Preparation and application of porous framework materials for bio-separation

Importance and urgency of bioseparation

In the context of rapid advancements in life sciences and biotechnology, bioseparation has emerged as a crucial bridge^[1-3]. Efficient and precise bioseparation technologies are indispensable for ensuring the quality, safety, and efficacy of bioproducts - ranging from the extraction of high-purity bioactive compounds from natural sources to the large-scale production of genetically engineered therapeutics^[4,5]. With growing global demands for innovative pharmaceuticals^[6], sustainable biomaterials^[7-9] and functional green foods^[10], the complexity of biological samples has significantly increased. This complexity, coupled with the urgent need to isolate low-abundance^[11], high-value biomolecules (e.g., post-translationally modified peptides) from highly intricate matrices, emphasizes the limitations of conventional separation methods^[12]. Consequently, overcoming these bottlenecks through next-generation separation platforms is not only crucial for advancing biotechnological innovation^[13] but also essential for addressing pressing challenges in human health^[14] resource sustainability^[15] and environmental protection^[16].

To meet these diverse separation challenges, porous materials have emerged as transformative tools due to their exceptional structural and chemical tunability^[17-20]. Their high specific surface area^[21-23], controllable pore architecture^[24-26], and customizable surface functionality^[27-29] make them uniquely suited for both macromolecular (e.g., proteins, nucleic acids, viruses)^[30,31] and small-molecule (e.g., metabolites, drug candidates, signaling molecules)^[32-34] separations from complex biological environments. For macromolecules, the well-defined pore size and surface charge of porous frameworks enable size-exclusion effects and electrostatic interactions, facilitating high-resolution separation and enrichment of target biomacromolecules. In contrast, for small molecules, the designable pore chemistry and host–guest recognition capabilities allow selective adsorption based on polarity, hydrogen bonding, or π–π interactions - critical for isolating trace bioactive compounds or removing toxic impurities. This dual capability positions porous materials as universal platforms capable of addressing the full spectrum of bioseparation needs, from proteomics and genomics to metabolomics and pharmaceutical purification.

Among these advanced porous platforms, metal–organic frameworks (MOFs), covalent organic frameworks (COFs), and hydrogen-bonded organic frameworks (HOFs) represent a powerful triad of functional materials, each offering distinct yet complementary advantages. MOFs feature highly tunable pore environments and abundant metal-coordination sites, enabling precise recognition and efficient adsorption of biomolecules^[35,36] based on size, charge, and affinity. This makes MOFs particularly effective in discriminating structurally similar species - a persistent challenge in traditional chromatography. COFs, built from strong covalent linkages, exhibit exceptional chemical and thermal stability, along with ordered porous structures and easily modifiable pore surfaces^[37-40]. These features allow COFs to maintain integrity under harsh conditions while facilitating selective interactions through tailored functional groups, thereby enhancing separation selectivity and reusability. More recently, HOFs have gained attention for their mild synthesis conditions, intrinsic biocompatibility, and dynamic yet directional hydrogen-bonding networks^[41-43]. These characteristics not only facilitate easy processability and recyclability but also enable selective binding of polar biomolecules, making HOFs especially promising for aqueous-phase separations and bio-related applications. Notably, all three frameworks share ultra-high surface areas and potential for regeneration, significantly improving separation capacity per unit volume while reducing operational costs and environmental footprint. Together, MOFs, COFs, and HOFs are driving a paradigm shift in bioseparation - transforming it from a bottleneck into a high-performance, sustainable, and intelligent process capable of handling both macromolecular complexity and small-molecule precision.

Key parameters for the development of porous materials for bioseparation

Porous materials like MOFs, COFs, and HOFs have emerged as pivotal tools in the field of bioseparation due to their high specific surface areas^[44], tunable pore structures, and versatile surface functionalities. Their ability to achieve precise molecular sieving through size-exclusion effects, coupled with selective interactions facilitated by surface modifications, makes them ideal candidates for various biological separations. This section highlights the development trajectory of these materials and their application potential in bioseparation.

Several critical attributes contribute to the rapid progress of porous materials in bioseparation: 1. Tunable Pore Structures: The adjustable pore sizes enable efficient separation based on the dimensions of biomolecules ranging from small metabolites to large macromolecular complexes. 2. Surface Functionalization Flexibility: The grafting of diverse functional groups allows for selective interactions with target biomolecules via mechanisms such as hydrogen bonding and electrostatic adsorption. 3. Chemical/Thermal Stability: Robust stability ensures that these materials maintain their structural integrity under various biological conditions. 4. Morphological Versatility: Innovations in synthesis methods, including 3D printing^[45] and hierarchical assembly^[46,47], allow for the creation of different forms suitable for a range of separation platforms. 5. Biocompatibility and Stimuli Responsiveness: Modifications can enhance biocompatibility^[48,49] while stimuli-responsive properties^[50,51] enable dynamic control over separation and release processes.

Although MOFs, COFs, and HOFs all show great potential in bioseparation, they differ significantly in structure, stability, and functionality. MOFs offer high porosity and tunable metal–ligand interactions but often suffer from limited hydrolytic stability; COFs feature robust covalent frameworks and precise pore engineering yet can be challenging to process; HOFs exhibit excellent biocompatibility and reversible assembly but generally have lower mechanical strength and narrower pore size distributions. A concise comparison of their advantages, disadvantages, and representative bioseparation applications is provided in Table 1, offering practical guidance for material selection under specific separation requirements.

Brief introduction of applications of porous materials in bioseparation

In protein separation, MOFs leverage moderate pore environments and surface charge control to achieve selective adsorption and separation of proteins, such as enzymes^[30,52]. COFs minimize protein denaturation during adsorption through hydrophilic pores and inert surfaces, making them suitable for purifying large molecules like antibodies. HOFs, with their unique hydrogen-bonding networks, offer excellent solution processability and biocompatibility, ideal for gentle handling and enrichment of proteins.

In glycopeptide enrichment, MOFs enhance hydrophilic interactions by introducing amino, carboxyl, or hydroxyl groups on their surfaces, facilitating stronger binding with glycan chains^[53]. Additionally, metal sites within MOFs can form coordination bonds with the hydroxyl groups of glycopeptides. COFs achieve strong hydrophilicity and high affinity for glycopeptides through the introduction of functional groups like polyethyleneimine (PEI) and boric acid. HOFs utilize specific hydrogen-bonding patterns to achieve efficient recognition and enrichment of glycopeptides, particularly in aqueous media.

For phosphopeptide enrichment, metal nodes in MOFs or post-modified metal ions interact with phosphate groups to form stable chelates. Polar pores in MOFs facilitate adsorption via hydrogen bonding or electrostatic interaction^[54]. COFs primarily use introduced metal sites to form coordination bonds with phosphate groups or achieve specific binding through electrostatic attraction between functionalized groups and phosphorylated peptides. HOFs, with their inherent hydrophilicity and modifiable pore environments, show promise for enriching phosphopeptides and enhancing selectivity through rational functionalization.

In summary, MOFs, COFs, and HOFs have emerged as transformative platforms in bioseparation, offering structurally programmable frameworks^[55], well-defined porosity^[56-58], and tunable interfacial functionalities^[59,60] that surpass the limitations of conventional materials. These attributes not only address long-standing challenges in selective recognition and efficient separation within complex biological matrices but also enable a shift from passive adsorption toward intelligent, stimuli-responsive, and multifunctional separation systems. In this review, we systematically examine the recent advances of these porous framework materials in key bioseparation applications - particularly in the selective separation of proteins and the high-efficiency enrichment of glycopeptides^[61,62] and phosphopeptides^[63,64]. We further highlight cutting-edge developments in stimuli-responsive separation platforms, biomimetic recognition interfaces, and synergistic purification strategies. A comparative analysis of performance across different material systems is provided, along with discussions on current challenges including stability under physiological conditions, scalability, pre- and post-modification, and biocompatibility. Finally, we offer perspectives on future directions, emphasizing their potential in precision medicine and high-throughput biomolecular separation and analysis.

In order to provide experimental support and theoretical guidance for the design of high-performance separation materials, this study methodically clarifies the synergistic regulation mechanism of pore geometric characteristics and local chemical microenvironments of porous framework materials (including MOFs, COFs, and HOFs) on the biomolecule separation performance. Based on the correlation between key structural parameters (e.g., pore size, functional group type, and charge distribution) and separation performance parameters (e.g., selectivity and adsorption capacity) established in this study, the development of standardized machine learning datasets and the further expansion of data-driven methods for the rational design of bioseparation materials are anticipated to emerge as a highly promising research direction in this field.

Separation of chiral molecules

Chirality, as a fundamental property of nature, plays a primary role in life activities and pharmaceutical research^[83,84]. Approximately 40%-50% of drugs exist in the form of chiral molecules, and their enantiomers often show significant differences in biological activity, one enantiomer may possess the expected pharmacological effects, whereas the other may be accompanied by loss of activity, toxicity, or adverse effects. These differences directly affect the therapeutic effects and safety of drugs. Therefore, efficient separation of chiral compounds has become a critical technical bottleneck in the screening of bioactive molecules, drug synthesis, and quality control^[85,86]. In the field of separation science, owing to their unique enantiomeric recognition and separation capabilities, chiral MOFs have demonstrated significant technical advantages, becoming a frontier hotspot in the research of chiral separation materials.

In 2020, Yu et al. reported the application of D-his-ZIF-8@SiO₂ core-shell microspheres as a chiral stationary phase (CSP) for the enantio-separation in high-performance liquid chromatography (HPLC), demonstrating high enantio-selectivity and effective chemo-selectivity^[87]. Figure 1A is the schematic diagram of the preparation process for D-his-ZIF-8@SiO₂. The structural and morphological properties of ZIF-8@SiO₂ nanocrystals were characterized using powder X-ray diffraction (PXRD), scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). PXRD patterns [Figure 1B] confirmed that the synthesized D-his-ZIF-8 nanocrystals showed characteristic diffraction peaks consistent with pristine ZIF-8, indicating their structural similarity. The coexistence of distinct signals from SiO₂−COOH and D-his-ZIF-8 in the spectra validated the successful formation of D-his-ZIF-8 on the SiO₂ microspheres. SEM was applied to characterize the morphological features of SiO₂−COOH, D-his-ZIF-8 nanocrystals, and D-his-ZIF-8@SiO₂ core-shell microspheres [Figure 1C]. The SiO₂−COOH microspheres demonstrated a smooth surface topography with an average diameter of approximately 5 mm. In contrast, the synthesized D-his-ZIF-8 nanocrystals displayed uniform polyhedral shapes with an average particle size of 200 nm [Figure 1C]. For the D-his-ZIF-8@SiO₂ core-shell structure, the average diameter increased from 5 mm (naked SiO₂ microspheres) to 5.5 mm, providing direct evidence for the successful immobilization of D-his-ZIF-8 nanocrystals onto the SiO₂ microsphere surface. the successful synthesis of the D-his-ZIF-8@SiO₂ core-shell structure was further confirmed by FT-IR analysis [Figure 1D]. As shown in Figure 1E, the homochiral structure of D-his-ZIF-8 was characterized by circular dichroism (CD) spectroscopy, compared with ZIF-8, a clear positive dichroic signal appeared at 235 nm, indicating that the chiral ligand was successfully embedded in the ZIF-8 framework through self-assembly of Zn²⁺, 2-methylimidazole, and d-histidine as chiral ligands. To evaluate the enantioselective separation capability of D-his-ZIF-8@SiO₂, 18 pairs of racemic compounds, including 1-(1-naphthyl)ethanol, 1-(4-chlorophenyl)ethanol, 1,1′-bi-(2-naphthol), 3,5-dinitro-N-(1-phenylethyl)benzamide, praziquantel, zopiclone, and flurbiprofen, naproxen, were resolved on the material-packed column using binary mobile phases of n-hexane and isopropanol at different volume ratios (99:1, 95:5, 9:1, 8:2, v/v). Table 2 summarizes the chromatographic parameters obtained from the column, including retention factor (k), separation factor (α), and resolution (Rₛ). For the racemates tested, the corresponding chromatograms of these racemic mixtures appearing in Figure 1F-H showed excellent enantiomer separation. Compared to the commercial AD column (Daisel Corp.), the D-his-ZIF-8@SiO₂ column offered complementary resolution for these racemic compounds.

