to get Zn-MOF-on-Zr-MOF. As we know the organic ligands in MOFs commonly have specific functional groups that can provide an available sites for immobilization of biomolecules via π−π interactions, covalent attachment, hydrogen bonding, or electrostatic interactions.¹²¹ Since the high affinity of biomolecule binding from Zr-based MOF, the immobilization of the aptamer onto the Zn-MOF-on-Zr-MOF can be achieved. To evaluate the electrochemical characteristics of the developed aptasensors, the EIS method was used. After aptamer immobilization, the impedance of modified electrodes was significantly increased due to the limitation of the access of redox ion mobility to the electrode surface. In the presence of PTK7, the diameter of the Nyquist plot increased due to the G-quadruplex interaction between aptamer and PTK7. To evaluate the sensitivity performance, the EIS method was used again which results in the limit of detection (LOD) on PTK7 was 0.84 pg ml⁻¹ within the concentration range of 0.001–1.0 ng ml⁻¹.⁴⁴

Meanwhile, the quantification of MicroRNA (miRNAs) also plays an important role in many clinical diagnoses of dangerous diseases, including cancers, heart diseases, neurological diseases, and many more. Sun et al. developed Black Phosphorus nanosheet and metal organic framework composite (BPNSs/MOF) for accurate detection of miR3123 as a biomarker for gastric cancer. They prepared 2D MOF via the solvothermal method, using Cu(II) as metal ion and PTTBA as organic ligand, as shown in Fig. 10. On the other hand, thionine (TH) was used as doping material and it was combined with BPNSs to immobilize the aptamer. Moreover, the successful synthesis process of modified electrodes was evaluated by CV and EIS methods. The performance detection of miR3123 was obtained by plotting linearity between the peak current of ferrocene (IFc) and the peak current of thionine (ITH). The ratio of IFc/ITH is known as ratiometric electrochemical. Due to the increasing of CmiR3123 concentrations, IFc regularly reduced and ITH hardly changed. This strategy resulting in a linear the range of miR3123 from 2 pM to 2 μM and limit of detection (LOD) of 0.3 pM.¹²²

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Besides the strategies for developing MOF on MOF 2D materials as mentioned in the previous paragraphs, the growth 2D MOF on COF (Covalent organic frameworks) is also a fascinating topic. Due to the combination between sensitivity and selectivity of aptasensors, easy operation of electrochemical techniques, as well as excellent electrochemical activity and strong bio-affinity towards biomolecules, makes it very interesting to be explored as novel biosensors.¹²³ Liu et al. designed MOF on COF composites based Co–MOF and TPN–COF as label-free aptasensor for ampicillin (AMP) detection. AMP is a contaminant which is often found in agricultural products and waters that can have an impact on allergic reactions, difficulty breathing, and spasms in the human body The aptamer immobilization can be obtained through a very strong bio-affinity between the aptamer strand and the Co-MOF@TPN-COF matrix via π-π stacking and hydrogen bonding. The electrochemical characteristic for this developed aptasensor was evaluated using the EIS method. The limit of AMP detection reached 0.217 fg ml⁻¹ with the linearity from 1.0 fg ml⁻¹ to 2.0 ng ml⁻¹.¹²⁴

By using the electrochemical luminescent technique, demonstrated by Shao et al., synthesized Ru–MOF nanosheet for labeled miRNA-141 detection. In their study, Ruthenium-based MOF was used as a signaling unit because of the excellent catalytic activity of Ru–MOF nanosheet. The immobilization of labeled signaling of DNA (sDNA) on the Ru–MOF surface was achieved by zero-length crosslinking agent for activated carboxylate group on Ru–MOF and forming an amide bond with sDNA. Meanwhile, the capturing DNA (cDNA) was attached on Fe3O4@SiO2@Au composite surface that has a function as a capturing unit, shown in Figs. 11a–11c. After that, cDNA will hybridize with miRNA-141 target following with the dropping of Ru-MOF labeled sDNA as a signal unit above it. This resulted in a significant enhancement of sensing performance and sensitivity of the sensor. This strategy achieved a wide linear range for miRNA-141 detection at 1 fM–10 pM with LOD of 0.3 fM.¹²³

