Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors

Accepted Manuscript Title: Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors Author: Kinga Meller Paweł Pomastowski Michał Szumski Bogusław Buszewski PII: DOI: Reference:

S1570-0232(16)30662-6 http://dx.doi.org/doi:10.1016/j.jchromb.2016.08.032 CHROMB 20223

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

13-5-2016 17-8-2016 20-8-2016

Please cite this article as: Kinga Meller, Paweł Pomastowski, Michał Szumski, Bogusław Buszewski, Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Preparation of an improved hydrophilic monolith to make trypsin-immobilized microreactors Kinga Meller1, Paweł Pomastowski1,2, Michał Szumski1,2, Bogusław Buszewski1,2* 1

Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarina 7, 87-100 Toruń, Poland 2 Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus University in Toruń, Wileńska 4, 87-100 Toruń, Poland *Corresponding author: Bogusław Buszewski, Chair of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Gagarin 7 87-100 Toruń, Poland. e-mail: [email protected] tel. (48)(56)6114308, fax. (48)(56)6114837

Highlights

Highly hydrophilic HEMA-MBA monoliths prepared with high monomer content reduce protein adsorption HEMA-MBA monoliths with new porogen composition allow high density of functional groups for trypsin immobilization Trypsin-immobilized microreactors show excellent homogeneity across 8 cm based on hydrolysis activity. Trypsin immobilized via spacer arm shows greater digestion ability and specificity then trypsin immobilized directly on the HEMA hydroxyl groups

Abstract In the present work the preparation of capillary-based microreactors with immobilized trypsin was investigated. The monolithic support was synthesized from 2-hydroxyethyl methacrylate (HEMA) as a functional monomer and N,N’-methylenebis(acrylamide) (MBA) as a hydrophilic crosslinker. Two monomers contents in the polymerization mixture (27% and 35%) at the ratio of HEMA:MBA=3:2 were tested. The results indicated that the heated mixture of the above monomers and the porogen consisting of 40% 1-decanol, 40% 2propanol and 20% water was the most appropriate polymerization mixture for preparation of HEMA-MBA copolymer. The elaborated novel procedure of HEMA-MBA hydrophilic monolith preparation allowed for the introduction of higher monomers content compared to earlier literature data. The obtained monolith exhibited lower adsorption of BSA in comparison with the commonly used glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) material. In the second step, the covalent enzyme attachment on the hydroxyl groups via 1,1’-carbonyldiimidazole activation was performed. Two immobilization techniques were tested. The first approach involved direct trypsin attachment to the hydroxyl groups of two-carbons HEMA chain (microreactor T1). In the other strategy, 5-amino-1pentanol was employed to form a twelve-atom spacer arm (microreactor TS1). The prepared microreactor s activities and specificities were evaluated using low molecular weight trypsin substrate (Nα-benzoyl-L-arginine ethyl ester (BAEE)) and bovine serum albumin (BSA). The chromatographic analysis of the eluates from T1 and TS1 microreactors indicated that the microreactor T1 showed higher activity toward the BAEE than the microreactor TS1. However, the BSA digestion and subsequent MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of the collected eluates resulted in a sequence coverage of 43.9% and 35.7% in the case of TS1 and T1 microreactors, respectively.

Keywords: trypsin, immobilized enzyme microreactor, proteomics, monolithic bed, 2-hydroxyethyl methacrylate, N,N’-methylenebis(acrylamide)

