MOFs-Based Biosensors for the Detection of Carcinoembryonic Antigen: A Concise Review

1. Structure and Function of CEA

CEA is a glycosylated protein of a high molecular weight (180-200 KDa), as shown in Figure 1a. It is involved in the cell adhesion and recognition mechanisms (PDB entry 1E07). [5] Gold and freedman were the pioneers for describing CEA sixty years ago. They realized that CEA was related to human colon cancer tissue extracts and later to digestive system cancer. [4] CEA has immunoglobulin-like structural features and many glycosylation modification sites. [6] Particularly, CEA belongs to the immunoglobulin superfamily of cell adhesion molecules (IgCAMs). IgCAMs are highly glycosylated surface proteins whose function is fundamental in cell-cell adhesion. [2]

The function of CEA in a normal individual is not well understood. Naturally, CEA is secreted in the fetal intestinal tissue, and its serum level can also be elevated in non-malignant diseases such as inflammatory bowel diseases. In the tissues of adults, the CEA-related cell surface molecules are expressed primarily in different epithelia, vessel endothelia, and hematopoietic cells. [7] Conversely, in healthy adults CEA serum level is usually found in low concentrations. As a result, its abnormal elevation is associated to gastric, breast, ovarian, pancreatic, and most importantly in colorectal cancer. [8,9]

The family of the CEA-related cell adhesion molecules (CEACAM), which are expressed from the CEA gene family, consists of a single N-terminal domain and a maximum of six disulfide-linked internal domains. The group contains 12 proteins (CEACAM1, 3–8, 16, 18–21) as shown in Figure 1b. [10] The extracellular domains of the CEACAM group function are homophilic and heterophilic cellular adhesion molecules or receptors. Members of the CEACAM group have variable functions as they can act as dimers or oligomers with other membrane molecules. [6] Anchorage to the membrane is a characteristic property of CEACAM proteins. CEACAM5 and CEACAM6 are attached to the cell membrane through a glycosylphosphatidylinositol (GPI) linkage. A small sequence of 26 hydrophobic amino acids is involved in the CEA membrane attachment. However, phospholipase can induce the release of CEA from the cell membrane to the extracellular lumen. [10]

As a result of the ability of CEA in the cell adhesion on the surface of epithelial cells, it was found that these properties play a crucial role in the growth of tumor through the development of CEA–CEA bridges between tumor cells or tumor–normal cells, as shown in Figure 2. In tis regard, the homotypic binding between two CEA molecules takes place through the attachment between the N and A3 domains. [11] Interestingly, this feature has been found to help the immune system to recognize the presence of cancerous cells. Examining the adhesion binding domains of CEA for therapeutic intervention has led to some pertinent findings. Blocking the binding ability of CEA on purified human cells via producing monoclonal antibodies (anti-CEA-mAb), which will bind specifically to the CEA, has led to the lysis of the tumor cells. Therefore, the anti-CEA mAb, named CC4, has been recently recognized as a promising treatment for cancer. [12]

2. Why using MOFs for biosensors

In the same context, MOFs have recently been recognized as potential materials for enhancing the sensitivity and selectivity of biosensors for the detection of cancer biomarkers. A typical MOF structure essentially consists of metal nodes and organic linkers known as struts (Figure 3). The arrangement of metal nodes and struts in an orderly fashion in their geometrical configuration results in uniform porous crystalline structures of MOFs with tunable outstanding porosity. [19] The hierarchical structure of MOFs provides unique features, such as high surface area that facilitates the interaction with more analytes thru high selectivity, and a tunable porosity that enables the capture of biomarkers with diverse structure and reactivity. Moreover, MOF can be fabricated in various dynamic geometrical shapes (0D, 1D, 2D, 3D), hence provide more options for capturing more analytes within the framework. [20] Additionally, the facile synthesis of multifunctional MOFs has extended its applications in the fields of energy storage, waste-water treatment, photovoltaics and biosensing. [18,21]