Figure 1. (A) Schematic demonstration for the preparation of D-his-ZIF-8@SiO₂ core−shell microspheres; (B) Comparison of PXRD patterns of the prepared SiO₂−COOH, D-his-ZIF-8, and D-his-ZIF-8@SiO₂; (C) SEM image of D-his-ZIF-8@SiO₂; (D) FT-IR spectra of SiO₂, SiO₂−COOH, D-his-ZIF-8, and D-his-ZIF-8@SiO₂ core−shell microspheres; (E) CD patterns of ZIF-8 (red) and D-his-ZIF-8 (black); (F-H) HPLC chromatograms on the D-his-ZIF-8@SiO₂ column (column A, 25 cm length × 2.1 mm i.d.) for the separation of racemic compounds: trans-stilbene oxide (F), praziquantel (G), and 1-(1-naphthyl)ethanol (H). (A-H) are reprinted with permission from Ref.^[87], Copyright © 2023 by American Chemical Society. PXRD: Powder X-ray diffraction; SEM: scanning electron microscope; FT-IR: Fourier transform infrared spectroscopy; CD: circular dichroism; HPLC: high-performance liquid chromatography.

However, it is widely recognized that pure MOFs-packed HPLC columns suffer from high backpressure due to the small sizes of the MOF particles and their inner pores, which significantly hinder their practical applications in HPLC chiral separations. To tackle these difficulties, Yu et al. utilized silica gel as the substrate to prepare a novel chiral core-shell composite [Zn₂(D-Cam)₂(4,4′-bpy)]_n@SiO₂, which was employed as a stationary phase for column packing^[87]. This composite-packed column not only effectively mitigated the issue of excessive column pressure but also significantly improved chiral separation efficiency. The developed stationary phase exhibits satisfactory separation performance toward disubstituted benzene isomers and a variety of chiral compounds, including alcohols, amines, ketones, esters, epoxides, and alkaloids, enabling highly efficient HPLC-based enantioseparation [Figure 1F-H].

Furthermore, Yuan’s group has carried out chromatographic separations using various chiral MOFs as CSPs, including [Cu(S-mal)(bpy)]_n]^[88], [{[Cu(sala)]^[89], Cu(S-mal)(bpe)]_n^[90], [Co₂(D-cam)₂(TMDPy)]@SiO₂^[91] and [Zn(L-tyr)]_n(L-tyrZn)^[92].

Enrichment of peptides

Enrichment of glycopeptide

Protein glycosylation, a highly prominent and diverse post-translational modification (PTM), significantly alters protein functions and profoundly influences multiple biological activities^[93]. Variations in protein glycosylation can govern inflammatory responses, allow viruses to evade immune detection, facilitate dissemination of cancer cells, and modulate programmed cell death^[94]. To understand the processes of protein glycosylation within complex biological systems, efficient enrichment and selective analysis of glycopeptides is essential. However, the complexity of biological sample matrices, low abundance of glycopeptides, and diversity of glycan structures together comprise core technical bottleneck in glycoproteomics research. As a result, the specific enrichment of glycopeptides is essential for their accurate analysis. Research showed that MOFs with strong hydrophilicity displayed meaningful advantages in the efficient capture of glycopeptides^[95].

For example, Zhou et al. developed a novel dual-hydrophilic MOF material based on UiO-66, which exhibited significantly enhanced hydrophilicity and excellent hydrophilic interaction chromatography (HILIC) enrichment performance for glycopeptide analysis^[96]. The synthesis procedure is shown in Figure 2A. As shown in Figure 2B, all 2θ characteristic peaks in PXRD pattern of modified UiO-66 MOF are consistent with those of the parent UiO-66-NH₂ reported in literature^[97]. FT-IR further confirmed the successful modification of boronic acid and phosphate functional groups on UiO-66 MOF [Figure 2C]. MOF was characterized by transmission electron microscope (TEM) [Figure 2D], and these nanoparticles (NPs) exhibited uniform shapes and their topological structures were comparable to those of unmodified UiO-66 MOF. The above results jointly confirmed the successful preparation of postmodified UiO-66 MOF. To evaluate the glycopeptide enrichment performance and reusability of the UIO-PBA&FDP material, 100 μg of this material was used to conduct glycopeptide enrichment experiments on a 100 fmol/μL immunoglobulin G (IgG) digest, with a total of 3 repeated cycles. The results are presented in Figure 2E. Experimental results demonstrated that the UIO-PBA&FDP probe could effectively capture glycopeptides. Even after 3 repeated uses, the enriched glycopeptides still exhibited high signal intensity and signal-to-noise (S/N) ratio, which confirmed that the UIO-PBA&FDP material possesses favorable reusability. The complementarity and overlap of the three distinct approaches to N-glycopeptide identification are graphically depicted in this Venn diagram. The UIO-PBA&FDP method showed the highest recognition ability, while the parts commonly identified by the three methods represent the most reliable results [Figure 2F]. This WebLogo describes the amino acid sequence features surrounding the N-glycosylation sites identified under specific conditions (UIO-PBA&FDP). When enriching N-glycopeptides, the UIO-PBA&FDP method can effectively recognize glycosylation sites that adhere to the traditional N-X-S/T motif. The preference for amino acids in the vicinity provides additional insights into the selectivity of N-glycosylation sites, which is conducive to understanding the sequence specificity during the glycosylation process [Figure 2G]. The highly hydrophilic UIO-PBA&FDP, demonstrated high-sensitivity enrichment of N-linked glycopeptides with a detection limit of 0.5 fmol/mL. The innovation of this material is that PBA&FDP’s hydrophilic design, The hydrophilic design of PBA&FDP is achieved by introducing boronic acid groups at the ligand termini and modifying FDP with metal sites, thus creating a MOF interface that is both synergistically functionalized at both ends and highly hydrophilic, which in turn efficiently drives the HILIC-based enrichment of glycopeptides. Specifically, 359 N-linked glycopeptides corresponding to 104 glycoproteins were verified from only 1 mL of human serum, illustrating its superior enrichment performance for glycopeptides and analysis by UIO-PBA&FDP from biological sample.

Figure 2. (A) Post-modification of UIO-PBA&FDP and the procedure of HILIC enrichment for glycopeptides and analysis from biological Sample; (B-D) XRD patterns, FT-IR spectra and TEM image of as-synthesized UIO-PBA&FDP; (E) MALDI-TOF MS of peptide mixtures of 100 fmol/μL tryptic digests of IgG with enrichment by UIO-PBA&FDP for 3 cycles. The red stars represent glycopeptides; (F) Venn diagram showing the overlapping of unique glycosylated peptides; (G) The motif analysis of glycosylation sites identified from human serum after enrichment by UIO-PBA&FDP. (A-G) are reprinted with permission from Ref.^[96], Copyright © 2023 by Springer Nature. HILIC: Hydrophilic interaction chromatography; XRD: X-ray diffraction; FT-IR: Fourier transform infrared spectroscopy; TEM: transmission electron microscope; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; IgG: immunoglobulin G.

Based on the molecular size and hydrophilic properties of glycopeptides, ideal enriched materials for biological samples must have suitable pore structures and highly hydrophilic surfaces. MOFs emerge as promising candidate materials in this context, nonetheless, their practical applications are frequently impeded by inherent constraints, including small micropore sizes and inadequate chemical stability. In response to the above challenges, Pu et al. developed an innovative strategy to combine collaborative etching with surface functionalization processes and introduce phytic acid (PA) to construct hydrophilic mesoporous MOFs^[98]. Furthermore, by introducing polyvinylpyrrolidone (PVP) during MOF synthesis, the modified metal organic framework material exhibited extremely high stability [Figure 3A]. TEM, FT-IR, SEM, and X-ray photoelectron spectroscopy (XPS) were used to confirm the successful synthesis of material [Figure 3B-G]. In particular, Figure 3E is intended to illustrate the effect of surface modification on the zeta potential of the material more precisely, it demonstrates that the zeta potential of Ce-MOF shifts from positive to negative after the introduction of a PA, which affects the subsequent glycopeptide enrichment. The etching process can control the pore size well and is suitable for glycopeptide enrichment. The as-prepared PA-modified cerium-based MOFs feature expanded hydrophilic mesoporous channels, thereby endowing Ce-MOF@PA with superior glycopeptide enrichment performance. Figure 3H and I describe the efficient performance of Ce-MOF@PA nanocomposites in glycopeptide enrichment. Before enrichment, only one glycopeptide with low mass spectral intensity was detected, and non-glycopeptides dominated the mass spectrometry spectrum. After capture with Ce-MOF@PA, 39 non-interfering glycopeptides were clearly detected, and the signal intensity of glycopeptides was significantly enhanced. Specifically, 422 glycopeptides were selectively captured from 2 mL of trypsin-digested human serum, which contains 155 glycoprotein [Figure 3J and K].

Figure 3. (A) Schematic illustration of the synthesis of Ce-MOF@PA and the enrichment procedure of glycopeptides; (B) XPS survey spectra of Ce-MOF@PA; (C) High-resolution XPS measurements of P_2p for Ce-MOF@PA; (D) The FT-IR spectra of Ce-BTC, Ce-MOF, and Ce-MOF@PA; (E) Zeta potential of Ce-BTC, Ce-MOF, and Ce-MOF@PA; (F and G) SEM and TEM images of Ce-MOF@PA; MALDI-TOF MS of IgG digests (6 μg) (H) before enrichment and after enrichment by (I) Ce-MOF@PA. The peaks of glycopeptides are marked with “In”; Overlapping of identified (J) glycopeptides and (K) glycoproteins in human serum after enrichment by Ce-MOF@PA and commercial HILIC material. (A-K) are reprinted with permission from Ref.^[98], Copyright © 2019 by American Chemical Society. MOF: Metal–organic framework; PA: phytic acid; XPS: X-ray diffraction; FT-IR: Fourier transform infrared spectroscopy; SEM: scanning electron microscope; TEM: transmission electron microscope; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; IgG: immunoglobulin G; HILIC: hydrophilic interaction chromatography.

Enrichment of phosphopeptides

Among the diverse regulatory mechanisms of organisms, protein phosphorylation is one of the most universal mechanisms. Studies have confirmed that it is associated with cell signaling and communication, cell proliferation, differentiation, survival and homeostasis maintenance, whereas regulating protein degradation, translation, transcription and metabolic processes^[99]. Although Phosphorylated proteins are extensively found in all cells of organisms and are essential to life activities, their natural abundance is usually low^[100]. This problem can only be solved by efficient enrichment and separation techniques in analytical methods such as mass spectrometry.

He et al. produced Zr-MOF in situ on the surface of Ti₃C₂T_x in an open glass tube using dielectric barrier discharge (DBD) technology^[101]. During the growth of Zr-MOF, Ti₃C₂T_x was simultaneously oxidized by DBD, resulting in the formation of TiO₂ NPs on its surface. Oxid-Ti₃C₂T_x/UIO-66-NH₂ composites were also prepared [Figure 4A]. In addition, the synthesis technique enables the surface of Ti₃C₂T_x to form abundant -O-, -F and -OH terminal groups, resulting in a ζ potential of -20.9 millivolts [Figure 4B], this negative surface charge facilitates the fixation of metal ions. Consequently promoting the growth of MOF Ti₃C₂T_x/UIO-66-NH₂. SEM images [Figure 4C and D] demonstrated that the material morphology changes from block to transparent reticular nanosheets. TEM further confirmed the successful synthesis of the material [Figure 4E and F]. The corresponding energy-dispersive X-ray spectroscopy (EDXS) mapping image [Figure 4G] further confirmed the homogeneous distribution of C, O, Ti, and Zr elements throughout the Oxid-Ti₃C₂T_x/UiO-66-NH₂ composite. High-resolution transmission electron microscopy (HRTEM) characterization [Figure 4H] revealed that the 0.24 nm lattice fringes match the (103) crystal plane of Ti₃C₂T_x, confirming the successful formation of the nanosheets. The composite’s crystal structure was also analyzed using X-ray diffraction (XRD) [Figure 4I]. The characteristic peak of TiO₂ was not observed, which can be ascribed to its low content and weak diffraction signal intensity, but its presence was verified by XPS [Figure 4J] and Raman spectroscopy [Figure 4K], confirming that the crystal structure of each component remains well. Notably, this study achieved rapid Zr-MOF growth on the surface of Ti₃C₂T_x under normal temperature conditions through discharge plasma (DBD) technology, and was accompanied by controllable oxidation of Ti₃C₂T_x to TiO₂. The composite has a high specific surface area of 905.1 m²/g, a porous structure with an average pore size of 1.3 nm, and multiple active sites (Ti-O and Zr-O chain clusters, exposed Ti sites), thereby demonstrating excellent sensitivity (0.1 fmol/L), specificity [100:1 ratio of bovine serum albumin (BSA) to α-casein] and reproducibility. In complex biological samples, it enables the enrichment of 24 phosphopeptides from the standard bovine α-casein [Figure 4L and M], 15 endogenous phosphopeptides from human saliva, and 4 endogenous phosphopeptides from human serum, showing phosphopeptide enrichment capability. The innovation of this research is that a new synthesis method is proposed to grow Zr-MOF and TiO₂ in situ on the surface of Ti₃C₂T_x, while simultaneously realizing precise control over the oxidation degree and composition.