1. Introduction Proteins are among the most important compounds occurring in living organisms. Electrophoretic approach is commonly used for proteins separation while the mass spectrometry is mostly employed for protein identification [1]. For this purpose, two different strategies may be involved: top-down and bottom-up. In the first case, the intact proteins are analyzed while for the bottom-up strategy, the proteins are firstly digested into peptides by a specific enzyme [2-3]. At that time, analyte identification may be performed using Peptide Mass Fingerprint (PMF) technique [4]. However, additional fragmentation of the peptides within the mass spectrometer is necessary for identification of unknown proteins by primary sequencing [5]. The classical protein digestion is performed in-solution with dissolved enzyme or in-gel after target protein isolation [6]. However, in such cases, the post-reaction mixture also contains the fragments from protease autolysis. In the case when too high protease concentration is used, these fragments from enzyme autodigestion complicate the MS spectrum interpretation and reduce the detection sensitivity due to low intensity of the peptides signals compared to the recorded autolysis peaks [7]. On the other hand, when the low protease concentration is used, the proteolysis requires very long incubation time to achieve efficient protein fragmentation. One of the relatively novel approaches making progress in this field is the application of microfluidic reactors with immobilized proteases (Immobilized Enzyme Reactors – IMERs) [8]. Such quasi-isolation of enzyme molecules prevents them from making contact with each other and thus also prevents their autodigestion. Moreover, IMERs may be reused as long as the protease retains its activity. In turn, the miniaturized capillary dimensions allow for the analysis of a low volume sample and it improves the digestion efficiency due to the high local protease concentration. The monolithic beds [9-15], particles/beads [16-19] or internal capillaries/channel surface [20-23] may be used as supports for covalent enzymes attachment. The monolithic polymers seem to be the most preferable supports due to their advantageous properties such (a) low back pressure, (b) rapid mass transfer, (c) a relatively high surface area, (d) a simple preparation process, and (e) possibility of in situ polymer synthesis within desired capillary/channel fragments [24].

The main problematic issue connected with protein digestion using protease-based μIMERs is the adsorption of hydrophobic peptides on the support surface. This situation results in the retention of reaction products in the microreactor and therefore limitation of the detection sensitivity. Moreover, the adsorbed fragments may be eluted during subsequent analysis which can lead to results misinterpretation and incorrect protein identification. To overcome this disadvantageous effect, the support should exhibit a hydrophilic character. Alternatively, it may be hydrophilically functionalized before enzyme immobilization. For example, N-vinyl-2-pyrrolidinone was used for commercially available acrylic beads modification to improve their hydrophilicity [25]. In another approach, polyethyleneimine was covalently attached to methacrylic acid-contained hybrid monolith which allowed not only for improved hydrophilicity but also for the elimination of residual silanol group effect [26]. In the case of organic monolithic beds, their hydrophilization may be achieved via surface photografting in an additional support preparation step [14]. The non-specific adsorption problem may be also avoided by employing hydrophilic monomers during support polymerization. For example, hydrophilic poly(ethylene glycol)diacrylate was used as a crosslinker to obtain proper support properties while the Nacryloxysuccinimide served as a functional monomer enabling the trypsin covalent immobilization [27]. In another research, two different hydrophilic monomers (2hydroxyethyl methacrylate (HEMA) and acrylamide (Aam)) were tested as additional components besides the ethylene dimethacrylate (EDMA, crosslinker) and 2-vinyl-4,4dimethylazlactone (functional monomer) [15]. At that time, it was found that HEMAcontaining microreactor showed greater maximum velocity (VMAX) than IMER containing Aam. It has been also proved that significantly higher bovine serum albumin (BSA) sequence coverage was achieved using Aam-MBA monolithic support than in the case of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) polymeric bed [28]. It may be attributed to the higher Aam-MBA support hydrophilicity compared to GMA-EDMA which, in turn, resulted in lower peptides retention. Taking into account the above mentioned literature reports concerning favorable effects of HEMA and MBA components, the main aim of the presented study was the preparation of capillary-based microreactor with trypsin immobilized on HEMA-MBA monolithic support. Thus far, the HEMA-MBA monolithic copolymer has been reported only once and it was applied for the preparation of a separation column for capillary electrochromatography [29]. The application of 1-decanol, 2-propanol and water as components of polymerization mixture,