The first attempts to design biosensors were carried out in 2008. [22] A significant exponential increase in the interest of biosensors in general and MOF-based biosensors in particular was observed since 2008, as shown in Figure 4. This was also accompanied with a consistent increase in the development of MOF nanostructures with various functionalities, morphologies and capabilities. Moreover, hybrid nanostructure involving MOFs and other nanomaterials, such as gold nanoparticle (Au NPs), Graphene oxide, carbon nanotube (CNT) have shown promising results as composite biosensors. This is attributed to the enhanced optical or electrochemical signals generated during the sensing process. [23,24]

Interestingly, the unique surface functionality and the highly interconnected porosity of MOF nanostructures can also serve as sites for the immobilization of bioactive molecules that can help in the biosensing process, such as oligonucleotides, antibodies, and enzymes. [25] Figure 5 shows typical MOF nanostructures that have been explored for the development of biosensors and as drug delivery vehicles for cancer treatment. Immobilization of these bioactive molecules usually takes place through any of the approaches shown in Figure 6. Choice of the immobilization approach depends to a large extent on the size of the molecules as well as the MOF’s surface functionalities and the average pore size. [26,27] Moreover, other materials such as polymer, carbon materials, metal nanoparticles could be considered in the design of the biosensor in order to contribute to the sensing mechanism. Recently, abundant studies have been described the biosensing applications of MOFs either as a stand-alone candidate or combined with other structures, which have shown promising potential applications. [24]

MOF nanostructures have been prepared by different methods, including hydrothermal [28], solvothermal [29], ultrasonication [30], electro-chemical [31], mechanochemical [32], and microwave assisted heating [33]. Facile methods of synthesizing MOFs provide the opportunity to optimize the composition and crystalline arrangement of metal nodes and struts in order to obtain desired porosity, surface area and reactivity for an application of interest. [19] In the following sections, examples of the most recent advances in the design and fabrication of MOF-based biosensors for the detection of CEA will be illustrated. Table 1 lists the most studied MOF nanostructures that have been developed for the detection of CEA biomarker as well as the method of structure modification, the type of sensing and the limits of detection.

3. Progress in MOF-based biosensors for the detection of CEA

Recently, many research papers described the design of MOF- based biosensors for CEA detection via different routes, namely electrochemical, optical, photoelectrochemical, etc. The application of MOF-based biosensors for the detection of CEA using these approaches will be explained in the following sections.

(a).

Electrochemical approach

The electrochemical detection mainly depends on the transduction of the chemical reactions to electrical signal (e.g., current, voltage, impedance, etc.). By incorporating this kind of detection to biosensors, the biochemical reactions such as enzyme substrate reaction and antigen-antibody can be translated to electrical signals. Many electrochemical sensors have proven their efficiency and have been commercialized. In these biosensors, the working electrode is a key component, which is employed as a solid support for the of immobilization of biomolecules (enzyme, antibody and nucleic acid) and electron movement. [34,35] The electrochemical cell used in these sensors is composed of three electrodes submerged in an electrolyte of the required analytes. One of the electrodes is a reference electrode (such as Ag/AgCl or Hg/Hg₂Cl₂), which is used to maintain a constant potential when compared to a working electrode. The other electrode is known as the working electrode, where the reaction of interest takes place. The third electrode, known as the auxiliary electrode, is composed of an inert conducting substance (e.g., platinum or graphite). A considerable interest in the development MOF-based biosensors has been shown, which is attributed to the high thermal and chemical stabilities of MOFs. This results in an excellent electrochemical biosensor performance, where the MOF nanostructure is immobilized on the working electrode. [36,37] Many electrochemical analysis techniques have been used in these biosensors, such as Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV), Square Wave Voltammetry (SWV) and Electrochemical Impedimetric Signal (EIS). Examples of MOF-based biosensors are described in the following sections.