Figure 4. (A) Synthesis of Oxid-Ti₃C₂T_x/UIO-66-NH₂ composites; (B) Zeta potential of Ti₃C₂T_x; (C) SEM image of Ti₃C₂T_x; (D) SEM images of Oxid-Ti₃C₂T_x/UIO-66-NH₂; (E) TEM images of Ti₃C₂T_x; (F)TEM images of Oxid-Ti₃C₂T_x/UIO-66-NH₂; (G and H) EDXS (G), HRTEM (H) images of Oxid-Ti₃C₂T_x/UIO-66-NH₂; (I) XRD patterns of Ti₃AlC₂, Ti₃C₂T_x, and Oxid-Ti₃C₂T_x/UIO-66-NH₂ composites (J) XPS spectra of Ti₃C₂T_x, and Oxid-Ti₃C₂T_x/UIO-66-NH₂ composites Ce-MOF@PA; (K) Raman spectra of Ti₃C₂T_x treated by DBD with different times; (L) Before enrichment and (M) after enrichment by Oxid-Ti₃C₂T_x/UIO-66-NH₂. (A-M) are reprinted with permission from Ref.^[101], Copyright © 2023 by Elsevier Ltd. Permission is not required for this non‑commercial use. SEM: Scanning electron microscope; TEM: transmission electron microscope; EDXS: energy-dispersive X-ray spectroscopy; HRTEM: high-resolution transmission electron microscopy; XRD: X-ray diffraction; XPS: X-ray photoelectron spectroscopy; MOF: metal–organic framework; PA: phytic acid; DBD: dielectric barrier discharge.

Du et al. proposed a novel strategy for phosphopeptide enrichment based on the complementary effects of (PEI) functionalized magnetic nanospheres and bimetal ions^[102]. The synthesis process is shown in Figure 5A.

Figure 5. (A) Synthetic procedure for Fe₃O₄@PDA@MIL(Ti)-PEI-Nb; (B) FT-IR spectra of (i) Fe₃O₄, (ii) Fe₃O₄@PDA, (iii) Fe₃O₄@PDA@MIL(Ti)-PEI, (iv) Fe₃O₄@PDA@MIL(Ti)-PEI-Nb; (C) XPS spectrum of Fe₃O₄@PDA@MIL(Ti)-PEI-Nb; (D) SEM image of Fe₃O₄@PDA@MIL(Ti)-PEI-Nb; (E) TEM image of Fe₃O₄@PDA@MIL(Ti)-PEI-Nb; (F) EDX spectrum of Fe₃O₄@PDA@MIL(Ti)PEI-Nb. The inset in (F) is the primary elements content table of EDX; Mass spectra of human saliva: (G) before and (H) after enrichment with Fe₃O₄@PDA@MIL(Ti)-PEI-Nb. (A-H) are reprinted with permission from Ref.^[102], Copyright © 2021 by Elsevier Ltd. FT-IR: Fourier transform infrared spectroscopy; XPS: X-ray diffraction; SEM: scanning electron microscope; TEM: transmission electron microscope; EDX: energy-dispersive X-ray spectroscopy.

Characterization results like SEM, TEM, XRD and FT-IR [Figure 5B-F] verified that the material is successfully synthesized, and it is then used for the efficient enrichment of phosphopeptides. The material showed extremely high detection sensitivity (0.1 fmol) and excellent selectivity, and can effectively capture phosphopeptides even at a molar ratio of 1:5,000 and successfully extract 34 phosphopeptides from saliva samples [Figure 5G and H]. The novel point of this research lies in its research method, through the synergistic effect of complementary bimetal ions (Ti⁴⁺ and Nb⁵⁺) and PEI, efficient enrichment of monophosphorylated peptides and polyphosphorylated peptides is significantly achieved. The material achieves comprehensive and efficient enrichment for the following reasons. Firstly, Nb⁵⁺ has a greater predilection for multiphosphopeptides, while Ti⁺ has a high affinity for monophosphopeptides. The material can concurrently capture both kinds of phosphopeptides by integrating both metal ions into the same framework, guaranteeing wide coverage across various phosphorylation states. Secondly, the amino groups on the branching PEI become extensively protonated under the acidic enrichment conditions, creating a thick positively charged layer. Strong electrostatic attraction toward negatively charged phosphopeptides is made possible by this high cationic density, which significantly increases enrichment efficiency. Thirdly, the material integrates the metal ion affinity chromatography (IMAC) mechanism - through coordination between Ti⁴⁺/Nb⁵⁺ and phosphate groups - with electrostatic attraction from the cationic field generated by PEI. This dualcapture mechanism collectively improves sensitivity, selectivity, and loading capacity.

Co-enrichment of glycopeptides and phosphorylated peptides

MOFs have shown extensive application potential in proteomics and peptidomics research with their unique pore structure and rich surface chemical properties^[103]. Protein phosphorylation and glycosylation are the two most common and critical types of PTMs in eukaryotes^[104]. By regulating a variety of cellular activities such as signal transduction^[105], immune response^[106], and cell proliferation^[107], they participate in the precision regulatory network of living organisms. Notably, the abnormal expression of these two modified types is closely related to the occurrence and development of multiple diseases. Particularly, the interaction between them can regulate the hyperphosphorylation of tau protein, which is considered to be one of the core pathological features of Alzheimer’s disease (AD)^[108]. However, achieving simultaneous characterization of these two types of PTMs in complex biological sample matrix remains a very challenging topic in current proteomics research. In the current research, multifunctional MOFs integrating hydrophilic ligands and phosphate-affinity metal centers are designed using HILIC and metal oxide affinity chromatography (MOAC) as core enrichment techniques. Such MOFs’ structure enables the simultaneous enrichment of glycopeptides and phosphopeptides by combining the synergistic effects of MOAC-mediated metal coordination and HILIC-based hydrogen bonding.

Wu et al. developed a multifunctional MOF composite (mMIL-125@Au@L-Cys) for the efficient enrichment and identification of N-linked glycopeptides and phosphopeptides in human lens tissues^[109]. The synthesis reaction is as follows [Figure 6A]. The material integrated the hydrophilic properties of L-Cys, the strong phosphopeptide-binding affinity of Ti-O clusters, the large specific surface area characteristic of MOFs, and the superparamagnetic behavior of Fe₃O₄ NPs. SEM, TEM characterization confirmed the successful stepwise synthesis [Figure 6B], including the formation of Fe₃O₄@PDA core-shell, the grafting of MIL-125 (evidenced by surface roughness), the deposition of uniform Au NPs measuring less than 10 nm, and the final L-Cys functionalization via Au–S bonds. FT-IR further confirmed the successful synthesis of this material [Figure 6C]. XRD analysis [Figure 6D] was performed to characterize the crystalline structures of both mMIL-125 and Au NPs. TEM [Figure 6E] characterization also confirmed the successful stepwise synthesis. These results confirmed the coexistence of mMIL-125 and Au NPs in the composite material. The developed material demonstrated good enrichment performance for both glycopeptides and phosphopeptides from standard proteins, achieving limit of detection of 0.1 fmol/mL and maintaining stability over 5 reuse cycles. The material successfully identified numerous N-linked glycopeptides and phosphopeptides in human lens proteins [Figure 6F and G], highlighting its potential for glycoproteomic and phosphoproteomic research. Furthermore, this study established a novel method for constructing functional MOF materials through post-modification strategies, providing a valuable framework for advancing proteomic studies.

Figure 6. (A) The synthetic strategy of mMIL-125@Au@L-Cys and (B) TEM images of mMIL-125@Au@L-Cys; (C) FT-IR spectra of Fe₃O₄@PDA, mMIL-125 and mMIL-125@Au@L-Cys; (D) XRD of mMIL-125@Au@L-Cys; (E) SEM images of mMIL-125@Au@L-Cys: magnified for 30,000 times; (F and G) MALDI-TOF MS for the phosphopeptides from a mixture of β-casein and BSA tryptic digests with a mass ratio of 1:100: (a) before enrichment, (b) after enrichment. for the glycopeptides from a mixture of HRP and BSA tryptic digests with a mass ratio of 1:100: (c) before enrichment, (d) after enrichment; Peaks of glycopeptides and phosphopeptides are marked with red stars and red triangles indicate the losses of phosphoric acid (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article); (H) The Synthetic route for Fe₃O₄@PDA@UiO-66-NH₂; (I) Workflow of glycopeptide or phosphopeptide enrichment from biological samples using Fe₃O₄@PDA@UiO-66-NH₂; (J) SEM images of Fe₃O₄@PDA@UiO-66-NH₂; (K) XRD patterns of Fe₃O₄@PDA@UiO-66-NH₂. MALDI-TOF MS for the glycopeptide enrichment from 250 fmol/mL; HRP tryptic digest: (L) after treatment with the first-time Fe₃O₄@PDA@UiO-66-NH₂; (M) for the phosphopeptide enrichment from 200 fmol/mL β-casein tryptic digest: after treatment with the first-time Fe₃O₄@PDA@UiO-66-NH₂. (A-G) are reprinted with permission from Ref.^[109], Copyright © 2019 by Elsevier B.V. (H-M) are reprinted with permission from Ref.^[110], Copyright © 2017 by Springer Nature. TEM: Transmission electron microscope; FT-IR: Fourier transform infrared spectroscopy; XRD: X-ray diffraction; SEM: scanning electron microscope; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; BSA: bovine serum albumin; HRP: horseradish peroxidase.

Xie and Deng needed a dual-functional hydrophilic magnetic amino-modified MOF for the selective enrichment of glycopeptides and phosphopeptides^[110]. The fabrication process began with the solvothermal synthesis of magnetic Fe₃O₄ cores, followed by the self-polymerization of dopamine to form a polydopamine (PDA) coating on their surfaces. The hydroxyl and amino groups of PDA facilitated the immobilization of Zr³⁺ ions, which subsequently reacted with amino ligands via a one-pot MOF synthesis, yielding Fe₃O₄@PDA@UiO-66-NH₂ [Figure 6H and I]. The resultant material was characterized using SEM, XRD [Figure 6J and K]. SEM images revealed a thin polymer shell on the spherical Fe₃O₄ microspheres. The UiO-66-NH₂ modification imparted MOFs with crystalline surfaces, contracting with the smoothness of Fe₃O₄@PDA. XRD displayed the characteristic peaks, confirming the successful synthesis. As shown in Figure 6L and M, horseradish peroxidase (HRP, a typical glycoprotein) and β-casein (a typical phosphoprotein) were enriched using Fe₃O₄@PDA@UiO-66-NH₂ material, demonstrating the high efficiency and selectivity of this material in enriching glycopeptides and phosphopeptides.