its subsequent heating in ultrasonic bath and capillary filling in heated water bath allowed for obtaining a hydrophilic HEMA-MBA monolith of proper porous structure and higher monomer content than in the previously reported approach [29]. For relative evaluation of the hydrophobic protein retention on the prepared bed, the BSA solution was passed through the HEMA-MBA support and GMA-EDMA copolymer. GMA-EDMA monolithic bed is one of the most common supports used for enzyme immobilization and therefore it was used as a reference material [28]. Two immobilization strategies including direct trypsin attachment and attachment through a spacer arm created from 5-amino-1-pentanol were tested. Microreactors activities were evaluated using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a low-molecular mass trypsin substrate. Then, the microreactors specificities were tested through digestion of bovine serum albumin (BSA) as a hydrophobic protein which is resistant to enzymatic hydrolysis. 2. Material and Methods 2.1 Materials Fused-silica capillaries (200 μm i.d. x 360 μm o.d.) were purchased from a Polymicro representative, CM Scientific Ltd. (Silsden, United Kingdom), Trypsin from bovine pancreas, 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS), 2-hydroxyethyl methacrylate (HEMA), N,N’-methylenebis(acrylamide) (MBA), 1-decanol, 2-propanol, 1,1’-carbonyldiimidazole (CDI), 5-amino-1-pentanol, DL-dithiothreitol (DTT), iodoacetamide (IAA), bovine serum albumin (BSA), benzamidine, Nα-benzoyl-L-arginine ethyl ester (BAEE), trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Steinheim, Germany). Storage solution (with sodium azide) was obtained from MACS Miltenyi Biotec (MEDianus Sp.z o.o., Kraków, Poland). Acetone, toluene, methanol, dichloromethane, sodium hydroxide, sodium bicarbonate, sodium phosphate monobasic dihydrate, ammonium bicarbonate (analytical reagent grade), and acetonitrile (HPLC ultra gradient grade) were purchased from Polskie Odczynniki Chemiczne (POCh, Gliwice, Poland). Deionized water was obtained from a miliQ ultrapure water producing system (Millipore, Bedford, MA, USA). Azobisisobutyronitrile (AIBN) and all chemicals for the MALDI-MS analyses were supplied at the highest commercially available purity by Fluka Feinchemikalien (Neu-Ulm, Germany; a subsidiary of Sigma-Aldrich). Anchor targets (Bruker Daltonics, Bremen, Germany) were used for sample deposition. α-Cyano-4-hydroxycinnamic acid (HCCA) was employed as matrix for MALDI

analysis of tryptic digest (dried droplet method). Trypsin-digested bovine serum albumin (BSA) MS Standard from Bruker Daltonics was used for external calibration [30]. 2.2 Instrumentation A syringe pump NE-1002X (New Era Pump Systems, Farmingdale, NY) was used to pass the reagents (during the modification) and the substrate solution through the μ-IMER. For carrying out the reaction at a constant temperature the thermostat (Julabo, type F25) connected to a specially designed heat exchanger was used. A pneumatic HPLC pump (Knauer GmbH, Berlin, Germany) was used for column flushing (after monolith synthesis) and flow rate measurements. All chromatographic experiments were performed in a nanoLC system consisting of a 1260 Capillary Pump (Agilent Technologies, Waldbronn, Germany), a 10-port nanoLC valve (model C72MX-6690D Vici-Valco, Schenkon, Switzerland), and a Crystal 100 UV-Vis detector (Thermo Separation Products, San Jose, CA, USA). The system was controlled with the Clarity software (DataApex, Prague, Czech Republic). All the MS spectra were obtained using MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a modified neodymium-doped yttrium aluminium garnet (Nd:YAG) laser (smartbeam II) operating at the wavelength of 355 nm and a frequency of 2 kHz. The evaluation of hydrophobic protein retention in the prepared monolithic capillary was performed using gel electrophoresis (Mini Gel Tank, Novex, Poland) and DirectDetect® Infrared Spectrometer (Merck Millipore, Germany). 2.3 Capillary pre-treatment and modification Initially, the capillaries were flushed with acetone and dichloromethane and then dried by flushing with nitrogen. Subsequently, the capillaries were etched with 1 M NaOH solution at 100°C for 3 h and silanized using 10% γ-MAPS solution in toluene at room temperature for 2 h. 2.4 Preparation of HEMA-MBA monolithic support The weight ratio of functional monomer (HEMA) to crosslinking monomer (MBA) was kept constant and equaled 3/2 (w/w) [31]. Different masses of 2-propanol, water and 1decanol were added to a mixture of both monomers (HEMA and MBA) to obtain final monomers concentration of 27% and 35% at varying percentage content of each porogen solvent component (Table 1). The prepared polymerization mixtures were heated and