Zhang et al. presented a CEA electrochemical biosensor that was based on the modification of a glassy carbon electrode (GCE) using polyaniline nanofibers. Polyaniline nanofibers provided high surface area and good electrical conductivity. The modified GCE was investigated for the simultaneous determination of the cancer biomarkers CEA and alpha-fetoprotein (AFP) by using particles of MOFs prepared from Pb(II) or Cd(II). The MOFs nanoparticles were labeled by secondary antibodies for both biomarkers to form a sandwich assay. The respective detection limits were 0.03 pg/mL and 0.1 pg/mL, respectively. [38]

A similar sandwich-type electrochemical biosensor was proposed by Li’s group. [39] In their work, Ce-MOF was synthetized and loaded with silver nanoparticles (Ag NPs) and horseradish peroxidase to catalyze H₂O₂, hence amplify the current signal. This MOF-based assembly was then coated with hyaluronic acid. The designed immune-sensor further used Au NPs to enhance the ability of attaching the antibody based on the high electrical conductivity of the Au NPs. The immune-sensor assembly was tested for the detection of CEA, and showed a limit of detection (LOD) of 0.2 pg/ml. Moreover, the proposed immune-sensor was found to possess high reproducibility, selectivity, and stability. [39]

Liu and his coworkers successfully used a Ag-based MOF as a decoration of an electrode and showed that it provided stable electrochemical signals in a voltametric technique alone. Using the Ag-MOF decorated electrode, it was able to detect CEA with a low LOD of 8.0 fg/ml. It should be mentioned that the voltametric immunoassay has been proven to be stable, inexpensive, sensitive, and selective. [40] Zhou et al in 2017 fabricated a Cu-MOF to be used as an electrochemical biosensor, where EIS measurements were used for the detection. [41] The prepared Cu-MOF nanostructures were further functionalized with Pt nanoparticles, aptamer, hemin and glucose oxidase (Pt@CuMOFs-hGq-GOx). This biosensor assembly was found to mimic the peroxidase activity. Based on the cascade catalysis amplification driven by glucose oxidase, CEA was detected at a LOD of 0.023 pg/ml. as described in Figure 7. [41]

UiO-66, a well-known MOF, was also used in combination with Ag nanoclusters (Ag NCs) and CEA aptamer (AgNCs@Apt@UiO-66) for the detection of CEA. [42] The synthesized AgNCs@Apt@UiO-66 sensor was found to utilize both electrochemical and SPR techniques, as showed in Figure 8. Results showed that the proposed electrochemical AgNC@Apt@UiO-66-based aptasensor exhibits high sensitivity with a LOD of 8.88 and 4.93 pg/ml, as deduced from electrochemical impedance spectroscopy and differential pulse voltammetry, respectively. Meanwhile, the developed SPR biosensor exhibited a slightly high LOD of 0.3 ng/mL. [42]

In a novel approach, self-polymerized dopamine-decorated Au NPs that are also coordinated with Fe-MOF (Au@PDA@ Fe-MOF) was designed for the detection of CEA. [43] This nanocomposite was employed as a transducer and showed good electrochemical signals. In addition, this nanocomposite assembly was used to immobilize the recognition element (CEA-aptamer) due to abundant COOH groups embedded in Fe-MOFs, as shown in Figure 9. This sensor showed a high sensitivity and excellent selectivity. [43]

Bao et al used UiO-66 as a nanocarrier of electroactive molecules (methylene blue, MB). DNA was further immobilized onto this composite (MB@DNA/MOFs), and was used as a platform for electrochemical biosensor, as described in Figure 10. [44] The biosensor presented good performance for CEA detection ranging from 50 fg/ml to 10 ng/ml with a detection limit of 16 fg/ml. [44] In an earlier study, a type of dendritic DNA scaffold labeled with a Pb-based MOF, synthetized by hybrid chain reaction as signal tags, was used for the detection of CEA. A detection limit of 0.333 pg/ml was obtained. [45]

