Physical adsorption refers to the way mesoporous silica nanoparticles connect with proteins through physical interactions such as van der Waals forces, hydrophobic interactions, hydrogen bonds, and ionic interactions. This method is relatively simple but leads to weak binding forces between enzymes and carrier molecules, causing enzymes to easily detach from the carrier. This often results in protein desorption and loss, and strict control of the enzyme-to-silica ratio is required to avoid enzyme aggregation, which can reduce enzyme activity^[4].

Unprotected thiol-functionalized silica surfaces can lead to cross-linking problems due to the sensitivity to oxidative dimerization. A protective pyridylthiol group must be grafted onto the silica surface to avoid cross-linking. However, in the presence of thiol-containing proteins, the protective group can be released and react with the protein to form disulfide bonds. During the reaction, side reactions can occur due to the reactivity of the thiol group in the peptide chain, affecting the activity of functional proteins ^[5].

To introduce NH₃ groups onto the silica surface, the system must be calcined at 550°C for 20 hours, followed by incubation with propargyl bromide and triethylamine in a methanol solution to obtain MSN-alkyne^[8]. Further incubation with the prepared Azido-pep-Tf results in Tf-Res-MSN, which takes a long time and consumes significant energy.

Using 3-aminopropyltriethoxysilane (APTES) to amino-modify mesoporous silica nanoparticles for binding to other proteins, such as common fluorescein and Rhodamine B isothiocyanates, seems straightforward. However, obtaining the desired surface coverage is highly sensitive to synthesis conditions, including silane concentration, reaction time, temperature, solvent polarity, and water content. Even after optimizing reaction conditions, a labor-intensive process involving sedimentation-redispersion is required to wash the particles in an appropriate reaction solvent at least 25 times^[7].

Hydrazide linker is another method for silica surface modification, where the formation of oxime bonds between aldehydes and hydrazines can occur on silica nanoparticles^[9]. While these bonds can degrade under low pH conditions, they can be stabilized using aromatic aldehydes. However, the explosive nature of hydrazides and their derivatives in the solid state poses significant risks when applied to nano-silica.

A new fusion protein, FlipHOB, was used to achieve precise and delicate immobilization of glucose sensor proteins on SiNP surfaces^[6]. This method relies on protein design, chemical ligand coordination, and the capture of single-stranded nucleic acids. While protein design is unique, it requires specific target protein (glucose sensor) design and connection and lacks generality and universality.

RAFT polymerization is a commonly used method for grafting zwitterionic layers onto silica nanoparticle surfaces^[9]. It has good grafting capabilities for various monomer chains (OH, NH₂, COOH, etc.) and can be conducted in aqueous conditions. However, RAFT has some weaknesses, such as limited range of graftable initiators and slower reaction times compared to ATRP, which may pose challenges for drug-loaded MS nanoparticles.