This study successfully designed and synthesized Fe₃O₄@PDA@UiO-66-NH₂, a material that effectively enriches both glycopeptides and phosphorylated peptides. The idea of a “dual-application” material that combined HILIC and immobilized IMAC technologies to expedite experimental procedures is a significant innovation. The material demonstrated high sensitivity and selectivity in tests with standard proteins and human serum, achieving detection limits as low as 0.2 fmol/mL for glycopeptides and 0.02 fmol/mL for phosphorylated peptides, along with maximum binding capacities of 4 and 0.8 mg/g, respectively. A successful application in complex biological samples, including human serum, revealed 307 N-glycosylated peptides and 33 phosphorylated peptides, demonstrating its good potential in glycoproteomics and phosphoproteomics research. Experimental validation also verified its stability and reproducibility.

Separation of protein

Protein purification serves as a crucial foundational technique for proteomics research in the life sciences^[111,112]. Therefore, the preparation of high-performance protein separation materials holds significant importance. Taking into account the substantial size and intricate structural nature of proteins, a digestion approach exhibiting site-specific selectivity is highly ideal.

Qian et al. initially synthesized carboxyl-functionalized carbon nanoparticles (CNs) as the substrate^[113]. Subsequently, UIO-66 crystals were grown in situ on these substrates to construct CN@UIO-66 composite carriers. To minimize non-specific protein adsorption, zwitterionic monomers were incorporated during the molecular imprinting process. Using cytochrome c as the template protein, the team successfully prepared CN@UIO-66@MIPs via surface imprinting technology. The preparation process is illustrated in Figure 7A. The effective synthesis of the composite material was confirmed by its characterization using SEM and TEM [Figure 7B and C]. Figure 7D and E presents the effects of two key parameters - protein-immobilizing carrier type and the loading of zwitterionic monomer DMAPS [3-dimethyl-(methacryloyloxyethyl) ammonium propane sulfonate] - on both the recognition ability and selectivity of molecularly imprinted polymers (MIPs). Collectively, these results confirm that the CN@UIO-66 composite carrier offers distinct advantages when constructing high-performance protein-imprinted materials.

Figure 7. (A) Illustration of the synthesis of CN@UIO-66@MIPs; (B) SEM image of CN@UIO-66@MIPs; (C) TEM image of CN@UIO-66@MIPs; Influence of the protein immobilization carrier (D) and DMAPS (E) content (the white and black numbers representing IF and β values, respectively) on the recognition and selectivity of the MIPs; (F) Fabrication of magnetic carbonized PDA@F127/ZIF-67 hollow nanocages; (G) High resolution XRD spectrum of carbonized PDA@F127/ZIF-67 NPs; (H) SEM image of carbonized PDA@F127/ZIF-67 NPs; (I) TEM image of carbonized PDA@F127/ZIF-67 NPs; (J) Influence of pH on the adsorption properties of the carbonized PDA@F127/ZIF-67 nanocages; (K) Spiked fetal calf serum analyzed by SDS-PAGE. Lane 1, protein molecular weight marker; lane 2, spiked fetal calf serum diluted 40-fold; lane 3, the supernatant after the enrichment by carbonized PDA@F127/ZIF; lane 4, the elute; lane 5, 0.2 mg·mL^-1 BHB standard solution; MALDI-TOF MS analyses for (L) 40-fold dilution of spiked fetal calf serum and (M) the eluate. (A-E) are reprinted with permission from Ref.^[113], Copyright © 2021 by American Chemical Society. (F-M) are reprinted with permission from Ref.^[114], Copyright © 2020 by American Chemical Society. SEM: Scanning electron microscope; TEM: transmission electron microscope; DMAPS: 3-dimethyl-(methacryloyloxyethyl) ammonium propane sulfonate; MIPs: molecularly imprinted polymers; XRD: X-ray diffraction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BHB: bovine hemoglobin; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This study successfully prepared raspberry-like cytochrome C surface-imprinted NPs based on CN@UIO-66 composites, demonstrating excellent adsorption capacity (815 mg∙g^-1), rapid equilibrium time (< 40 min), and remarkable recognition specificity (IF = 6.1). The innovative strategy of combining MOF composites with zwitterionic monomers can effectively overcome mass transfer limitations and non-specific binding problems in traditional methods, thus enabling efficient enrichment and specific recognition of targets in complex protein mixtures and biological samples.

Tan et al. prepared a novel MOF-derived polymer-mediated magnetic hollow carbon nanocage for the selective enrichment of bovine hemoglobin (BHB) proteins^[114]. The preparation process is illustrated in Figure 7F. The as-prepared nanocages were systematically characterized using XRD, SEM, TEM, and vibrating sample magnetometry (VSM) [Figure 7G-I]. All characterization results collectively confirmed the successful synthesis of the nanocages. Figure 7J is mainly used to analyze the surface charge properties of nanomaterials and their influence on the adsorption behavior of the target protein (BHB) under different pH conditions. Figure 7K-M shows the capture and enrichment of BHB protein by carbonized PDA@F127/ZIF-67 nanocages in real biological samples (e.g., fetal bovine serum and bovine serum). This confirms the feasibility and efficiency of this material for enriching target proteins in complex biological samples. This study validated that magnetic hollow carbon nanocages exhibited excellent adsorption ability on BHB in complex biological samples. The novelty of this study is the development of a stress-induced oriented contraction method to prepare magnetic hollow carbon nanocages with graded pore structures, which effectively overcoming the limitations of the inefficient adsorption ability and cumbersome separation process of traditional MOF-derived materials. This stratified hollow porous structure significantly increases the specific surface area, reduces mass transfer resistance, and reinforce the high affinity adsorption capacity of the target protein BHB. Experimental results revealed that the adsorption capacity of this material to BHB is up to 834.3 mg·g^-1, which is significantly more than other methods reported in existing literature, and provides broad application prospects for the separation and purification of complex biological sample.

Separation of peptides

Separation of glycopeptides

In recent years, COFs have been widely utilized for the efficient separation and enrichment of glycopeptides, making them ideal candidates for glycoproteomic analysis in complex biological samples. These materials achieve strong hydrophilicity and high affinity for glycopeptides through the incorporation of hydrophilic functional groups, such as PEI, boronic acid, and glutathione (GSH)^[125-128]. Moreover, by integrating with NPs - such as gold or silver - the number of available binding sites is significantly increased, thereby enhancing interactions with glycopeptides and improving capture efficiency^{[125,126,129]}.

In 2022, Ji et al. developed a hierarchical flower-like hollow composite (HFH-COFs@Au@GSH) by anchoring Au NPs via sodium citrate reduction and functionalizing with GSH through Ag–S bonds [Figure 8A]^[127]. TEM confirmed the preservation of the hollow morphology (450-550 nm) after Au NP and GSH loading, with a specific surface area retention of 634.7 m²/g (vs. 2,324.8 m²/g for pristine HFH-COFs). The flower-like hollow architecture of the material, endowed with hierarchical porosity and internal cavities, not only confers structural stability and abundant binding sites but also facilitates glycopeptide accessibility and shortens diffusion pathways, thereby enabling rapid adsorption [Figure 8B]. After enrichment with HFH-COFs@Au@GSH, 49.59% of the sequences were identified as the NXT sequence, which is the primary site for N-glycosylation in eukaryotes. Additionally, this material captured 8.94% of the NXC sequence. This indicates that the material possesses the ability to enrich atypical N-glycosylation sites and exhibits high coverage [Figure 8C]. The -SH groups in GSH directly interact with sialic acid residues on N-glycopeptides, enabling highly selective enrichment with a detection limit of 0.1 fmol/μL - superior to most reported materials. The WebLogo shows that within the broad range of “-7 to +7”, the occurrence frequency of hydrophilic amino acids [such as serine (S), threonine (T), etc.] is significantly higher. This reflects the enrichment mechanism of the material, where the hydrophilic GSH modified on the material surface binds to N-glycopeptides through “HILIC” [Figure 8D]. GO functional annotation of glycoproteins was performed from the dimensions of cellular component, biological process, and molecular function, respectively, which clarified the biological functions of N-glycoproteins [Figure 8E-G]. This selectivity was further validated by the successful identification of 308 N-glycopeptides in human serum, demonstrating the material’s practical utility in glycoproteomics. The synergy between the hierarchical structure (for high capacity) and GSH’s specificity (for molecular recognition) establishes this composite as a robust platform for sensitive glycopeptide analysis in complex biological matrices.

Figure 8. (A) Preparation process of HFH-COFs@Au@GSH with TEM image; (B) Workflow of N-glycopeptide enrichment using the HFH-COFs@Au@GSH; (C) N-Glycosylation sites identified from human serum; (D) Web-Logos of N-glycosylation sites identified from human serum; GO analysis by DAVID for the identified glycoproteins from human serum, including the cellular component (E), biological process (F), and molecular function (G); (H) Synthetic schematics of TbBD@PEI@Au@4-MPBA composites and Enrichment procedure of glycopeptides by TbBD@PEI@Au@4-MPBA; (I and J) SEM (I) and TEM (J) images of the TbBD@PEI@Au@4-MPBA. (A-G) are reprinted with permission from Ref.^[127], Copyright © 2022 by Royal Society of Chemistry. (H-J) are reproduced with permission from Ref.^[125], Copyright © 2022 by Elsevier. GSH: Glutathione; TEM transmission electron microscopy; GO: Gene Ontology; DAVID: database for annotation, visualization, and integrated discovery; TbBD: 1,1,4,4-tetraphenyl-1,3-butadiene; PEI: polyethyleneimine; MPBA: mercaptophenylboronic acid; SEM: scanning electron microscope.

Xie et al. constructed a boronic acid-functionalized COF composite (TbBD@PEI@Au@4-MPBA) for highly selective glycopeptide enrichment^[125], where the COF substrate provides a high surface area and stable pore structure, PEI enhances hydrophilicity and serves as a stabilizer for in-situ growth of Au NPs, and 4-MPBA, anchored via Au–S bonds, introduces abundant boronic acid groups that specifically bind glycopeptides through reversible boronate ester bonds under alkaline conditions, with release achieved under acidic elution [Figure 8H]. Structural characterization (SEM/TEM) revealed spherical aggregates (~25 nm) embedded with Au NPs (~15 nm) [Figure 8I and J], whose regular and tunable pore architecture enables selective sieving of target glycopeptides. This design enabled ultra-sensitive enrichment (detection limit: 5 amol/μL) and high selectivity (1:1,000, HRP/BSA), with minimal nonspecific adsorption due to PEI’s hydrophilic shielding. The composite successfully identified 56 endogenous glycopeptides in saliva and 513 disease-related glycopeptides in laryngeal cancer serum, showing excellent reusability (10 cycles) and stability (2 months at room temperature).

Magnetic COF materials enable efficient glycopeptide enrichment with rapid separation/recovery capabilities for high-throughput operations. In 2020, Wu et al. developed a magnetic COF material (mCTpBD) designed to enhance the selective enrichment of glycopeptides through multiple synergistic mechanisms, including hydrophilic interactions and size-exclusion effects^[116]. The material was synthesized by interfacial deposition, with an average diameter of 398 nm [Figure 9A]. The TEM images of mCTpBD show that compared with Fe₃O₄−NH₂, mCTpBD still maintains a spherical morphology, which intuitively verifies the successful construction of the “magnetic core-hydrophilic COF shell” core-shell structure [Figure 9B]. The detection results of HRP digests, obtained by comparing the “pre-enrichment” samples with those “after enrichment using mCTpBD”, showed that after enrichment with mCTpBD, the number and intensity of glycopeptide characteristic peaks in the spectrum were significantly increased. The core reason for this lies in the inherent hydrophilicity of mCTpBD [Figure 9C and D]. The introduction of CTp endowed the material with intrinsic hydrophilicity, facilitating efficient binding to glycopeptides in complex biological matrices. The microporous structure further improved selectivity by excluding macromolecular interferences, while the imine bonds (C=N) and aromatic rings (C=C) in the COF layer promoted π–π stacking interactions with glycopeptides. In a 1:50 mixture of HRP and BSA digests, mCTpBD successfully enriched 15 glycopeptides, with no significant performance loss observed over five reuse cycles. The material exhibited a high enrichment sensitivity of 0.5 fmol/μL for standard glycopeptides, outperforming previously reported COF-based materials. When applied to real-world samples, mCTpBD enabled the identification of 28, 32, and 49 endogenous glycopeptides from saliva samples of three healthy individuals, and 27, 39, and 40 glycopeptides from patients with inflammatory bowel disease, using nano-LC-MS/MS analysis. These results highlight its practical applicability in complex biological systems. Additionally, the material retained strong magnetic responsiveness (saturation magnetization: 50.49 emu/g), enabling rapid separation under an external magnetic field. Then in 2022, Su’s team^[128] developed a magnetic COF composite (Fe₃O₄@TpBD@Au@GSH) for efficient and selective glycopeptide enrichment, combining rapid separation capability with high adsorption capacity. The material was constructed by coating 400 nm Fe₃O₄ spheres with TpBD to form a 150 nm smooth layer, followed by in-situ modification with Au NPs and GSH via Au–S bonds, resulting in a final particle size of ~900 nm with a coarse surface morphology confirmed by SEM [Figure 9E and F]. The synergistic effect of TpBD’s hydroxyl/amino groups and GSH’s carboxyl/amino groups created dual hydrophilic binding sites, achieving a high adsorption capacity of 160 mg/g and excellent selectivity with resistance to protein interference at a ratio of 1:2,000 [Figure 9G and H]. This structural design enabled ultra-sensitive glycopeptide enrichment and highly specific capture, as demonstrated by the detection of 21 HRP glycopeptides even after 3 months of storage at room temperature, along with stable performance over six reuse cycles, highlighting its practical applicability in complex biological samples.