sonicated in an ultrasonic bath at 70°C to dissolve MBA. Subsequently, the initiator was quickly added to the solutions and the vials were tightly capped using aluminum crimp seals with septum. The content of initiator (AIBN) was always 1.25% (w/w) with respect to the total weight of the monomer (HEMA and MBA). The end of each capillary was pushed through the rubber stopper of vial and immersed in the polymerization solution. The empty capillary connected to a syringe was also pushed through a rubber stopper and, subsequently, the air was injected into the vial through pressing the plunger. During this process, the vials and capillaries were placed in water heated to a temperature of 70°C to avoid the MBA precipitation (Fig. 1). After a few drops of the polymerization mixture flowed out through the capillary, its ends were sealed with pieces of GC septa. Finally, the capillaries were placed in a water bath and polymerization was carried out at 80°C for 24 h. Fig. 1 2.5 Characterization of HEMA-MBA monolithic support The monoliths permeability (𝐾𝐹 ) was evaluated on the basis of water flow rate through the capillaries and calculated using the following formula (Eq. (1)): 𝐹𝜂𝐿

𝐾𝐹 = 𝛥𝑃𝜋𝑟 2 [m2 ]

(1)

Where: 𝜂 – viscosity of water [Pa∙s], 𝐹 – water flow rate [μl/min], 𝐿 – length of the capillary [dm], 𝑟 – radius of the capillary [dm], 𝛥𝑃 – the pressure set on the pneumatic pump [Pa].

The bed homogeneity in a capillary cross-section was evaluated by scanning electron microscope (SEM). To evaluate the adsorption of a hydrophobic protein on the HEMA-MBA monolithic bed, two BSA solutions of different concentration were passed through the capillary at F = 1 μl/min (C1 was about 1.0 mg/ml and C2 was 100-fold diluted). For a comparison, the same solutions were also passed through the glycidyl methacrylate-coethylene glycol dimethacrylate (GMA-EDMA) monolithic column at the same flow rate. Both monolithic capillaries were 7 cm in length. The BSA solutions, as well as the collected eluates, were analyzed using gel electrophoresis (C2 solution) and DirectDetect Infrared Spectrometer (C1 solution). The GMA-EDMA monolithic bed was synthesized at 70°C for 24 h using the mixture of cyclohexanol (70%) and 1-dodecanol (30%) as a porogen solvent, AIBN as an initiator (1% with respect to the total weight of the monomers) with GMA/ EDMA ratio of 3/2 (w/w) [32]. 2.6 Immobilization of trypsin