In a recent study by Zhang et al, both AuNPs and ordered mesoporous carbon (OMC) were incorporated with ZIF-8, and an electrochemical biosensor was prepared thereof. [46] Zif-8 served as a support nanocarrier for the immobilization and increased loading of the antibody onto its large surface area. The OMC was dropped on a glassy carbon electrode to improve electrochemical signals due to its intrinsic electrical conductivity. DPV was carried out to record the electrochemical responses. The sensor demonstrated excellent performance with a LOD of 1.3 pg/ml. [46]

Liu et al revealed the synthesis of Cu-MOFs by a hydrothermal method for establishing a sandwich-type sensor, as shown in Figure 11. [47] Toluidine blue (TB) loaded mesoporous Cu-MOFs with PDA coating were employed as a signal transducer probe. The suggested sensor was fabricated by Cu-MOFs-TB/PDA, chitosan and multiwall carbon nanotubes (MWCNTs) on a glassy carbon electrode with a large surface area to immobilize primary antibodies (Ab1). [47] An excellent conductivity was achieved, and CEA was quantitatively detected with LOD of 3.0 fg/mL under optimal conditions. [47]

A platform for the ultrasensitive detection of three analytes: thrombin, kanamycin, and CEA, was also established by Zhang et al. [23] A series of Zr-based MOF composites embedded with three kinds of aptamer strands were achieved by a one-step de novo synthetic approach. [23] The label-free electrochemical aptasensors based on Zr-MOF@Apt composites demonstrated high stability, repeatability, applicability and showed an excellent sensitivity to these analytes with detection limits of 0.40, 0.37, and 0.21 pg/mL for CEA, thrombin, and kanamycin, respectively. [23]

In another study, three Sm-based MOF nanostructures were synthesized with different organic linkers, resulting in three different morphologies including rod-shaped, cubic consisting of stacked 2D layers, and spherical made of small cubic structures. [48] These Sm-MOFs were used to fabricate immunosensors for the detection of CEA, where the CEA antibodies were immobilized onto the working electrode. [48] The exhibited reproducibility and selectivity of the immunosensors were proven with low limit of detection. They were tested with human serum samples, proving the promising results. [48]

(b).

Chemiluminescence Approach

Chemiluminescence (CL) has grown into a well-established luminometry method in analytical chemistry and has been widely applied to liquid phase samples for over 30 years. CL is defined as the production of light through a chemical reaction that is accompanied by energy release. It has numerous advantages such as superior sensitivity, rapidity, safety and controllable emission rate. However, CL-detection acquires extra facilities, such as on-line sample processing and in-line multi-detector installment. [49] For CL-based biosensors the most utilized design is a flow-through biosensor one. [50]

A novel preparation of MIL-88B (Fe) by hemin a metalloporphyrin Fe-based MOF, called as hemin@MIL-88B (Fe), has been reported in 2020 by Han et al [51] to be used for the CL biosensing of CEA. The hemin@MIL-88B (Fe) was conjugated with CEA aptamer1 (hemin@MIL-88B (Fe)-apt1) as the large signaling strategy. The ssDNA was immobilized on the surface of Fe₃O₄@SiO₂ magnetic material, and hemin@MIL-88B (Fe)-apt1 was adsorbed on the Fe₃O₄@SiO₂ by the complementary pairing of the partial bases between ssDNA and CEA apt1. The luminol CEA aptamer2 (L-apt2), which can generate the CL signal, was separately prepared, and was adsorbed on magnetic carbon nanotubes (M-CNTs) by an electrostatic adsorption. Interestingly, hemin@MIL-88B (Fe)-apt1, L-apt2 and CEA formed by sandwiched-like ternary complexes. The positively charged hemin@MIL-88B (Fe) easily accumulated near the hydroperoxide anion (HO²⁻) through electrostatic adsorption, thereby catalyzing the luminol transient chemiluminescence system. LOD was successfully reported to be 1.5 × 10^-3 ng/ml. [51]

(c).

