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.

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.

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.

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.

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].

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.

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.

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.

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].

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.

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].

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.

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.

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.

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].

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.

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].

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.

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.