Two different immobilization strategies were tested: a) direct immobilization on the hydroxyl groups present in the copolymer and b) enzyme attachment via spacer arm. Firstly, the capillaries were flushed with anhydrous acetonitrile (ACN) at F = 1 μl/min for t = 30 min. Then, the hydroxyl groups present in the copolymer were activated using 0.2 M 1,1’carbonyldiimidazole (CDI) solution in ACN at room temperature (F = 1 μl/min, t = 2 h). Subsequently, the capillaries were flushed with ACN (F = 1 μl/min for t = 30 min) to remove the unreacted CDI and dried by flushing with nitrogen (20 min). For direct immobilization strategy (without spacer arm), the trypsin solution (2.5 mg/ml) containing benzamidine (50 mM) in 50 mM sodium bicarbonate buffer of pH 9.5 was passed through a capillary at 4°C for 24 h (F = 0.5 μl/min). For spacer arm formation, the 5-amino-1-pentanol solution (0.5 M) in 100 mM bicarbonate buffer of pH 11.0 was passed through a capillary at room temperature (F = 0.5 μl/min, t = 14 h) after CDI activation. Then, the trypsin was immobilized on the hydroxyl groups ending the attached 5-amino-1-pentanol as in the case of the first strategy. After immobilization, the unreacted imidazole carbamate groups were blocked by the flushing capillaries with 50 mM TRIS buffer of pH 9.5 (F = 1 μl/min, t = 1 h). The microreactors were flushed (F = 1 μl/min, t = 30 min) and stored in a storage buffer at 4°C after their preparation and performing the experiments. 2.7 Evaluation of immobilized trypsin activity and microreactor homogenity The total mass of the immobilized enzyme was not measured due to the fact that some of the immobilized trypsin molecules can lose their activity after immobilization e.g. because of autolysis or deformation of the enzyme active sites. That is why the amount of immobilized proteases does not reflect the final efficiency of IMER which has already been proven [33]. Instead of this, the efficiencies of prepared microreactors were evaluated directly by measurement of trypsin activities using Nα-benzoyl-L-arginine ethyl ester (BAEE) for this purpose. Trypsin catalyses the hydrolysis of BAEE ester bond which results in the formation of Nα-benzoyl-L-arginine (BA) and ethanol. The BAEE solutions in 0.05 mM sodium phosphate buffer (pH 7.5) of different concentrations (30, 50, 100 mM) were passed through both μ-IMERs at room temperature and flow rate of 1.00 μl/min set on the syringe pump. The reaction product (BA) was separated from the substrate (BAEE) using a nanoLC system equipped with a homemade column packed with octadecyl stationary phase. The elution was performed at a gradient mode using mixture of water and acetonitrile (ACN) as a mobile phase (both with 0.1% TFA addition). The chromatographic process started with 20% ACN in a mobile phase whose content was increased to the final ACN concentration of 40%. The

detection was performed at the wavelength of λ = 223 nm according to ref. [34]. For the calibration curve preparation, standard BAEE solutions of concentrations between 10 ÷ 100 mM were directly injected on the separation column with the same chromatographic conditions. Immobilized trypsin activity was expressed as a degree of substrate hydrolysis, %H (Eq. (2)). The amount of unreacted BAEE in the eluate from a μ-IMER was calculated based on its peak area and using the calibration curve. %𝐻 =

𝐶𝐵𝐴𝐸𝐸,𝑠𝑡 −𝐶𝐵𝐴𝐸𝐸 𝐶𝐵𝐴𝐸𝐸,𝑠𝑡

∙ 100 [%]

(2)

Where: 𝐶𝐵𝐴𝐸𝐸,𝑠𝑡 – concentration of standard BAEE solution before passing through a μ-IMER [mM], 𝐶𝐵𝐴𝐸𝐸 – concentration of BAEE in eluate from a μ-IMER calculated using calibration curve [mM].

To verify the microreactor homogeneity over the entire capillary length, one of the prepared μ-IMERs was sequentially shortened after each reaction by cutting off 1.0-1.5 cm then retesting the remaining microreactor activity on the basis of BAEE hydrolysis degree. 2.8 Protein digestion and MALDI-TOF identification Bovine serum albumin (BSA) was dissolved in a 50 mM ammonium bicarbonate buffer of pH 8.05 (0.2 mg/ml). The protein solution was heated at 90°C, then the disulfide bridges present in BSA molecules were reduced with DTT, and finally the thiol groups were alkylated with IAA. The pre-treated sample was passed through μ-IMERs using syringe pump at 37°C and flow rate of F = 0.25 μl/min. Each of the eluates was collected in a plastic vial for 15 min. Subsequently, 1.5 μl of eluate was mixed with a matrix at the ratio of 1:1, and finally three spots of 0.5 μl volume were deposited on the MALDI target. A saturated solution of HCCA in mixture of acetonitrile and 0.1% trifluoroacetic acid in water at a volumetric ratio of 30:70 was used as the matrix. Mass spectra of trypsin-digested BSA MS standard and eluates from microreactors were recorded manually in the positive mode using acceleration voltage of 25 kV within m/z range of 500-3000 Da (500 shots for one acquisition to sum of 2000). The resulted mass spectra were acquired and processed using dedicated software, flexControl and flexAnalysis, respectively (both from Bruker Daltonik). The obtained MS peak lists were submitted to a Mascot search with SwissProt database for protein identification with the use of BioTools and ProteinScape software (both from Bruker Daltonik). 3. Results and discussion 3.1 The preparation and characterization of HEMA-MBA monolithic support

