Preparation of porous graphene oxide–poly(urea–formaldehyde) hybrid monolith for trypsin immobilization and efficient proteolysis

Abstract

Porous graphene oxide–poly(urea–formaldehyde) (GO–PUF) hybrid monolith was prepared by the in situ polycondensation of the urea–formaldehyde prepolymer solution in the presence of GO sheets and ammonium chloride. The amide groups in the residues of asparagine and glutamine in trypsin was allowed to condense with the residual N-hydroxymethyl groups of PUF in the monolith at low pH to fabricate bioreactors for proteolysis. The morphologies and structures of the prepared materials were investigated by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and Raman spectroscopy. The results indicate that both GO–PUF hybrid and trypsin-immobilized GO–PUF hybrid mainly consist of irregular micrometer size flakes and pores that are interconnected to form 3D porous architectures. In addition, the feasibility and performance of the novel capillary bioreactors were demonstrated by the digestion and identification of bovine serum albumin, lysozyme, ovalbumin, and cytochrome c. The digestion time was significantly reduced to less than 10 s. The digests were identified by mass spectrometry with results that were comparable to those obtained by 12-h conventional in-solution tryptic digestion.

Introduction

As a fascinating member of carbon family, graphene owns a two-dimensional nanostructure of sp²-bonded carbon atoms that are arranged in a honeycomb pattern [1], [2], [3]. It has become a rapidly rising star in the fields of material sciences since Novoselov and Geim successfully isolated and characterized it in 2004 [4], [5]. Because of its high surface area, excellent thermal and electric conductivity and strong mechanical strength, it holds great promise for potential applications in many technological fields such as drug delivery [6], cell imaging [7], sensors [8], [9], [10], proteomics [11], [12], water treatment [13], electronics [14], supercapacitors [15], fuel cells [16], catalyst [17], hydrogen storage [18], etc.

To date, a variety of approaches have been developed for producing graphene. Among them, the commonly used method is chemical reduction of graphene oxide (GO) [19], [20]. Graphite powder is oxidized with strong oxidants to form oxidized graphite that is subsequently exfoliated to form GO sheets that can be reduced to prepare graphene. GO is basically a single atomic layer of carbon covered with a great deal of oxygen-containing groups, including hydroxyl, epoxy, carbonyl, and carboxyl groups [21]. Because GO sheets are hydrophilic, they can be well dispersed in aqueous solution and mixed with other materials to prepare functional hybrids.

Poly(urea–formaldehyde) (PUF) is a commonly used thermosetting polymer. It is usually used as an adhesive in manufacturing plywood, medium density fiberboard, and other non-structural wood products. To prepare PUF, the mixture solution of urea and formaldehyde is allowed to react in basic mediums to produce water-soluble urea–formaldehyde (UF) prepolymer. It can further polycondense to form water-insoluble crosslinked PUF network with the aid of curing catalysts such as ammonium chloride [22], [23]. UF prepolymer solution is a reactive polymer mixture. It can be mixed with other matters to prepare functional PUF-containing materials. In the past decade, a variety of PUF-inorganic material hybrids have been prepared by in situ polycondensation. The inorganic materials includes iron oxides, silica, tin, and zinc [24], [25], [26], [27]. In addition, we have dispersed graphene and carbon nanotubes (CNTs) in UF prepolymer solution to prepare graphene-PUF and CNT-PUF composite electrodes for electrochemical sensing [28], [29].

In proteome research, protein digestion is an important procedure prior to subsequent mass spectrometry-based peptide mapping [30]. However, the typical time of the commonly used in-solution proteolysis is in the range of several hours to half a day that is incompatible with high-throughput protein identification [31]. In addition, the autolysis of trypsin may interfere with the identification of the target proteins. To solve these problems, proteases were immobilized on particles, monolithic supports, fibers, and the inner surface of microchips or capillaries to minimize the autolysis of protease and to increase the amount of protease during heterogeneous proteolysis [32], [33], [34].

In 2013, we developed a microchip bioreactor based on trypsin-immobilized GO–PUF hybrid coating for efficient proteolysis [35]. The mixture solution of UF prepolymer and GO sheets was allowed to flow through the channel of a poly(methyl methacrylate) (PMMA) microchip. The channel was dried so that a layer of compact GO–UF prepolymer coating formed on its wall. After the modification layer was treated with ammonium chloride solution, it polycondensed to form GO–PUF hybrid coating. In addition, the hybrid coating was prepared on a PMMA plate following the same procedure. It was observed that its color was brown. The content of GO sheets in the coating was estimated to be ∼13% w/w. The carboxyl groups of the GO sheets in the channel were subsequently linked to the primary amino groups of trypsin to realize covalent immobilization [35].

Based on this work, we tried to add ammonium chloride directly into the mixture solution of UF prepolymer and GO sheets to initiate the polycondensation of the prepolymer. It was surprisingly found that the brown mixture cured within 15 min to form porous grey monolith. The color and density of the GO–PUF hybrid monolith was much different from the brown GO–PUF hybrid film prepared in our previous work [35]. To minimize the volume shrink of the porous monolith and enhance its mechanical strength, the amount of GO was reduced by two-thirds in this work. The content of GO in the prepared GO–PUF hybrid monolith was ∼4.1% w/w. In addition, GO–PUF hybrid monolith was also prepared in fused silica capillaries. Trypsin was further covalently immobilized in the in-capillary monoliths via the condensation reaction between the residues of asparagine (Asn) and glutamine (Gln) in the enzyme and the residual N-hydroxymethyl groups of PUF to fabricate bioreactors for proteolysis. The novel capillary bioreactors were coupled with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the digestion and peptide mapping of bovine serum albumin (BSA), lysozyme (LYS), ovalbumin (OVA), and cytochrome c (Cyt-c). To the best of our knowledge, there is no early report on the preparation and application of 3D porous GO–PUF hybrid monolith. The preparation details and characterization of GO–PUF hybrid monolith and trypsin-immobilized GO–PUF hybrid monolith as well as the feasibility and performance of the novel capillary bioreactors are reported in the following sections.