Metal–Organic Frameworks-Based Immobilized Enzyme Microreactors Integrated with Capillary Electrochromatography for High-Efficiency Enzyme Assay

Abstract

Enzyme assays are important for studying enzymemediated biochemical reactions and for clinical diagnosis and drug development. The technique of an immobilized enzyme microreactor (IMER) integrated with capillary electrophoresis (CE) has been frequently utilized in online enzyme assays. However, the traditional approaches for IMER-CE enzyme analysis have some defects such as low loading capacity and poor stability. Herein, metal−organic frameworks (MOFs), which have enormous potential in the fields of enzyme immobilization and capillary electrochromatographic (CEC) separation, were first explored as novel support materials with good enzyme immobilization performance and stationary phases with excellent separation abilities to construct an integrated MOFs-IMER-CEC microanalysis system for a high-efficiency online enzyme assay. As a proof-of-concept demonstration, acetylcholinesterase (AChE) was immobilized on a densely packed UiO-66-NH 2 nanocrystal coating on a capillary inner surface with abundant intercrystalline mesoporosity and was employed as a highly effective and robust IMER for CEC-integrated online enzyme analysis. The excellent separation performance of the UiO-66-NH 2 -modified capillary was verified by high-efficiency separation of three types of neutral, acidic, and basic compounds. The Michaelis−Menten constant and enzyme inhibition kinetics of UiO-66-NH 2 −IMER were systematically assessed, exhibiting distinct advantages such as remarkably increased enzyme loadability, superior affinity for substrates, and greatly improved stability and repeatability compared to CE-integrated IMERs prepared by the traditional covalent bonding method. Furthermore, the developed method was successfully utilized for detecting organophosphorus pesticides in leguminous vegetable samples, demonstrating its strong practicality. The study not only proposed a novel support material and construction strategy for a highperformance microchannel-based IMER but also can be widely used in bioanalysis and biosensing research.

Ultrasensitive Detection of Organophosphorus Pesticides Using Single-Molecule Conductance Measurement

“…Organophosphorus pesticides (OPs) have been used extensively to eradicate insects in agricultural areas across the world in recent decades. , And OPs are an ensemble of strictly regulated toxic compounds that have harmful effects on humans and animals when the residues are in low amounts of 55.2 pM (10 –12 mol/L). , The toxic effects of OPs make them inhibit the activity of acetylcholinesterase, which causes interruption of neurotransmitter transmission and eventually death. − Although colorimetric systems were widely employed to detect OPs, the severe interference of sensitivity and selectivity about OPs caused by color limited their applications in practical samples. − To overcome the interference by color, lots of efforts had been made in the past years, such as microarray, , capillary electrophoresis, liquid chromatography, nuclear magnetic resonance, and mass spectrometry . However, most of these methods required time-consuming sample pretreatment, the use of other materials for signal amplification, low sensitivity, and sophisticated instrument manipulation.…”

“…5−7 Although colorimetric systems were widely employed to detect OPs, the severe interference of sensitivity and selectivity about OPs caused by color limited their applications in practical samples. 8−10 To overcome the interference by color, lots of efforts had been made in the past years, such as microarray, 11,12 capillary electrophoresis, 13 liquid chromatography, 14 nuclear magnetic resonance, 15 and mass spectrometry. 16 However, most of these methods required timeconsuming sample pretreatment, the use of other materials for signal amplification, low sensitivity, and sophisticated instrument manipulation.…”

Homomesoporous Metal–Organic Framework for High-Performance Electrochromatographic Separation

“…As a novel class of crystalline porous materials, metal–organic frameworks (MOFs) are polymers with porous structures formed by the self-assembly of metal ions (or ion clusters) and organic ligands through coordination interactions . Owing to their ultrahigh porosity, extraordinary large surface area, abundant active sites, and uniform and adjustable pores, MOFs have received tremendous attention in a wide range of fields such as gas adsorption and storage, , sensing, , photocatalysis, , drug delivery, , and chromatographic separation. − In particular, depending on their excellent porosity and unique modifiable functionality of the organic ligands, MOFs are particularly suitable for the preparation of porous stationary phases with highly specific surface area for chromatographic separation.…”

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis

“…Recently, a noncovalent approach to enzyme immobilization based on a metal organic framework mesoporous support was reported which maintained enzyme stability and activity. 19 In this report, the immobilization is achieved through permanent modifications to the internal capillary surface rather than the enzyme. 19 In a different approach, enzymes were non-covalently immobilized with the use of a thermally responsive nanogel.…”

“…19 In this report, the immobilization is achieved through permanent modifications to the internal capillary surface rather than the enzyme. 19 In a different approach, enzymes were non-covalently immobilized with the use of a thermally responsive nanogel. 20−23 The use of nanogel provided a straightforward means to integrate the enzyme in the capillary and a simple method to ensure that the reagents and enzyme are co-located in a reaction zone.…”