MBA is a hydrophilic crosslinking monomer of low solubility in water and most organic solvents. That is why the MBA-contained monoliths usually suffer from low total monomers content in polymerization mixture because of the need to use a high amount of solvent to completely dissolve MBA. The low monomers concentration allows for obtaining the bed of high permeability but, on the other hand, it contributes to low efficiency of immobilization [31]. The increase of the amount of immobilized molecules may be achieved through increasing the functional monomer to crosslinking monomer ratio but this, in turn, may decrease the monolith rigidity [31]. These considerations lead to the conclusion that high monomers content in polymerization mixture at a reasonable functional monomer to crosslinker ratio is the most appropriate approach for the preparation of a bed of proper properties. The monolithic copolymers containing MBA are usually prepared using dimethyl sulfoxide (DMSO) as a component of polymerization mixtures. For example, AAm-co-MBA monolithic copolymer was prepared using 35 mg AAm, 40 mg MBA, and the porogen solvent consisted of 150 mg 1,4-butanediol, 330 mg DMSO, and 185 mg dodecanol [28]. Consequently, the total monomer content in the polymerization mixture was only 10%. The solution of DMSO, N,N-dimethylformamide (DMF), and water saturated with Na2HPO4 was used for the preparation of other MBA-based monoliths copolymerized with 4vinylphenylboronic acid [35] or HEMA and 2-acrylamido-2-methyl-1-propanesulfonic acid [29]. In the mentioned HEMA-MBA copolymer, 400 μl of DMSO/DMF/H2O mixture was used to dissolve a crosslinker. Additionally, 400 μl of dodecanol and toluene solution of different volumetric proportion was used as a porogen solvent. Consequently, the total monomer percentage content corresponded to about 15 % or 22 % for 70 μl or 140 μl of HEMA, respectively. To increase the total weight percentage of monomers in the polymerization mixture, the higher MBA solubility is necessary. The improvement of MBA solubility in water may be achieved through 2-propanol addition as it as previously reported [36]. Therefore, 2-propanol was tested as a polymerization mixture component. Unfortunately, a colorless, gelatinous, and non-porous polymer was obtained in this case. Similar results were also obtained using cyclohexanol as a co-solvent. Such a situation was changed when 1-decanol was used besides 2-propanol and water. It suggests that 1-decanol is a component which enables obtaining a proper HEMA-MBA porous structure. The possible proportions of heated polymerization mixtures which did not lead to phase separation and allowed for MBA dissolving are

presented in the Table 1. Under such conditions, after heating to temperature of 70°C, the polymerization mixture was homogenous but the MBA quickly precipitated after slight cooling. That is why the polymerization temperature was 10°C higher in order to avoid potential possibility of MBA precipitation. This situation also required the capillary filling using the above described method, (Section 2.4.) because, after introduction of the polymerization mixtures into a syringe, the MBA rapidly precipitated from the solutions. Table 1 The permeability of the obtained beds at constant water concentration (%H2O = 20%) increased with the 1-decanol content but only to %D = 40%. For higher 1-decanol concentration, the permeability rapidly decreased for both monomers contents (Table 1). In turn, at constant 1-decanol concentration (%D = 20%), the beds permeability increased with water concentration but this effect was significantly observable only for %M = 27 %. From the beds prepared at constant water concentration (%H2O = 20%) and monomers content of %M = 27%, the greatest permeability was exhibited by the monolith synthesized using %D = 40% (KF = 28·10-15 [m2], Fig. 2B). Both lower and higher 1-decanol concentration led to the decrease in permeability. In the case of %D = 20% (impermeable, Fig. 2C), it may be explained by copolymer shrinking and formation of extremely small globules. In turn, at higher 1-decanol concentration, the diameter of the flow-through pores decreased which is observable on the SEM images, %D = 60% (KF = 9.1·10-15 [m2], Fig. 2A). From the beds synthesized at constant 1-decanol concentration (%D = 20%), the greatest permeability was shown by the beds synthesized using 50% content of water in porogen solvent (KF = 49·10-15 [m2]). Unfortunately, such high permeability probably resulted from the voids consistently occurring between the monolith and the capillary inner wall (Fig. 2F). However, at %H2O = 45%, each of the prepared monoliths (n = 5) exhibited a homogenous structure in the capillary cross section and the large size of the flow-through pores (KF = 20·10-15 [m2], Fig. 2E). In turn, slightly lower water concentration caused significant decrease in permeability due to bed shrinking, %H2O = 40% (KF = 0.4·10-15 [m2], Fig. 2D). Fig. 2 The beds prepared from polymerization mixture with 35% monomers content were expected to be more suitable for enzyme immobilization due to the higher density of functional groups on their surface. For these columns (%M = 35%), at constant water

