Less common applications of monoliths: I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports

This review summarizes the recent contributions to the rapidly growing area of immobilized enzymes employing both silica and synthetic polymer-based monoliths as supports. Focus is mainly on immobilized proteolytic enzyme reactors designed for studies in proteomics. Porous monoliths emerged first as a new class of stationary phases for HPLC in the early 1990s. Soon thereafter, they were also used as supports for immobilization of proteins and preparation of both stationary phases for bioaffinity chromatography and enzymatic reactors. Organic polymer-based monoliths are typically prepared using a simple molding process carried out within the confines of a "mold" such as chromatographic column or capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials. In contrast, silica-based monoliths are first formed as a rigid rod from tetraalkoxysilane in the presence of PEG and subsequently encased with a plastic tube. Both types of monolith feature large through-pores that enable a rapid flow-through. Since all the solutions must flow through the monolith, the convection considerably accelerates mass transfer within the monolith. As a result, reactors including enzyme immobilized on monolithic support exhibit much higher activity compared to the reactions in solution.     

蛋白质组学中的固定化酶反应器制备的研究进展

固定化酶反应器作为蛋白质组学研究中"bottom-up"策略重要的组件,具有酶解快速、酶解效率高、酶稳定性和活性高、简单易操作、能够与多种检测方式联用等优点,对于发展高效快速的蛋白质组学分析方法具有重要意义。本文就固定化酶反应器的制备方法及其在蛋白质组学中的应用做简单的概述,着重介绍酶的固定方法、固定化酶的载体、用于固定的酶的种类。近几年固定化酶反应器的研究集中于提高固酶量、保持酶活性、增加酶解效率、减小非特异性吸附等方面。研究结果表明,采用纳米材料、整体材料等新型载体,提高载体亲水性,采用多酶同时酶解等方法能够有效改善固定化酶反应器的性能,提高蛋白质的鉴定效率。     

Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries

Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.     

Recent advances in the design of organic polymer monoliths for reversed-phase and hydrophilic interaction chromatography separations of small molecules

Owing to their favorable porous structure with pore size distribution shifted towards large flow-through pores, organic polymer monoliths have been mainly employed for the separation of macromolecules in gradient elution liquid chromatography. The absence of significant amounts of small pores with a stagnant mobile phase and the resulting low surface area were considered as the main reason for their poor behavior in the isocratic separation of small molecules. Several recent efforts have improved the separation power of organic polymer monoliths for small molecules offering column efficiency up to tens of thousands of plates per meter. These attempts include optimization of the composition of polymerization mixture, including the variation of functional monomer, the cross-linking monomer, and the porogen solvents mixture, adjustment of polymerization temperature, and time. Additionally, post-polymerization modifications including hypercross-linking and the use of carbon nanostructures showed significant improvement in the column properties. This review describes recent developments in the preparation of organic polymer monoliths suitable for the separation of small molecules in the isocratic mode as well as the main factors affecting the column efficiency.     

Fabrication and characterisation of gold nano-particle modified polymer monoliths for flow-through catalytic reactions and their application in the reduction of hexacyanoferrate

Polymer monoliths in capillary (100 μm i.d.) and polypropylene pipette tip formats (vol: 20 μL) were modified with gold nano-particles (AuNP) and subsequently used for flow-through catalytic reactions. Specifically, methacrylate monoliths were modified with amine-reactive monomers using a two-step photografting method and then reacted with ethylenediamine to provide amine attachment sites for the subsequent immobilisation of 4 nm, 7 nm or 16 nm AuNP. This was achieved by flushing colloidal suspensions of gold nano-particles through each aminated polymer monolith which resulted in a multi-point covalent attachment of gold via the lone pair of electrons on the nitrogen of the free amine groups. Field emission scanning electron microscopy and scanning capacitively coupled conductivity detection was used to characterise the surface coverage of AuNP on the monoliths. The catalytic activity of AuNP immobilised on the polymer monoliths in both formats was then demonstrated using the reduction of Fe(III) to Fe(II) by sodium borohydride as a model reaction by monitoring the reduction in absorbance of the hexacyanoferrate (???) complex at 420 nm. Catalytic activity was significantly enhanced on monoliths modified with smaller AuNP with almost complete reduction (95 %) observed when using monoliths agglomerated with 7 nm AuNPs.

