Immobilization of trypsin via graphene oxide-silica composite for efficient microchip proteolysis
Introduction
One of the most important tasks of proteomics is to develop high throughput approaches to separating and identifying a large number of proteins from a wide variety of biological sources [1], [2]. In proteome research, protein digestion is an important procedure prior to subsequent peptide mapping based on mass spectrometry (MS). 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 [3], [4]. In addition, the autolysis of trypsin may interfere with the MS identification of the target proteins. To solve these problems, proteases were immobilized on particles, monolithic supports, fibers, and the inner surface of microchannels to minimize the autolysis of protease and to increase the amount of protease during heterogeneous proteolysis [5], [6], [7], [8], [9], [10], [11].
Microfluidic chips are of considerable recent interest owing to their high degree of integration, portability, minimal sample/reagent consumption, high performance and speed since the pioneering work of Harrison et al. [12], [13]. It is particularly suitable for the low volume samples in the field of biomedical analysis where the available volumes are usually small. Microfluidic devices are powerful platforms for handling small-volume samples (nL to μL) in microchannels to perform enzymatic reactions [14], immunoassay [15], etc. Microfluidic chips can dramatically change the speed and scale of biomedical analysis and should find a wide range of applications in protein identification [11], [16]. To date, trypsin has been immobilized in the channels of microchips to fabricate bioreactors by covalent linking [17], [18], sol–gel embedding [19], and layer-by-layer assembly [20]. The biocompatible channel wall in microchips offered trypsin mild environment so that its denaturation was minimized. Because the autolysis of trypsin reduced after immobilization, high amount of trypsin could be used so that the digestion efficiency was significantly improved.
Graphene is an important allotrope of carbon with a two-dimensional nanostructure of sp2-bonded carbon atoms that are arranged in a chicken wire or honeycomb pattern [21]. Since Novoselov and Geim successfully isolated graphene using adhesive tape in 2004, it has attracted tremendous scientific and technological attention because of its unique nanostructure and properties [22], [23]. It indicates great promise for a variety of applications such as drug delivery, electronics, sensors, batteries, solar cells, fuel cells, supercapacitors, hydrogen storage and nanocomposites because of its excellent thermal and electric conductivity, strong mechanical strength, and high surface area [24], [25], [26], [27], [28], [29], [30], [31], [32].
Graphene oxide (GO) is basically a single atomic layer of carbon covered with epoxy, hydroxyl, carbonyl and carboxyl groups [33]. It can be facilely prepared by chemical oxidation of graphite and subsequent sonication exfoliation. GO has been employed to prepare nanocomposites [34], antibacterial paper [35], chemically modified graphene [36], conjugate with proteins [37], etc. Zhang et al. modified GO sheets with amine-functionalized Fe3O4 nanoparticles via covalent bonds. Trypsin was immobilized on the prepared material via π–π stacking and hydrogen bonding for efficient microwave-assisted proteolysis. The novel trypsin-immobilized nanocomposite was successfully employed for the digestion of bovine serum albumin, myoglobin (MYO) and proteins extracted from rat liver with satisfactory results [38]. Because hydrophilic GO bears abundant oxygen-containing functional groups, it can be well dispersed in aqueous solution and should find a wide range of applications in the fabrication of microchip bioreactors for highly efficient proteolysis. Proteases can be immobilized in microchips via GO-based materials assembled in the channels. Recently, we immobilized trypsin in the layer-by-layer coating of GO and chitosan on glass fibers by adsorption to fabricate in channel fiber bioreactors for efficient protein digestion [39]. GO bears a great amount of carboxyl groups that can be employed to immobilize trypsin via amide bonds, indicating great promise for the fabrication of microchip bioreactors.
In this work, trypsin was covalently immobilized in the GO-silica composite coating on the channel wall of poly(methyl methacrylate) (PMMA) microchips for efficient proteolysis. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used as carboxyl activating agents to crosslink the primary amino groups of trypsin to the carboxyl groups of the entrapped GO sheets in the coating to realize immobilization. Moreover, the novel bioreactors were coupled with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the digestion and peptide mapping of several proteins and human serum. The fabrication details, characterization, feasibility, and application of the novel microchip bioreactors are reported in the following sections.
Section snippets
Reagent and solutions
Ammonium bicarbonate (NH4HCO4), tetra ethyl oxysilane (TEOS), acetonitrile (ACN), acetic acid, graphite powder, sodium nitrate, potassium permanganate, hydrogen peroxide solution (30 wt.%), and sulfuric acid (98 wt.%) were all purchased from SinoPharm (Shanghai, China). Hemoglobin (HEM) from bovine blood, cytochrome c (Cyt-c) from horse heart, MYO from horse heart, ovalbumin (OVA) from chicken egg, trypsin from bovine pancreas, 4-morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS),
Results and discussion
The PMMA microchips used in this work were fabricated by in situ polymerization of a molding solution on a template [40]. Prior to modification, a layer of silica gel was prepared on the PMMA channel wall by the in situ hydrolysis of the adsorbed TEOS to obtain hydrophilic surface [41]. This layer of silica was crucial for the stable construction of GO-silica network on the PMMA surface because it was beneficial to not only the wetting of the aqueous coating solution but also the adhesion force
Conclusions
In summary, GO was successfully employed to prepare biocompatible coating in the channels of microchips via sol-gel approach for the covalent immobilization of trypsin. The novel microchip bioreactors have been employed for the rapid digestion and identification of several standard proteins and human serum albumin in real sample. It was demonstrated that the GO-based microchip bioreactors coupled with MALDI-TOF MS was a promising strategy for the efficient proteolysis and peptide mapping. The
Acknowledgements
This work was financially supported by NSFC (21075020 and 21375023), State Oceanic Administration (201105007), Shanghai Science Committee (12441902900), and Education Ministry of China (20090071120011 and NCET-08-0134).
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