Electrochemiluminescence Approach

On the other hand, electrochemiluminescence (ECL), which is defined as electrogenerated CL, possesses high sensitivity and broad dynamic range. [52] ECL differs from most other electrochemical techniques. [52] In fact, ECL relies on the generation of high energy electro-active species in the vicinity of electrode surface. As a result, ECL techniques have been widely used in the immunoassays as a direct optical readout of electrochemical reactions. The ECL intensity of the targeted nanomaterials considerably depends on the corresponding electrocatalytic activity. In recent years, extensive research has been performed on ECL in combination with various nanomaterials, including MOFs. The brilliant features of MOFs, make them promising candidates in ECL bioanalysis. [53] In a unique study, Hf-based 2D MOF was fabricated and used in CEA detection, as described in Figure 12. The proposed 2D nanostructure exhibited higher ECL intensity and efficiency and was used to load aptasensor for the ultrasensitive detection of CEA. A detection limit of 0.63 fg/ml was achieved. It should be mentioned that these findings strongly provide new prospects for the development of novel ECL materials and will provoke more interests in the use of 2D MOF nanostructures for ECL sensing. [54]

Graphene oxide (GO)-doped ZIF-8 were also used for the fabrication of an ultrasensitive sandwich-type ECL immunosensor. The pyrolyzed nanocomposites were loaded with Au NPs, which were immobilized with antibodies. These nanocomposites were further loaded onto ruthenium (Ru)-labelled silica nanoparticles (RuSi NPs), and were used as ECL signaling units as it provided numerous luminophores that enhanced the ECL as shown in Figure 13. [55]

Through the combination of MIL-101 and CdSe quantum dots (QDs), an ECL sensor has been successfully synthesized, as described in Figure 14. The as-prepared nanocomposites were applied to immobilize the antibody of the CEA, and a high ECL activity and sensing selectivity were obtained, where a detection limit of 0.33 fg/ml was observed. [56]

(d).

Fluorescence Approach

Fluorescence is by far the most commonly used method in the design of biosensors, as described in many studies. Peculiarly, fluorescent MOFs were used in in these research directions. The fluorescence properties of MOFs are generated by metal ions and organic ligands. Moreover, other fluorescent molecules are encapsulated within the MOF channels and can produce emission fluorescence. [57] These characteristics make MOF materials suitable candidates for the fabrication of MOF-based fluorescence sensors for specific targeted species such as metal ion, anion and small molecule. [58]

A promising fluorescence-based microfluidic sensor for the quantification of CEA was developed by Zhao et al, as schematically represented in Figure 15. [59] In this work, a synergetic fluorescence enhancement strategy based on micro/nanostructure optimization of ZnO nanorod arrays and the in situ ZIF-8 coating was proposed. A glass capillary was chosen as the microfluidic channel, and controllable construction of ZnO nanorod arrays on the inner wall of the microchannel was conducted via the intermittent reaction method. The fluorescence enhancement characteristics of ZIF-8 towards organic fluorescence labels were investigated and were successfully applied to protein marker detection. The LOD reached was as low as 0.01 pg/ml. [59]

In a different study, an innovative and powerful visible fluorescence immunoassay method was fabricated through a wet NH₃- triggered structural change of NH₂-MIL-125(Ti) impregnated on paper for the detection of CEA. [60] Au NPs heavily functionalized with glutamate dehydrogenase and secondary antibody were used for the generation of wet NH₃ with a sandwiched immunoassay format. Paper-based analytical device (PAD) coated with NH₂-MIL-125(Ti) exhibited good visible fluorescence intensity through wet NH₃-triggeried structural change with high accuracy and reproducibility. Moreover, the NH₂-MIL-125(Ti)-based PAD displayed two visual modes of fluorescence color and physical color with the naked eye and allowed for the detection of CEA at a concentration of as low as 0.041 ng/ml. Importantly, the PAD-based assay provides a promise for the mass production of miniaturized devices and opens new opportunities for protein diagnostics and biosecurity. [60]

(e).