Preparation of a novel polymer monolith with functional polymer brushes by two‐step atom‐transfer radical polymerization for trypsin immobilization

Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]‐co‐glycidyl methacrylate} brushes were fabricated via two‐step atom‐transfer radical polymerization and used as a trypsin‐based reactor in a continuous flow system. This is the first time that atom‐transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface‐initiated atom‐transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes.     

Recent advances in immobilized enzymatic reactors and their applications in proteome analysis

Immobilized enzymatic reactors recently have drawn much attention because of the striking advantages, such as high substrate turnover rate and ease in coupling with the separation and detection systems. Carrier materials, which have great effects on the development of the immobilized enzymatic reactors, have always being the focus of study. In this paper, the contributions, mainly in the last 5 years, on the enzymatic reactors and their applications in proteome study are reviewed, with some newly developed inorganic and organic carriers for enzyme immobilization described in details. Moreover, the hyphenation of immobilized enzymatic reactors with the separation and identification systems is also summarized. By reviewing these achievements, it could be seen that enzymatic reactors have very bright future, especially in proteome analysis.     

载体对包埋酶微环境的影响分析

包埋法固定化酶过程中,酶固定化载体的选择和设计是酶固定化过程的关键因素,适宜的载体微环境对酶活性和稳定性的影响尤为重要。论文首先分析并提出了影响固定化酶所处载体微环境的主要因素,包括载体的亲疏水性、结构形态和反应活性。载体的亲疏水性决定固定化酶微环境中的水分含量。载体的结构形态对酶形成的笼效应,以及载体的反应活性,包括共价键合、静电和氢键等的结合能力,影响酶构象的稳定性和运动性。另外,底物／产物的扩散速率和酶的可及性也同样受到载体的孔结构、孔分布以及载体反应活性的影响。本文介绍了常用的酶包埋载体,包括sol-gel二氧化硅、高分子水凝胶以及高分子－二氧化硅杂化凝胶固定化酶过程,结合上述影响酶微环境的因素,分析比较了三类载体固定化酶的包埋率、活性和稳定性,综述了为改善固定化酶微环境所进行改进研究的进展。     

Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperometric detection

The advantages to be gained by conducting enzyme assays of carbohydrates in liquid chromatography are discussed. The enzymes are contained in immobilized enzyme reactors and used in the post-column mode. The product formed in the reactor is selectively detected amperometrically at a chemically modified electrode mounted in a flow-through detector. Selected examples are given to illustrate the advantages obtained.     

Metal–Organic Framework Disintegrants: Enzyme Preparation Platforms with Boosted Activity

An enzyme formulation using customized enzyme activators (metal ions) to directly construct metal–organic frameworks (MOFs) as enzyme protective carriers is presented. These MOF carriers can also serve as the disintegrating agents to simultaneously release enzymes and their activators during biocatalysis with boosted activities. This highly efficient enzyme preparation combines enzyme immobilization (enhanced stability, easy operation) and homogeneous biocatalysis (fast diffusion, high activity). The MOF serves as an ion pump that continuously provides metal ion activators that greatly promote the enzymatic activities (up to 251 %). This MOF–enzyme composite demonstrated an excellent protective effect against various perturbation environments. A mechanistic investigation revealed that the spontaneous activator/enzyme release and ion pumping enable enzymes to sufficiently interact with their activators owing to the proximity effects, leading to a boost in biocatalytic performance.     

Metal–Organic Framework Disintegrants: Enzyme Preparation Platforms with Boosted Activity

An enzyme formulation using customized enzyme activators (metal ions) to directly construct metal–organic frameworks (MOFs) as enzyme protective carriers is presented. These MOF carriers can also serve as the disintegrating agents to simultaneously release enzymes and their activators during biocatalysis with boosted activities. This highly efficient enzyme preparation combines enzyme immobilization (enhanced stability, easy operation) and homogeneous biocatalysis (fast diffusion, high activity). The MOF serves as an ion pump that continuously provides metal ion activators that greatly promote the enzymatic activities (up to 251 %). This MOF–enzyme composite demonstrated an excellent protective effect against various perturbation environments. A mechanistic investigation revealed that the spontaneous activator/enzyme release and ion pumping enable enzymes to sufficiently interact with their activators owing to the proximity effects, leading to a boost in biocatalytic performance.     