concentration (%H2O = 20%), the obtained beds were impermeable below 32% of 1-decanol while at %D = 32% the bed permeability equaled only KF = 0.4·10-15 [m2]. It is caused by the formation of a large number of small, microporous, and tight globules (Fig. 3A). At %D = 40% the formed globules were significantly larger which, in connection with the presence of numerous flow-through pores, resulted in higher permeability (KF = 26·10-15 [m2], 3B). However, when the higher 1-decanol concentration (%D = 55%) was employed, the globule diameter increased, while the spaces between them (i.e. flow-through pores) decreased, which caused the decrease of permeability (KF = 3.5·10-15 [m2], Fig 3C). At constant 1-decanol concentration (%D = 20%), the obtained beds were impermeable at water percentage value of 40% (no mobile phase drop occurred at the outlet end of capillaries under the pressure of 200 bar after 15 min). It was caused by enormous monolith shrinkage (%D = 30%, Fig. 3D).This situation did not change even when 50% water concentration was used (Fig. 3E). Fig. 3 From the obtained beds of %M=35%, the greatest permeability was displayed by the monolith synthesized using polymerization mixture consisting of %D = 40 %, %H2O = 20 % and %P = 40 % (Fig. 3B). Moreover, the copolymer obtained at the above mentioned conditions exhibited repeatable homogenous structure (n = 5) and permeability (RSD = 2.1%). Therefore, it was used as a support for trypsin immobilization. To evaluate the effect of non-specific adsorption of hydrophobic protein on the prepared support, the BSA solution of about 1.0 mg/ml concentration was passed through the HEMA-MBA and GMA-EDMA monolithic columns. GMA-EDMA monolithic bed is one of the most common supports used for enzymes immobilization and therefore it was chosen as a reference material. The results obtained from DirectDetect Infrared Spectrometer indicated that the protein concentration in the eluate from HEMA-MBA monolithic bed practically did not change. However, the BSA concentration in the eluate from GMA-EDMA monolith significantly decreased (Fig. 4). It proved that the studied protein was less adsorbed (and also retained) in HEMA-MBA capillary compared to GMA-EDMA. The same conclusion can be drawn from the obtained gel electropherogram (Fig. 5). In the case of lower BSA concentration (about 0.01 mg/ml) passed through a GMA-EDMA monolithic bed, no band was observed on the gel (lane 3). It suggests that such a small amount of protein was completely retained in column. Fig. 4