Photoelectrochemical Approach

The photoelectrochemical (PEC) process is a promising low-cost approach to convert chemical energy to electricity under light illumination and applied potential. PEC biosensing has attracted huge attention because of its ability to detect biomolecules through the photocurrent generated from a biomolecule oxidation. The ability to couple the photoexcitation process with electrochemical detection renders PEC sensors unique. Moreover, the separation between the sources of excitation (light) and detection (photocurrent) in the PEC process offers high sensitivity with low background signal. [61]

Liu et al proposed a photoelectrochemical biosensor based on UiO-66 MOF. [62] This work established an immobilization-free PEC biosensor based on the DNA-functionalized MOF. Here UiO-66 served as a nanocarrier for the efficient encapsulation of electron donors, while a designed probe was employed as the recognition element (Figure 16). The proposed biosensor was found capable of ultrasensitive and highly selective determination of CEA with a detection limit down to 0.36 fg/ml. [62]

A recent PEC immunosensor based on Yb-MOF was designed for the high performance determination of CEA. [63] The surface of the Yb-MOF was integrated with AuNPs to improve the photoelectric conversion efficiency of the Yb-MOF in the near infrared region. Subsequently, this nanocomposite became a photoelectrochemical platform for loading the CEA antibody (anti-CEA). After exposing it to CEA, the photogenerated electron-hole pairs transfer was blocked thus leading to a decrease in the photocurrent response. The photocurrent variation can be used for determining CEA quantitatively. The range of detection was measured from 0.005 to 15 ng/ml. [63]

(f).

Colorimetric Approach

Colorimetric sensors and biosensors exhibit promising potential due to their simplicity and reliability as potential low-cost platforms. The main prospect of colorimetric sensors is based on the interaction of light and metal nanomaterials. In particular, the color is created by the change in absorbance due to the optical properties of the material. The change in absorbance can be measured as a function of the different concentrations of clinical biomarkers. [64] For CEA colorimetric detection, a group of researchers synthetized a novel biosensor based on PCN-222 MOF, which was prepared by grafting CEA aptamer incorporated DNA tetrahedral (TDN) nanostructures. [65] The synthesized CEA aptamer-TDN-MOF showed a high detection stability due to the iron porphyrin ring in the PCN-222 MOF which possess a significant horseradish peroxidase mimicking activity, which leads to a colorimetric reaction upon binding toward antibody-captured CEA. Ultra-sensitive detection of CEA can be achieved with a limit of detection as low as 3.3 pg/ml. In addition, they demonstrated a great potential upon the analysis of clinical serum samples. [65] In a latest study, Zeng and his colleagues have designed and fabricated a colorimetric immunosensor by taking advantage of 2D-MOF nanomaterials as enzyme mimics. [66] The nanomaterial showed a strong peroxidase mimetic activity, and good selectivity after being modified with a specific aptamer. They reported a CEA detection performance with a linear range from 1 pg/ml to 1000 ng/ml and LOD of 0.742 pg/ml. [66]

ELISA has been identified as a gold standard and is the most widely used immunoassay technique for detecting and measuring disease biomarkers. However, there are some inevitable limitations in the use of ELISA, including limited sensitivity, which requires novel strategies to improve the ELISA limit of detection. For the enhancement of ELISA researchers have introduced MOFs during the fabrication of an ELISA platform. The MOF-based ELISA provides large surface-to-volume ratios which promote the efficient immobilization of antibodies on the nanoscale surface that consequently increase the capture efficiency of substrate surfaces.

In one of the most interesting studies, ZIF-8 doped with carbon dots (CDs) and thymolphthalein was used as a platform for CEA detection via ELISA technique, as shown in Figure 17. In this nanocomposite sensor, the strong fluorescence intensity of CDs could be observed directly to achieve the sensitive detection of the target. After stimulation of alkaline solution, TP was released from ZIF-8 carriers and generated a color change with an obvious absorption, which was beneficial for an increased sensitivity of this ELISA due to the high loading of the thymolphthalein. [67]