Porogens and porogen selection in the preparation of porous polymer monoliths

Porogens are key components required for the preparation of porous polymer monoliths for application in separation science. Porogens determine the stability, selectivity, and permeability of polymer monoliths. This review summarizes the role of porogens in the preparation of porous polymer monoliths with a focus on clear understanding of effect of porogens on morphological properties, porosity, surface area, mechanical stability, and permeability of monoliths, particularly targeting the field of separation science. This review also includes the use of different types of porogens with the focus on various approaches used to set criteria for their systematic selection, including porogen‐free techniques recently used for synthesis of porous monoliths. It discusses the current state‐of‐the‐art applications of porogens in column preparation as well as where the future developments in this field may be directed.     

Agglomerated polymer monoliths with bimetallic nano-particles as flow-through micro-reactors

Polymer monoliths in capillary format have been prepared as solid supports for the immobilisation of platinum/palladium bimetallic nano-flowers. Optimum surface coverage of nano-flowers was realised by photografting the monoliths with vinyl azlactone followed by amination with ethylenediamine prior to nano-particle immobilisation. Field emission SEM imaging was used as a characterisation tool for evaluating nano-particle coverage, together with BET surface area analysis to probe the effect of nano-particle immobilisation upon monolith morphology. Ion exchange chromatography was also used to confirm the nature of the covalent attachment of nano-flowers on the monolithic surface. In addition, EDX and ICP analyses were used to quantify platinum and palladium on modified polymer monoliths. Finally the catalytic properties of immobilised bimetallic Pd/Pt nano-flowers were evaluated in flow-through mode, exploiting the porous interconnected flow-paths present in the prepared monoliths (pore diameter~1–2?μm). Specifically, the reduction of Fe (III) to Fe (II) and the oxidation of NADH to NAD⁺ were selected as model redox reactions. The use of a porous polymer monolith as an immobilisation substrate (rather than aminated micro-spheres) eliminated the need for a centrifugation step after the reaction.

Diverse Supports for Immobilization of Catalysts in Continuous Flow Reactors

The rapid development of continuous flow processes is driving innovations in various chemical syntheses and industrial productions. Immobilizing catalysts in flow reactors allows transformations with high-efficiency and excludes the subsequent separation procedures. This concept outlines the approaches to incorporate catalysts within flow reactors, with particular focus on the application of additional supports including inorganic materials like silica, zeolite and reduced graphene oxide, polymeric materials like polymer packings, monoliths, cross-linked gels and polymer brushes, and other materials for specific conditions like transparent glass fibers and glass beads. Furthermore, advanced methods to develop ordered micro-/nanoarrays from internal walls of flow channels for immobilization of catalysts as well as application of innovative vortex fluidic devices are discussed to inspire new designs of supports for novel fluidic reactors with broad applications.     

Flow-injection determination of sugars with immobilized enzyme reactions and chemiluminescence detection

Glucose is determined by reaction with gluocose oxidase to produce hydrogen peroxide which is quantified via a chemiluminescence reaction with luminol. Sucrose, maltose, lactose and fructose are determined by enzymatic conversion to glucose (using invertase, amyloglucosidase, lactase. and glucose isomerase, respectively) and subsequent determination of the glucose, All enzymes are immobilized on controlled-pore glass and contained in flow-through reactors. For glucose, sucrose, and maltose the linear log-log working range 0.2 μM-1 mM, with a detection limit of 0.1 μM; for lactose and fructose the linear working range is 3 μM-1 mM with a detection limit of 1 μM. Assay time is 2 min.     

Are we approaching a post‐monolithic era?

Thirty years after their introduction, monolithic stationary phases are an important member of chromatographic phases. When compared to conventional particulate materials, the continuous internal structure of both inorganic silica and organic polymer monoliths allows some hydrodynamic and analytical possibilities that are not provided by conventional particulate stationary phases. Polymer‐based monolithic stationary phases offer simple preparation and straightforward surface modification, which makes them very versatile materials that are applicable, for example, as chromatographic stationary phases, sample enrichment units, enzymatic reactors, and external trigger‐responding materials. On the other hand, current polymer monoliths cannot compete with efficiency provided by superficially porous and sub 2 µm particles. In this highlight article, I take advantage of the 30th anniversary of their introduction to discuss several concerns related to polymer‐based monolithic stationary phases. Particularly, I focus on preparation repeatability, porous properties, swelling of the polymers in organic solvents, column efficiency for small molecules, and heterogeneity of dominant flow‐through pores. In the end, I offer three possible approaches on how to overcome drawbacks related to stationary phases heterogeneity to further increase the applicability of polymer‐based monolithic stationary phases.     