Figure 9. (A) Graphical synthetic route of CTp and mCTpBD, respectively, and enrichment procedure of glycopeptides by mCTpBD; (B)TEM images of mCTpBD; MALDI-TOF MS of HRP tryptic digest (100 fmol/μL) (C) before the enrichment, and (D) after the enrichment by mCTpBD. The peaks of glycopeptides are marked with a red asterisk (*); (E) The synthetic route of Fe₃O₄@TpBD@Au@GSH; (F) SEM of Fe₃O₄@TpBD@Au@GSH; MALDI-TOF MS of the mixture of HRP tryptic digests and BSA proteins at various molar ratios after enrichment by Fe₃O₄@TpBD@Au@GSH (G) 1:1,000, (H) 1:2,000. (A-D) are reprinted with permission from Ref.^[116], Copyright © 2020 by American Chemical Society. (E-H) are reproduced with permission from Ref.^[128], Copyright © 2022 by Elsevier. CTp: Carboxyl-modified 1,3,5-triformylphloroglucinol; mCTpBD: magnetic carboxyl-modified 1,3,5-triformylphloroglucinol-benzidine covalent organic framework; TEM: transmission electron microscopy; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; HRP: horseradish peroxidase; Tp: 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde; BD: benzidine; GSH: glutathione; SEM: scanning electron microscope; BSA: bovine serum albumin.

COF and its composites have become cutting-edge tools in the field of glycopeptide separation and enrichment due to their highly adjustable structure and excellent hydrophilicity. They exhibit high sensitivity and selectivity in complex biological samples, advancing glycoproteomics and disease marker research. The synthesis steps of some materials are complex, and there is still room for improvement in the separation of very low-abundance glycopeptides in practical applications.

Separation of phosphorylated peptides

Protein phosphorylation is a key PTM involved in various biological processes, and its dysregulation is closely linked to diseases such as cancer and diabetes^[130,131]. Due to their low abundance and interference from complex biological matrices, efficient enrichment is essential for sensitive detection of phosphorylated peptides^[132]. COFs, with their high surface area, tunable pore structures, and versatile functionalization, have shown great promise in glycopeptide enrichment. These features make COFs a compelling platform for developing advanced materials for selective phosphopeptide capture, offering significant potential in proteomics and biomedical research.

By introducing functional groups such as metal ions such as Ti⁴⁺ or guanidine groups, COF materials can achieve high affinity enrichment of phosphorylated peptides, which, combined with magnetic NPs, facilitate rapid separation and recovery, making them suitable for high-throughput processing of complex biological samples. Wang’s team^[133] designed a titanium-functionalized 2D-COF (TpPa-2-Ti⁴⁺) for highly selective phosphopeptide enrichment [Figure 10A]. SEM/TEM revealed elongated sheet-like nanostructures offering abundant surface binding sites, and the mesoporous architecture (2.8 nm) effectively excluded large interferences like serum albumin while allowing selective access of small phosphopeptides. The Ti⁴⁺–phosphate coordination served as the primary driving force for enrichment, assisted by hydrogen bonding and electrostatic interactions between framework-bound polar groups and phosphopeptides. The chelation between metal cations and phosphate groups, along with the hydrophilic adsorption of PEG groups, inhibits nonspecific hydrophobic adsorption, synergistically enabling efficient and selective enrichment of phosphorylated peptides. The limit of detection is as low as 0.02 fmol/μL. The phosphorylated peptide is still specifically enriched with a 1:100 molar ratio of α-casein to BSA with a clean background signal. It identified 18 and 17 phosphopeptides from α-casein digests with and without BSA (1:50), respectively, outperforming commercial TiO₂ beads, and successfully enriched 12 from skim milk and 7,432 from HeLa cells, demonstrating high sensitivity, selectivity, and practical applicability in complex biological samples.

Figure 10. (A) Schematic illustration of synthesis of TpPa-2-Ti⁴⁺ through condensation and coordination with chemical structure of TpPa-2 and chemical structure of TpPa-2-Ti⁴⁺; (B) Representation of the strategy for preparing Zr⁴⁺-immobilized magnetic COFs through sequential post-synthetic modifications; (C) Schematic representation of preparation of TpTGCl CONs, and enrichment procedure of phosphopeptides with TpTGCl CONs; (D) Powder XRD patterns of simulated and as-synthesized TpTGCl CONs; (E) TEM image of TpTGCl CONs; (F) AFM image and height profile of TpTGCl CONs. MALDI-TOF MS of non-fat milk tryptic digest; Direct analysis (G), after enrichment by TpTGCl CONs in buffer A (H). MALDI-TOF MS of human saliva (5 μL); Direct analysis (I), after enrichment by TpTGCl CONs in buffer A (J) (“s” in blue indicates mono-phosphopeptide, “m” in red indicates multi-phosphopeptide. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article); (K) Distribution proportions of mono- and multi-phosphopeptides identified from rat liver protein digest by TpTGCl CONs in buffer A and buffer B, respectively. And distribution proportions of the integrated phosphorylation sites identified from rat liver protein digest by TpTGCl CONs in buffer A and buffer B; GO analysis by DAVID for the identified phosphoproteins from rat liver protein digest, including biological process (L), molecular function (M). (A) is reprinted with permission from Ref.^[133], Copyright © 2017 by Elsevier. (B) is reproduced with permission from Ref.^[134], Copyright © 2021 by American Chemical Society. (C-M) are reproduced with permission from Ref.^[135], Copyright © 2021 by Elsevier. Tp: 1,3,5-Triformylphloroglucinol; Pa-2: 2,5-dimethyl-1,4-benzenediamine; COFs: covalent organic frameworks; TGCl: triaminoguanidinium chloride; CONs: covalent organic nanosheets; XRD: X-ray diffraction; TEM: transmission electron microscopy; AFM: atomic force microscopy; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; GO: Gene Ontology; DAVID: database for annotation, visualization, and integrated discovery.

Gao et al. developed a magnetic three-layer composite material with a COF shell for highly selective phosphopeptide enrichment^[134]. The structure consists of magnetic colloidal nanocrystalline clusters (MCNCs) as the core, a COF layer synthesized from TfPb and DHBD as the middle shell, and Zr⁴⁺ ions anchored on the surface via coordination with phosphate groups introduced through succinic anhydride (SA) and PA modification [Figure 10B]. This “magnetic nucleus–functionalized COF shell–Zr⁴⁺ active site” design enables specific phosphopeptide capture via Zr⁴⁺–phosphate coordination under neutral conditions, with elution achieved under acidic conditions [2% trifluoroacetic acid (TFA)], while the mesoporous structure (2.7-3.3 nm) excludes large proteins. Therefore, it is suitable for the diffusion of small phosphorylated peptides while repelling large proteins (such as BSA) to achieve a size exclusion effect. The magnetic core provides rapid separation within 1 min under an external field (saturation magnetization: 42.4 emu/g), facilitating efficient handling in complex biological samples. With a positively charged surface minimizing nonspecific adsorption, the material achieved a detection capacity of 46.48 μg/mg and successfully enriched 3 phosphopeptides from 0.25 μg/mL β-casein digest at a BSA/β-casein molar ratio of 200:1. It also identified 14 phosphopeptides from skim milk and detected endogenous phosphorylated peptides from diluted human serum. In 2021, He et al. synthesized two-dimensional guanidine-rich covalent organic nanosheets (TpTGCI CONs) via a simple solvothermal Schiff-base reaction between 1,3,5-triformylphloroglucinol (Tp) and triaminoguanidine hydrochloride (TGCl), yielding layered nanosheets with abundant guanidine groups that enable pH-tunable phosphopeptide recognition [Figure 10C]^[135]. With a thickness of ~6 nm (3-6 layers), a diameter of ~2 μm, and a specific surface area of 251 m²/g, the material offers numerous accessible active sites for efficient binding. Structural characterization confirmed the successful formation of COF architecture: PXRD showed characteristic peaks at ~9.7° and ~27.3°, consistent with hexagonal layering and π–π stacking [Figure 10D]; TEM and SEM revealed thin, folded sheet-like morphology; atomic force microscopy (AFM) verified the nanosheet thickness [Figure 10E and F]. The interlayer spacing allows selective entry of small phosphopeptides while excluding larger proteins, significantly enhancing selectivity. Benefiting from the strong polar interactions between guanidine groups and phosphate moieties, the material achieved an ultralow detection limit of 0.05 fmol/μL for global phosphopeptides, demonstrating excellent potential for sensitive biomarker screening in complex biofluids such as serum and saliva. For the enrichment of phosphopeptides from the tryptic digest of non-fat milk, 8 mono-phosphopeptide peaks and 18 multi-phosphopeptide peaks were clearly identified [Figure 10G and H]. For the enrichment of endogenous phosphopeptides in human saliva, no phosphopeptide peaks were observed in the spectrum during direct detection. However, after enrichment with TpTCI CONs, 9 mono-phosphopeptide peaks and 12 multi-phosphopeptide peaks were identified [Figure 10I and J]. Through Gene Ontology (GO) analysis, the biological significance of phosphorylated proteins enriched from rat liver proteins by TpTGCI CONs was systematically illustrated from three dimensions [Figure 10K-M].

Co-enrichment of glycopeptides and phosphorylated peptides

Bifunctional COF materials introduce specific functional groups, such as MUBA, iron ions, amino groups, etc., and use hydrogen bonds, metal ion coordination and hydrophilic interactions to achieve synergistic recognition and enrichment of phosphorylated peptides and glycopeptides^[136,137].

In recent years, there have been several notable advancements in the application COFs for co-enrichment, showcasing their potential in various analytical and separation tasks. For instance, in 2021, Luo’s team^[138] developed a core-shell magnetic COF material, MCNC@Polymer@COF-MUBA, for efficient co-enrichment of glycopeptides and phosphopeptides [Figure 11A]. The hydrophilic COF surface facilitates hydrophilic interaction with glycopeptides, while the urea group in MUBA forms multiple hydrogen bonds with phosphate groups of phosphopeptides, reducing non-specific adsorption [Figure 11B]. The material allows either simultaneous or selective enrichment by tuning conditions. Under high interference (IgG:α-casein:BSA = 1:1:1,000), 21 glycopeptides and 18 phosphopeptides were identified [Figure 11C and D]. From rat liver digest, 1,717 unique glycopeptides (898 glycoproteins) and 1,997 phosphopeptides (1,100 phosphoproteins) were captured, including 229 dual-modified proteins, demonstrating high efficiency and coverage [Figure 11E]. Exosomes isolated from hepatocellular carcinoma patient plasma (confirmed by cup-shaped morphology in TEM and markers CD9/CD63/CD81 in Western blot, Figure 11F and G) yielded 293 glycopeptides (180 glycoproteins) and 116 phosphopeptides (76 phosphoproteins) after enrichment, highlighting its selectivity and applicability in complex biological samples [Figure 11H].