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Immunosensors based on antigen and antibody reactions can be considered as clinical analysis for sensitive detection of disease-related proteins.¹²⁵ Compared with conventional enzyme-linked immunosorbent assays (ELISAs), electrochemical immunoassays have much advantages, such as high sensitivity, high selectivity, low cost, easy to use, portability and fast analysis.¹²⁶ Dong et al. reported the detection of calprotectin (CALP) by using two-dimensional Cu-TCPP(Fe) doped PtNi nanosheet.¹²⁷ CALP is a biomarker for several melioidosis diseases that can be used to evaluate treatment responses to antibiotics.¹²⁸ CALP monitoring in patients is vital to evaluate the severity and effectiveness therapy of inflammatory diseases in clinical diagnosis. On the other hand, the bimetallic Cu-TCPP(Fe) nanosheets with their large active surface area permit more binding sites for PtNi attachment, resulting in the increasing electrochemical catalytic activity and also provide more active sites for antibody immobilization. Based on CV curve and the EIS plot, they claimed successful electrode preparation including immobilization of antibodies and the detection of CALP. This labeled immunosensors resulting in a low detection limit of 137.7 fg ml⁻¹ within the range of 200 fg.ml⁻¹ to 50 ng.ml⁻¹ towards CALP.¹²⁷

Similarly, Xiao et al. developed two-dimensional Cu-TCPP(Fe) and polyethyleneimine (PEI) composite for sulfonamide detection.¹²⁹ Sulfonamides (SAs) is generic level antibiotics that can become contaminants in the aquatic environment through feces and urine released by patients that can contribute to the evolution of antibiotic-resistant genes in the environment and potential damage to human health.¹³⁰ Interestingly, the Cu-TCPP(Fe) structure is destroyed when the introduction of polyethyleneimine (PEI) from PEI graphene and SAs antibody composite (PEI-GO/Ab) by the stronger affinity interaction between PEI and Cu²⁺ than that of Cu-TCPP(Fe). These phenomena result in a decreased electrochemical signal of immunosensor and can be used for SAs antibody detection. The electrochemical properties of the fabricated immunosensors were analyzed by EIS method under the optimum conditions. This strategy had a linear range of 1.186–28.051 ng ml⁻¹, with a low detection limit of 0.395 ng ml⁻¹.¹²⁹

In a different point of view, Li et al. proposed Zirconium-porphyrin composite as label-free immunosensors for enolase detection.¹³¹ This complex Zr-TAPP was established from Zr(III) as metal ion and tetra aminophenyl porphyrin (TAPP) as organic ligand. The neurons specific enolase (NSE) is the type of YY dimer enzyme that is widely distributed in mammalian tissues.¹³² NSE can be used as a biomarker for various diseases related to the nervous system such as Alzheimer's, neuroblastoma, and even neuroendocrine diseases because there will be an increase in NSE levels when a person is diagnosed by this disease.¹³³ Several advantages is possessed by the Zr-TAPP complex, including abundant N-related functional groups, high π-π stacking, and high hydrophobicity. These advantages not only can increase the antibody immobilization capacity which will increase efficiency in capturing antigens, but also can increase the stability of complex interactions between antibodies antigen in aqueous solution, as shown in Fig. 12. Under EIS measurement for NSE detection, the Zr-TAPP-based immunosensor showed a low detection limit of 7.1 fg ml⁻¹ with a linear range of 10.0 fg ml⁻¹ to 2.0 ng ml⁻¹.¹³¹

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On the other hand, combining electrochemical and luminescent techniques, i.e. electrochemiluminescent (ECL), is also an auspicious thing, which provides many advantages in an immunoassay such as wide linear range, dissolving low background signal problem, accessible to modified and adjustable performance. Generally, ECL technique uses luminol and noble metal nanoparticles (MNPs) composite as sensing platforms for many applications. Unfortunately, the classical problem in noble metal synthesis is the agglomeration process that can affect the immunosensor performance. Therefore, Wang et al. proposed a metal-organic framework with high porosity and large surface area for luminol-MNPs platform to prevent agglomeration phenomena of MNPs. The combination between Co/Ni MOF and luminol-AgNPs was used for the detection of alpha-fetoprotein (AFP). Low concentration of AFP in the human body is responsible for many liver cancer cases, so that early detection of AFP is very crucial, especially for humans that are infected by hepatitis B or Hepatitis C. This developed immunosensor resulting linear range from 1 pg ml⁻¹ to 100 ng ml⁻¹ with LOD of 0.417 pg ml⁻¹ towards AFP.¹³⁴

Lastly, the summary of 2D MOFs applications as electrochemical biosensors are shown in Table III.