Advances in the preparation of porous polymer monoliths in capillaries and microfluidic chips with focus on morphological aspects

Porous polymer monoliths have emerged as unique materials for many applications, including liquid-chromatographic analyses at an unrivaled speed, solid-phase extraction, and enzyme immobilization in capillary and microfluidic chip format. This article reviews the state of the art in the preparation of monoliths in narrow-bore capillaries and microfluidic chips and their miniaturization under conditions of spatial confinement. New developments in their preparation mainly using free radical polymerization techniques with a focus on morphological aspects in view of homogeneous porous materials are described. The suitability of monoliths for analysis of both large and small molecules is also discussed.     

Monolith and coating enzymatic microreactors of L-asparaginase: kinetics study by MCE-LIF for potential application in acute lymphoblastic leukemia (ALL) treatment

The study of enzyme immobilization using an extracorporeal shunt system is essential to eliminate the side effects of L-asparaginase (L-Asnase; including hepatic toxicity, allergic reaction, pancreatitis, central nervous system toxicity and decreased synthesis of blood clotting factors) when it was applied as an anticancer drug given directly to patients by injection. Thus, the novel monolith and coating enzymatic reactors of L-asparaginase were provided in this assay and a microchip electrophoresis-laser induced fluorescence (MCE-LIF) method was set up for the enzyme kinetics study. The enzymatic reactors would be a promising in vitro therapeutic method in an extracorporeal shunt system for acute lymphoblastic leukemia (ALL) treatment. For the first time, L-asparaginase was covalently bound to the polymer monolith and coating in the capillary and the activity characteristics of these enzymatic microreactors have been probed by Michaelis-Menten kinetic constants. Meanwhile, the D,L-amino acids were chirally separated using microchip electrophoresis with a laser induced detector and D,L-aspartic acid (D,L-Asp) were tested for the L-asparaginase enzymatic reactor kinetics study. Furthermore, human serum adding with L-asparagine (L-Asn) as the sample was hydrolyzed by the enzymatic microreactors. The results demonstrated that the developed enzymatic microreactor of L-asparaginase would be a potential therapeutic protocol for ALL treatment.     

Recent strategies to enhance the performance of polymer monoliths for analytical separations

This review summarizes recent developments made in the incorporation of functional materials into organic polymer monoliths, together with new monolithic forms and formats, which enhance their application as supports and stationary phase materials for sample preparation and chromatographic separations. While polymer monoliths are well‐known supports for the separation of large molecules, recent developments have been made to improve their features for the separation of small molecules. The selectivity and performance of organic polymer monoliths has been improved by the incorporation of different materials, such as metal‐organic frameworks, covalent organic frameworks, or other types of nanostructured materials (carbon nanohorns, nanodiamonds, polyoxometalates, layered double hydroxides, or attapulgite). The surface area of polymer monoliths has been significantly increased by polymer hypercrosslinking, resulting in increased efficiency when applied to the separation of small molecules. In addition, recent exploration of less conventional supports for casting polymer monoliths, including photonic fibres and 3D printed materials, has opened new avenues for the applications of polymer monoliths in the field of separation science. Recent developments made in these topics are covered, focusing on the strategies followed by the authors to prepare the polymer monoliths and the effect of these modifications on the developed analytical applications.     

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications

The use of enzymes as catalysts in chemical synthesis offers advantages in terms of clean and highly selective transformations. Galactose oxidase (GalOx) is a remarkable enzyme with several applications in industrial conversions as it catalyzes the oxidation of primary alcohols. We have investigated the wiring of GalOx with a redox polymer; this enables mediated electron transfer with the electrode surface for its potential application in biotechnological conversions. As a result of electrochemical regeneration of the catalytic center, the formation of harmful H₂O₂ is minimized during enzymatic catalysis. The introduced bioelectrode was applied to the conversion of bio-renewable platform materials, with glycerol as model substrate. The biocatalytic transformations of glycerol and 5-hydroxymethylfurfural (HMF) were investigated in a circular flow-through setup to assess the possibility of substrate over-oxidation, which is observed for glycerol oxidation but not during HMF conversion.