Figure 11. (A) Schematic representation of the synthesis of MCNC@polymer@COF-MUBA nanospheres; (B) Schematic diagram of the binding modes and controllable selective enrichment of the nanospheres for phosphopeptides and glycopeptides; (C) MALDI-TOF MS of peptide mixtures of tryptic digests of IgG, α-casein, and BSA with a molar ratio of 1:1:500 direct analysis; (D) MALDI-TOF MS of peptide mixtures of tryptic digests of IgG, α-casein, and BSA with a molar ratio of 1:1:1,000 after enrichment with the nanospheres. Glycopeptides are marked with red arrows and phosphopeptides are marked with blue arrows (For interpretation of the references to color in this Figure legend, the reader is referred to the Web version of this article); (E) The numbers of glycopeptides (glycoproteins) and phosphopeptides (phosphoproteins) identified using the nanospheres from mouse liver digests; (F) TEM negative staining analysis of the obtained exosomes; (G) Western blot analysis of the proteins collected from exosomes and three exosome markers (CD-9, CD-63, and CD81) were used; (H) Numbers of glycopeptides (glycoproteins) and phosphopeptides (phosphoproteins) in the exosome lysate identified using the material. (A-H) are reprinted with permission from Ref.^[138], Copyright © 2021 by Elsevier. MCNC: Magnetic colloidal nanocrystalline cluster; COF: covalent organic framework; MUBA: 4-(3-(2-(methacryloyloxy)ethyl)-ureido)benzoic acid; MALDI-TOF MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; IgG: immunoglobulin G; BSA: bovine serum albumin; TEM: transmission electron microscope.

In 2022, Xiong et al. developed Fe₃O₄@Thio-COF@Au@GSH, a magnetic zwitterionic COF designed for the efficient enrichment of glycopeptides and phosphopeptides [Figure 12A-C]^[139]. This material integrates multiple functional components. GSH provided strong hydrogen bonding with glycan chains and electrostatic interactions with phosphate groups in exosomal membranes, ensuring stable binding [Figure 12D and E]. The material demonstrated excellent sensitivity, detecting glycopeptides at 0.2 fmol and phosphopeptides at 0.4 fmol, and maintained high selectivity even in the presence of a 1,000-fold excess of BSA. It successfully identified 419 glycopeptides and 316 phosphopeptides from urinary exosomes, including 42 proteins with dual modifications involved in key cellular processes. Supported by theoretical calculations and protein interaction network analysis, this design integrates exosome capture with targeted peptide enrichment, offering a powerful platform for exosome proteomics and PTM analysis, with excellent reusability over five cycles. In addition to electrostatic action, the electron-rich P atom in the phosphate group forms intermolecular hydrogen bonds (marked with a blue dotted line), further enhancing binding stability. The interaction subnets predicted by STRING 11.0 show that they may play a synergistic role in translation, protein phosphorylation, glycosylation, and localization, indicating that glycosylation and phosphorylation modifications in exosomes may affect protein function and recognition, providing clues to understanding the role of exosomes in cellular activity [Figure 12F].

Figure 12. (A) Synthetic route of Fe₃O₄@Thio-COF@Au@GSH; (B) Workflow for the enrichment of glycopeptides and phosphopeptides in standard samples by Fe₃O₄@Thio-COF@Au@GSH individually; (C) Workflow of the consecutive enrichment of exosomes and exosomal glycopeptides/phosphopeptides from urine samples using Fe₃O₄@Thio-COF@Au@GSH; (D) Calculated electron density mapped with the electrostatic potential of GSH/phosphate head group; (E) Possible interaction model between GSH and the phosphate head group. Hydrogen bonds are represented by blue dashed lines; (F) Protein–protein interaction network of the identified 42 proteins with the co-existence of glycosylation and phosphorylation; (G) Preparation diagram of ferrocene COF; (H and I) TEM (H) and SEM (I) images of FE-1; (J) Mass spectrum of eluent obtained by enriching glycopeptides in IgG enzymatic hydrolysate with FE-1 COF material; (K) Mass spectrum of eluent obtained by enriching phosphopeptides in α-casein enzymatic hydrolysate with FE-1 COF material. (A-F) are reprinted with permission from Ref^[139], Copyright © 2022 by Royal Society of Chemistry. (G-K) are reproduced with permission from Ref.^[140], Copyright © 2024 by Multidisciplinary Digital Publishing Institute. Thio-COF: Thioether-functionalized covalent organic framework; GSH: glutathione; TEM: transmission electron microscopy; SEM: scanning electron microscope; IgG: immunoglobulin G.

The integration of HILIC and IMAC dual mechanisms allows for synergistic recognition and efficient co-enrichment of glycopeptides and phosphorylated peptides. In 2024, Wu’s team^[140] developed a ferrocene-based FE-1 COF via imine bonding, designed for dual recognition and selective enrichment of glycopeptides and phosphorylated peptides [Figure 12G]. Material characterizations (including XPS, FT-IR, Ms, TEM, and SEM) confirm that FE-1 COF has a well-defined structure, tunable microporous architecture (average pore size: 1.26 nm), and abundant functional groups, enabling selective enrichment of small peptides while excluding larger proteins [Figure 12H and I]. The rich nitrogen-containing groups support hydrophilic interactions for glycopeptide capture, while iron ions serve as coordination sites for phosphorylated peptides. Under optimized conditions (90% ACN, 1% TFA), it simultaneously enriched 4 endogenous phosphopeptides and 41 N-glycopeptides from human serum, demonstrating its capability in integrated analysis of PTMs with high selectivity and applicability. A considerable number of glycopeptides and phosphopeptides could still be detected, with their signal intensities remaining at a high level. This confirms that the material has excellent room-temperature storage stability [Figure 12J and K].

COF and its derivatives provide an efficient, sensitive, and controllable new strategy for the simultaneous enrichment of glycopeptides and phosphorylated peptides, which greatly promotes the joint omics study of PTMs of multiple proteins in complex biological samples.

Separation of proteins

Protein separation is of great importance in the life sciences, medicine, and food industry. Isolation and purification of proteins is a prerequisite for studying their structure and function, and only by obtaining high-purity proteins can their biological activity, enzymatic properties, and interactions with other molecules be accurately analyzed^[141].

By precisely designing the type of junction in the COF structure, the pore size can be finely tuned, resulting in efficient size-exclusion separations based on protein molecular weight differences^[119]. On this basis, according to the charge and hydrophobicity control mechanism, when COF is designed as a cationic material, it can be used as an ion exchange medium to achieve selective adsorption and separation of different proteins by coupling multiple parameters such as isoelectric point, hydrophobicity and molecular size of proteins^[142].

For example, Wang et al. developed two COFs, Azo-COF and Tp-COF, achieving fine-tuned pore sizes of 2.78 and 2.99 nm, respectively [Figure 13A]^[119]. PXRD confirmed their good crystallinity, with AA stacked models matching experimental data well. SEM revealed rod-shaped crystals, while HRTEM observed lattice fringes consistent with simulation models, indicating clear pore structures [Figure 13B-G]. CLSM images further validated protein adsorption inside the pores rather than on the surface. Model protein separations were performed in vials and in a COF-based device, respectively [Figure 13H and I]. By serially using Azo-COF and Tp-COF with finely tuned pore sizes, size-selective separation of low-molecular-weight proteins can be achieved [Figure 13J and K]. By leveraging size exclusion and weak interactions, this dual-COF system efficiently separates target proteins, achieving a recovery rate exceeding 75% from mixed protein samples. This work underscores the potential of precisely tuned COF structures for enhancing selectivity in protein enrichment by demonstrating effective size-based separation and high specificity for small molecules.

Figure 13. (A) Synthetic routes of Azo-COF and TP COF; (B and E) SEM images of Azo-COF (B) and Tp-COF (E); (C, D, F, G) HRTEM images of Azo-COF (C and D) and Tp-COF (F and G); (H and I) Schematic representation of model proteins separation in vials (H) and COF-based device (I); (J) Proteins separation by the tandem utilization of Azo-COF and Tp-COF: proteins mixture before treatment (1), the supernatant after (1) treated with Azo-COF (2), the supernatant after (2) treated with TpCOF (3), the eluate of Azo-COF (4), and the eluate of Tp-COF (5); (K) Proteins separation by the COF-based device. Proteins mixture before treatment (1), the eluate of Azo-COF-contained filter (2), the eluate of Tp-COF-contained filter (3), and the solution through the COF-based device (4). (A-K) are reprinted with permission from Ref.^[119], Copyright © 2022 by Springer Nature. COF: Covalent organic framework; SEM: scanning electron microscope; HRTEM: high-resolution transmission electron microscopy.

Mosleh et al. synthesized Py-BPy-COF using 2,2′-bipyridine (BPy) and pyrene (Py) as building blocks via a Schiff base reaction, followed by modification with quaternary ammonium from 1,2-dibromoethane to obtain positively charged Py-BPy²⁺−COF for specific separation of amino acid [Figure 14A and B]^[142]. TEM revealed clear lattice fringes, confirming high crystallinity even after modification [Figure 14C and D]. The quaternary ammonium introduction was aimed at enhancing electrostatic interactions for selective adsorption. Py-BPy²⁺−COF exhibited significant differences in amino acid adsorption, particularly favoring glutamic acid and aspartic acid due to their negative charges at pH 7.8, which interact strongly with the cationic sites. For proteins, cytochrome c (12 kDa, pI 10.7) showed the highest adsorption capacity of 5,800 μg/mg, attributed to its size and charge compatibility with the material’s pore size of 2.15 nm, allowing adsorption both on the surface and within the pores. In contrast, BSA could only adsorb minimally on the surface due to its larger radius. When tested with protein mixtures, negatively charged and size-matched cytochrome c was preferentially adsorbed, while lysozyme and BSA were excluded due to size or weaker charge interactions. HPLC quantification and SDS-PAGE analysis further validated the highly selective capture of cytochrome c, demonstrating efficient and specific separation [Figure 14E and F].

Figure 14. (A) Adsorption strategies of COFs toward low-molecular-weight biomolecules (amino acids) and adsorption of larger biomolecules (proteins); (B) Synthesis of Py-BPy–COF and transformation of Py-BPy–COF to the cationic Py-BPy²⁺–COF; TEM characterization of (C) Py-BPy−COF and (D) Py-BPy²⁺−COF; Selective protein separation using Py-BPy²⁺−COF, (E) HPLC curves, and (F) SDS-PAGE analysis of protein selectivity assay. (A-F) are reprinted with permission from Ref.^[119], Copyright © 2021 by American Chemical Society. COFs: Covalent organic frameworks; Py: pyrene; BPy: 2,2′-bipyridine; TEM: transmission electron microscope; HPLC: high-performance liquid chromatography; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Additionally, functionalizing COFs with GSH enhances protein interactions to improve binding affinity, optimizing separation efficiency. Bettada et al., synthesized a magnetic 3D COF–GSH MIPs material designed for selective protein recognition and separation, particularly aiming to enhance selectivity and applicability in complex biological systems relevant to glycopeptide enrichment^[143]. The structure features a Fe₃O₄ NP core for magnetic responsiveness, a 3D COF layer formed by imine bonding between COOH-1,3,5-triformylphloroglucinol (cTp) and tetra(p-aminophenyl)methane (TAM), and an outer MIP shell using BSA as a template [Figure 15A]. Adsorption experiments showed that the MIPs had a maximum BSA binding capacity of 429 mg/g, significantly higher than the 89 mg/g for non-imprinted polymers (NIPs), with an imprinting factor of 4.79 and separation factors of 2.76-3.68, demonstrating the effectiveness of molecular imprinting and GSH–BSA interactions in achieving selective recognition [Figure 15B]. UV-Vis spectroscopy showed that the supernatant treated with MIPs had lower absorbance at 280 nm than after NIPs, indicating that MIPs adsorbed BSA more efficiently [Figure 15C]. In SDS-PAGE analysis, BSA bands were obvious in L5 (eluent of MIPs), which verified the specific recognition and separation ability of MIPs to BSA, while the adsorption of NIPs was non-specific [Figure 15D]. By integrating magnetic separation, molecular imprinting, and GSH-based affinity, this 3D COF–GSH MIPs system offers a highly selective, stable, and reusable platform for protein separation, aligning well with the design goal of improving selectivity in protein enrichment and showing strong potential for application in complex biological samples.

Figure 15. (A) Preparation of magnetic 3D COF–GSH MIPs; (B) Rebinding selectivity of magnetic 3D COF–GSH MIPs and NIPs for different proteins; (C) UV-Vis analysis and (D) SDS-PAGE analysis for compatibility of magnetic 3D COF–GSH MIPs/NIPs. L1: Marker, L2: PBS with spiked proteins, L3; and L4: supernatant solutions after adsorption with magnetic 3D COF-GSH MIPs and NIPs respectively, L5; and L6: Eluted solutions from magnetic 3D COF-GSH MIPs and NIPs in 0.5% SDS and 0.1% HOAc (1:1) respectively. (A-D) are reprinted with permission from Ref.^[143], Copyright © 2022 by Multidisciplinary Digital Publishing Institute. COF: Covalent organic framework; GSH: glutathione; MIPs: molecularly imprinted polymers; NIPs: non-imprinted polymers; UV-Vis: ultraviolet-visible spectroscopy; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; HOAc: acetic acid.

Separation of small molecules

Small-molecule metabolites directly reflect the body’s metabolic status, and their changes are often closely associated with the occurrence and development of diseases, thus being widely used for early disease diagnosis, prognosis evaluation, and therapeutic effect monitoring^[144]. COFs have demonstrated high sensitivity, selectivity, and excellent anti-interference ability in the separation, enrichment, and detection of various small-molecule biomarkers. COF-based separation and detection platforms have been applied to complex biological samples such as serum and urine, enabling efficient screening and early diagnosis of disease-related small-molecule markers^[145].

COF can be modified with boric acid^[146], sulfonic acid^[147] and other groups or metal ions^[148] to specifically bind to the target small molecule biomarkers, and combined with mass spectrometry, electrochemistry and other detection methods, ultra-sensitive detection of low-abundance biomarkers can be realized.

Dan et al. constructed a core-shell structure (Fe₃O₄@TtDt@Cu²⁺) via hydrothermal and Schiff base reactions, with Fe₃O₄ as the magnetic core and COF as the mesoporous shell, with a specific surface area of 348.1 m²/g [Figure 16A]^[148]. The triazine ring forms π–π stacking interactions with the aromatic rings in mPAEs, while the nitrogen atom in the imine bond forms hydrogen bonds with the hydrogen atoms in mPAEs. The hydrophobic region of the catechol group interacts hydrophobically with the alkyl chains of mPAEs. The synergistic combination of these multiple interactions significantly enhances the material’s selectivity. This work presents a robust and selective platform for trace mPAE detection in biological samples, enabled by the synergistic integration of magnetic separation, structural porosity, and multifunctional binding mechanisms [Figure 16B].

Figure 16. (A)Schematic representation of the preparation of Fe₃O₄@TtDt@Cu²⁺ composites; (B) the MSPE process for mPAEs in mouse plasma samples; (C)Scheme of the NiFe₂O₄/COF preparation and MSPE procedure for QNs. (A and B) are reprinted with permission from Ref.^[148], Copyright © 2023 by Elsevier. (C) is reproduced with permission from Ref.^[147], Copyright © 2024 by Elsevier. TtDt: Tt (1,3,5-tris-(4-aminophenyl) triazine)-Dt (2,3- dihydroxyterephthalaldehyde) covalent organic framework; MSPE: magnetic solid-phase extraction; mPAEs: phthalate monoesters; COF: covalent organic framework; QNs: quinolones.

Lin et al. developed a magnetic NiFe₂O₄/COF composite with a mesoporous structure (pore size: 4.3 nm) and sulfonic acid functional groups, designed for efficient and selective enrichment of small molecules [Figure 16C]^[147]. The material combines super-paramagnetism (16.5 emu/g) for rapid magnetic separation (< 10 s), a mesoporous framework suitable for molecules ~1 nm in size, and multiple interaction sites - such as electrostatic, hydrogen bonding, and π–π interactions - provided by sulfonic acid groups, which enable a high adsorption capacity of 716.7 mg/g for ciprofloxacin. When applied to the enrichment of quinolones (QNs, ~0.8-1.2 nm), the material demonstrated excellent selectivity by allowing target molecules to diffuse into the pores while blocking larger interferences. Coupled with UHPLC-Q-Orbitrap HRMS, the method achieved a linear range of 0.01-100 ng/g, a low limit of detection (1.1 pg/g), and the recoveries ranged from 82.2% to 108.4% for 18 QNs in complex biological matrices such as pig and chicken viscera.

Additionally, COF nanosheets have also been applied in fluorescent sensing. In 2025, Ma et al. synthesized a Eu³⁺-functionalized COF (Eu³⁺@TAB–DFP–COF) via Schiff base reaction^[149]. The hexagonal mesoporous structure (pore size ~0.9 nm) retained its crystallinity and porosity after metal coordination, allowing selective molecular diffusion and interaction within the pores. The material enables specific recognition of L-lysine through a combination of electrostatic and hydrogen bonding interactions, as well as chiral spatial matching. Fluorescence response was ratiometrically measured (I₄₆₈/I₆₁₄), effectively reducing background interference. The sensor exhibited a linear range of 0.1-130 μM, a detection limit of 17.5 nM, and a selectivity factor of 3.16 for L-lysine over its enantiomer, with recovery rates of 95.9%-103.4% in spiked milk samples. This work demonstrates the potential of functionalized COF nanosheets in selective molecular sensing, particularly for chiral biomolecules, by integrating structural order with tailored recognition sites.

Separation of small molecules

Small-molecule biomarker detection serves as a crucial approach in modern biomedical research and disease diagnosis. By monitoring the dynamic changes of small-molecule metabolites in the body, it can reveal the molecular mechanisms underlying disease occurrence and progression, while providing potential biomarkers for the early diagnosis and treatment of diseases. Since most raw materials used for synthesizing HOFs contain conjugated aromatic rings, many HOF materials exhibit fluorescent properties. Especially when the raw materials include the organic building blocks contain mechanisms such as π–π* transitions, which give the material strong fluorescence properties making HOFs show great potential as fluorescent sensing probes^[154]. Mechanistically, fluorescence quenching in luminescent materials mainly originates from electron or energy transfer between analytes and emissive units. In HOF-based systems, a pronounced spectral overlap between the HOF emission and analyte absorption enables non-radiative energy transfer, leading to efficient fluorescence attenuation. Meanwhile, fluorescence recovery occurs upon removal or competitive displacement of the quenching species, accompanied by restoration of the electronic structure and ordered framework. Owing to their hydrogen-bond-driven self-assembly, HOFs can spontaneously reconstruct their ordered architectures after external perturbations, suppressing non-radiative decay pathways and thereby restoring fluorescence emission. This reversible on–off luminescence response underpins the utility of HOFs as robust and reusable fluorescent sensing platforms.

Chen et al. developed a HOF, named HOF-BTB, for the highly sensitive fluorescent detection of 3-methoxytyramine (3-MT), a tumor biomarker^[155]. Constructed from 1,3,5-tris(4-carboxyphenyl)benzene (H₃BTB) through carboxylic acid dimerization and π–π stacking, the material forms a complex framework with a two-dimensional (6,3) honeycomb structure and one-dimensional channels, enabling rapid diffusion and enrichment of 3-MT [Figure 17A and B]. Characterization including PXRD, FT-IR, and SEM confirmed the material’s structural stability and crystallinity, both before and after binding with 3-MT, indicating a non-covalent adsorption mechanism [Figure 17C]. Upon 3-MT addition, the fluorescence peak at 360 nm (HOF-BTB) was quenched, while a new peak at 312 nm emerged and intensified. By using the fluorescence intensity ratio I₃₁₂/I₃₆₀ as a ratiometric signal, a sensitive “turn-on” fluorescence sensor was established, achieving a wide linear range and a detection limit as low as 46 nM - superior to most fluorescent sensors and comparable to HPLC. This performance enables efficient detection of 3-MT in clinical urine samples, offering a promising tool for early diagnosis of pheochromocytoma and paraganglioma. Zhang et al. developed a fluorescent sensor based on an iron-modified Fe-HOF for the sensitive detection of ascorbic acid (AA)^[156]. Constructed from melamine (MA), BDC-(OH)₂, and H₃BTC through hydrogen-bonded self-assembly, Fe-HOF features porous structure with redox-active Fe³⁺ centers coordinated to phenolic hydroxyl groups [Figure 17D]. After Fe³⁺ modification, it can be seen that partial cracks appear in the original complete filamentous and rod-like structures, indicating that the addition of trivalent iron changes the morphology of the original HOF, but the overall micron rod-like structure did not change [Figure 17E and F]. The material exhibits fluorescence derived from the π–π* transitions of BDC-(OH)₂. Upon AA introduction, Fe³⁺ is reduced to Fe²⁺, leading to fluorescence recovery at 530 nm, which serves as the detection signal. The fluorescence intensity ratio (F/F₀) shows a linear response to AA concentrations from 0.5 to 8 μM, with a low detection limit of 0.14 μM - well below physiological AA levels - enabling sensitive detection in complex matrices. When there are multiple coexisting substances (such as Na⁺, lysine, GSH, etc.), the fluorescence intensity ratio (F/F₀) is almost unnoticed. And when AA is added, the rate increases significantly [Figure 17G]. AA Fe-HOF was successfully applied to AA quantification in vitamin C tablets with high accuracy (RSD = 2.1%). Its porous structure, redox activity, and fluorescence recovery mechanism make it a promising platform for biosensing. Similarly, Chen et al. reported a multi-responsive fluorescent HOF material, HOF-PyTTA, synthesized via solvent diffusion^[154]. Fluorescence is quenched by Fe³⁺ ions and subsequently restored upon binding with GSH, enabling selective GSH detection based on fluorescence recovery. This system highlights the versatility of HOFs in designing stimuli-responsive fluorescent sensors for small biomolecules.

Figure 17. (A and B) HOF-BTB has complex eight-fold interpenetrating structure; (B) HOF-BTB exhibits one-dimensional channels in the frame; (C) Different urine interfering substances were added to HOF-BTB with its SEM image; (D) The synthesis route of Fe-HOF and the experimental principle of Fe-HOF used in the detection of ascorbic acid; (E and F) SEM images of Fe-HOF at different magnifications; (G) Fluorescence response of HOF based assay system toward various coexisting species, including Na⁺, Ca²⁺, Zn²⁺, SO₄^2-, Mg²⁺, K⁺, and Cl^- (1 mM), Lys, Glu, Thr, His, Ala, and Leu (100 μM), AA, L-Cys, and GSH (5 μM). (A-C) are reprinted with permission from Ref.^[148], Copyright © 2023 by Academic Press Inc Press. (D-G) are reproduced with permission from Ref.^[147], Copyright © 2024 by Elsevier. HOF: Hydrogen-bonded organic framework; BTB: 1,3,5-tris (4-carboxyphenyl) benzene; SEM: scanning electron microscope; Lys: lysine; Glu: glutamic acid; Thr: threonine; His: histidine; Ala: alanine; Leu: leucine; Cys: cysteine; GSH: glutathione.

Sahoo et al. developed a three-dimensional porous HOF, named HOF (IITKGP-HOF-6), using the tetradentate carboxylic acid ligand 5-(bis(4-carboxyphenyl)amino)isophthalic acid (H₄L) [Figure 18]^[157]. The framework’s benzene rings and carboxylic acid groups form a stable network through hydrogen bonding, creating twisted tetrahedral conformations with dihedral angles of 43° and 82°, which establish 1D nanochannels along the a-axis (6.8 × 6.8 Ų). This structure, confirmed by XRD and PLATON analysis showing 28% solvent-accessible volume, exhibits excellent water and air stability, as evidenced by unchanged PXRD patterns after immersion in water for 7 days or exposure to air for 15 days. The material’s hydrophilic microporous structure, demonstrated by its high water vapor adsorption capacity (75 mg/g at 298 K), and thermal stability up to 250 °C make it ideal for capturing targets in aqueous solutions. H₄L shows strong fluorescence emission at 480 under 360 nm excitation, which is significantly quenched upon binding nitrofuran antibiotics (NFZ and NFT) due to hydrogen bonding and π–π interactions, achieving a quenching efficiency of 84%. Specifically, the binding energy between NFZ and HOF reaches -34.5 kcal/mol, significantly higher than other antibiotics, leading to a low detection limit of 0.75 μM. HOF (IITKGP-HOF-6) demonstrates high sensitivity and reliability for real-world water sample testing, providing a robust platform for on-site monitoring of antibiotic residues in environmental water.

Figure 18. A water-stable HOF (IITKGP-HOF-6) for aqueous medium antibiotic sensing for NFT and NFZ. Reprinted with permission from Ref.^[157], Copyright © 2025 by Wiley. HOF: Hydrogen-bonded organic framework.

What’s more, many HOFs are used for the separation and detection of small molecules. For example, FJU-360 is employed to detect aniline^[158], HOF-DBA is used for the detection of γ-aminobutyric acid and nitrofurazone^[159], Eu@HOF-GS-10 is used for the detection of quinolone antibiotics (QNs)^[160].

Separation of biomacromolecules

A biomarker is a measurable biological indicator that can be used as a potential index for evaluating normal or abnormal pathophysiological states or the pharmacological response to specific therapeutic regimens, and can be detected and quantified^[161]. Molecular biomarkers cover a wide range from small molecules to macromolecules, including peptides, proteins, lipid metabolites, nucleic acids (DNA and RNA), etc., and can be applied to disease diagnosis, prognostic assessment, therapeutic monitoring, and disease mechanism research^[162-165].

Li et al. constructed a luminescent HOF (Lumi-HOF@Tb) by assembling four hydrogen-bonding ligands–melamine (MA), cyanuric acid (CA), luminol, and H₃BTC– and incorporating Tb³⁺ ions to form a luminescent center [Figure 19]^[166]. The resulting 3D HOF features a regular nanocylinder morphology (~1 μm in length, ~50 nm in width), confirmed by SEM and TEM, and exhibits high crystallinity, as shown by XRD. Coordination of Tb³⁺ with H₃BTC was verified by FT-IR and XPS, which confirmed the formation of Tb–O bonds and successful functionalization of the framework. Lumi-HOF@Tb enables dual-ratiometric fluorescence and chemiluminescence sensing of α-glucosidase activity. Upon enzymatic hydrolysis of the substrate pNGP, p-nitrophenol (pNP) is generated, which competes with Tb³⁺ for inner filter effect (IFE), restoring Tb³⁺ fluorescence at 546 nm. Simultaneously, pNP forms a fluorescent complex with luminol at 466 nm. Additionally, luminol oxidation by H₂O₂ generates a chemiluminescent signal, enhancing detection sensitivity. This dual-signal strategy achieves ultra-low detection limits of 0.04 U/L (F₅₄₆/F₄₂₅), 0.07 U/L (F₄₆₆/F₄₂₅), and 0.005 U/L by CL, surpassing conventional single-signal methods. The sensor exhibits high selectivity, unaffected by common proteins, ions, or amino acids, and shows excellent recovery (93.0%-118.8%) in human serum, consistent with colorimetric assays. It has also been successfully applied to screen α-glucosidase inhibitors, such as flavonoids, with IC₅₀ values as low as 1.64 μM (by CL detection), demonstrating its potential for enzyme-related biomedical and pharmaceutical applications.

Figure 19. Scheme illustration of the dual-signal optical sensor based on Lumi-HOF@Tb for α-glucosidase detection. It was synthesized at room temperature and modified with Tb³⁺, integrates fluorescence and chemiluminescence for dual-signal detection of α-glucosidase activity. By monitoring fluorescence recovery at 546 nm (Tb³⁺) and H₂O₂-driven chemiluminescence, the sensor enables selective and sensitive detection, showcasing its potential in biosensing and enzyme inhibitor screening. Reprinted with permission from Ref.^[166], Copyright © 2022 by American Chemical Society. HOF: Hydrogen-bonded organic framework.

As two important PTMs, protein phosphorylation and glycosylation play a key role in cell signaling and disease diagnosis, but traditional methods struggle to enrich multiple PTMs simultaneously, especially monophosphorylated peptides that are often overlooked due to low affinity. Thus Xiong et al. developed a dual-ligand Fe₃O₄@HOF for the simultaneous enrichment of mono-phosphopeptides and glycopeptides^[163]. The framework is constructed via hydrogen bonding and electrostatic interactions between guanidine cations and borate anions, with superparamagnetic Fe₃O₄ NPs embedded for easy separation and recycling [Figure 20A]. Guanidine (GD) and boric acid (BA) are connected through hydrogen bonds (marked with blue dashed lines) to form a “guanidine-boric acid” synergistic unit [Figure 20B and C]. This unit not only enhances the structural stability of the HOF but also endows it with dual recognition capabilities for phosphopeptides and glycopeptides, respectively. In addition, the positive and negative charge regions in the HOF structure are evenly distributed and exhibit strong complementarity [Figure 20D]. The saturated adsorption isotherms of Fe₃O₄@HOF for phosphate ions and glycogen showed that the adsorption equilibrium of both was achieved within 5 min, and the maximum adsorption capacity for phosphate ions was higher than that for glycogen. This confirms the material’s efficient adsorption capacity for these two simulated target molecules [Figure 20E]. Fe₃O₄@HOF demonstrated exceptional performance in complex biological samples: it detected mono-phosphopeptides at 4 × 10^-10 M even in the presence of a 1,000-fold excess of BSA, and enriched glycopeptides from HRP digests down to 2 × 10^-10 M, with 17 highly abundant glycopeptides specifically enriched from HRP/BSA mixtures (1:1,000). Fe₃O₄@HOF was applied to the enrichment of glycopeptides and phosphopeptides in the serum of lung cancer patients and healthy people, and the results showed that 51 proteins in both groups of serum had both glycosylation and phosphorylation modifications, and more than 90% of the phosphopeptides were mono-phosphopeptides, verifying the high selectivity of the material for mono-phosphopeptides [Figure 20F and G]. PCA and heat maps show that the phosphorylated and glycosylated protein expressions in the two groups are clearly differentiated, and this material can be used to distinguish lung cancer patients from healthy people, providing a basis for disease-related PTM studies [Figure 20H-K].

Figure 20. (A) Fabrication process of Fe₃O₄@HOF; (B) Schematic illustration of HOF comprising borate ester anion and guanidinium cation via H-bonding (highlighted by dashed blue line with disordered Cl^- omitted for clarity); (C) Calculated electron density map of HOF (red, negative part; blue, positive part); (D) H-bonding (denoted as dashed blue line) between GD and BA; (E) Saturated adsorption isotherms for phosphate and glycogen adsorbed by Fe₃O₄@HOF; (F) Venn diagram of the overlap of glycosylation and phosphorylation at the protein level; (G) Distribution of mono-, and multi-phosphorylation events observed from human serum samples of lung cancer patients and healthy controls; Quantitative PCA of (Group A) healthy controls and (Group B) lung cancer patients of (H) phosphoproteins and (I) glycoproteins; Heat maps of the significantly regulated proteins between (Group A) healthy controls and (Group B) lung cancer patients of (J) phosphoproteins and (K) glycoproteins. Reprinted with permission from Ref.^[163], Copyright © 2024 by Elsevier. HOF: Hydrogen-bonded organic framework; GD: guanidine hydrochloride; BA: boric acid; PCA: principal component analysis.

Chen et al. successfully prepared a DS-HOF by self-assembling TAM and 4,4′-dithiobisbenzoic acid (DTBA) through intermolecular hydrogen bonding^[165]. This material enables efficient encapsulation and intracellular delivery of functional proteins, with over 95% encapsulation efficiency for model protein GFP [Figure 21A] The disulfide bonds (-S-S-) in DTBA render DS-HOF responsive to high levels of GSH in cancer cells, enabling selective degradation and controlled protein release. In vitro studies showed that 75% of the encapsulated GFP was released within 12 h under GSH-rich conditions. The size of DS-HOF NPs can be tuned by adjusting the TAM:DTBA ratio, with a 1:4 molar ratio yielding uniform spherical NPs (~100 nm). This size of the material favors cellular internalization, enabling tumor-targeted accumulation. Structural and morphological characterizations (SEM, TEM, PXRD) confirmed that protein loading had minimal impact on the framework’s integrity, preserving its crystallinity and porosity. DS-HOF exhibits excellent biocompatibility and efficient cellular uptake, with over 90% internalization efficiency in HeLa cells and cytoplasmic delivery confirmed by GFP fluorescence. To observe the degradation effect of DS-HOF delivery of bacterial effector protein DUF5 on mutant RAS. Western blot results showed that HCT-116 cells were treated with DUF5@DS-HOF, RAS degradation, p-ERK1/2 downregulation, and cell growth was inhibited [Figure 21B]. When loaded with DUF5, a protein degrader of mutant RAS, DS-HOF effectively reduced RAS levels, inhibited p-ERK1/2 signaling, and suppressed HCT116 cell viability to 40% in vitro, with tumor volume reduced to 20% of the control group [Figure 21C]. Experiments on tumor-bearing mice showed that DUF5@DS-HOF could reduce the tumor volume to only 20% of that in the PBS group [Figure 21D and E]. TUNEL and H&E staining showed that the apoptosis of tumor cells in the DUF5@DS-HOF-treated group was obvious. It was verified that DS-HOF efficiently delivers DUF5 to the cytosol and inhibits tumor growth by degrading mutant RAS [Figure 21F]. These features, combined with low in vitro and in vivo toxicity, highlight DS-HOF as a promising platform for targeted protein delivery in cancer therapy.

Figure 21. (A) Schematic illustration of the self-assembly of bio-reducible DS-HOF for intracellular protein delivery and mutant RAS signaling rewiring in cancer cells; (B) Western blot assay of RAS and p-ERK1/2 in HCT-116 cells following the treatment of DUF5@DS-HOF and DUF5@TAHOF NPs (DUF5 concentration: 400 nM); (C) DUF5@DSHOF delivery prohibited HCT-116 cell growth. HCT-116 cells were treated with GFP@DS-HOF or DUF5@DS-HOF at indicated concentrations before cell viability assay; (D) Tumor volume of HCT-116 tumor-bearing mice received different injections as indicated. Data are presented as means ± SD (n = 5); (E) Western blot assay of RAS and p-ERK1/2 in tumors harvested from mice that received different DUF5 and NP treatments; (F) TUNEL analysis (for cell apoptosis) and H&E staining of tumor tissues harvested from mice received different treatments as indicated. Scale bar: 100 μm. Reprinted with permission from Ref.^[165], Copyright © 2023 by American Chemical Society. DS: Disulfide; HOF: hydrogen-bonded organic framework; RAS: rat sarcoma viral oncogene homolog; p-ERK: phosphorylated extracellular signal-regulated kinase; HCT: human colon tumor; DUF5: domain of unknown function 5; NPs: nanoparticles; GFP: green fluorescent protein; SD: standard deviation; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Encapsulating proteins in HOF materials not only protects biomolecule activity and improves detection performance, but also expands functional boundaries through material-biomolecule synergies. What’s more, there are also many HOFs used to encapsulate proteins, such as BioHOF-1^[167], enzyme@TaTb^[168]. Moreover, HOFs have the greatest potential in bioseparation due to their excellent biocompatibility, mild synthesis conditions, and reversible self-assembly. Unlike MOFs, which may contain metal residues, and COFs, which are sensitive to water, HOFs are composed of small organic molecules and can be synthesized under mild, aqueous conditions. Their self-assembly allows for easy recovery and reusability, making them ideal for applications involving small-molecule drugs, natural products, or sensitive biomolecules. These properties make HOFs highly promising for bioseparation.