Fig. 5 3.2 Trypsin immobilization and microreactors' activity evaluation 1,1’-carbonyldiimidazole (CDI) is a coupling reagent which may be used for activation of hydroxyl as well as carboxyl functionalities for subsequent reaction with compounds containing amino groups [37]. One of the first CDI applications included the preparation of agarose-based affinity adsorbents [38] and proteins attachment on diol-bonded silica [39]. In recently published papers, trypsin was immobilized on dextran-coated capillary inner walls through CDI strategy [40]. CDI was also used for attachment of different amines on cellulose fibers [41] and for ovalbumin immobilization on the magnetic iron oxide nanoparticles containing hydroxyl groups [42]. CDI activation was also employed for enzymes attachment on the epoxy-functionalized supports using initial acidic hydrolysis of epoxy rings to diol forms [43-44]. Due to the fact that the hydroxyl groups are present on the HEMA-MBA monolithic bed, CDI activation is the most logical immobilization strategy. However, the hydroxyl group of HEMA terminates a 2-atom carbon chain. The small distance between the support and the immobilized enzyme has been shown to lower enzymatic activity and hence, reaction efficiency [33, 45]. It may be explained by partial deformation of enzyme active sites through the multipoint attachment and its limited accessibility to substrate molecules as well. That is why two immobilization strategies were tested to prepare two different microreactors (T1 and TS1). The first approach involved the direct activation of hydroxyl functionalities (T1, Fig 6 a) while the other one included the intermediate step for the spacer arm formation (TS1, Fig 6 b). As for the activation of hydroxyl groups with CDI, alkoxycarbonyl imidazole intermediates are formed [46]. This step should be performed using non-aqueous solvent to avoid the CDI hydrolysis. Subsequently, the amines react with introduced electrophilic substituents by the nucleophilic attack leading to stable carbamate bond formation. The higher the buffer pH used for the second step, the greater coupling efficiency is achieved [47]. Therefore, support modification with 5-amino-1-pentanol was performed at room temperature using the buffer of pH 11.0 to increase the reaction rate. However, trypsin immobilization was carried out at 4°C and pH of 9.5 to prevent the enzyme from deactivation. The final stage of both immobilization strategies was quenching of the residual active groups by using 2-amino2-hydroxymethyl-propane-1,3-diol (50 mM Tris buffer). Fig. 6

Firstly, the activities of T1 and TS1 microreactors were evaluated using BAEE as a low-molecular mass trypsin substrate. All the reactions were carried out at room temperature and flow rate of 1 μl/min. Both μIMER capillaries were 8.5 cm in length. In the case of 30 mM BAEE solution, the substrate was completely hydrolysed using T1 as well as TS1 μIMER. However, the reactions efficiencies for 50 and 100 mM BAEE concentrations slightly differed. For 50 mM BAEE, the degrees of hydrolysis were 98% and 94% when T1and TS1 was used, respectively. In the case of 100 mM BAEE solution, the reaction performed in T1 and TS1 resulted in %H = 60% and %H = 49% (Fig. 7).The obtained results indicated that the BAEE hydrolysis was less efficient in the case of a reaction performed in TS1 microreactor for a high substrate concentration. It may be explained by the attachment of a lower number of trypsin molecules due to the greater number of TS1 preparation stages. Fig. 7 To verify the reproducibility of the elaborated microreator preparation methods, four other μIMERs (T2, T3, TS2 and TS3) were prepared using the same protocols as in the case of T1 and TS1. The 50 mM BAEE solution was passed through the each of microreactors. The results expressed in hydrolysis degree per capillary centimeter units were 11.6, 12.0, 11.7 and 11.1, 10.4, 11.3 [%/cm] for T1, T2, T3, and TS1, TS2, TS3, respectively. It indicates that the reproducibility of the monolith synthesis as well as immobilization techniques is satisfactory. For the evaluation of homogeneity along the length (L) of the monolithic support, one of the microreactors (TS3) prepared by the spacer method shown in Fig. 6B was used. Its initial activity was 96% for L=8.5 cm, which corresponds to 11.3 %/cm. The microreactor was shortened by 1.0 or 1.5 cm, and its activity was tested again using the HPLC assay. Consequently, the calculated degrees of hydrolysis were 96, 72, 59, 51, 43 and 34 [%] for the capillary length of 8.5, 7.0, 5.5, 4.5, 3.5 and 2.5 [cm] which corresponds to 11.3, 10.3, 10.7, 11.3, 12.3 and 13.6 [%/cm], respectively. Consequently, the two extreme results correspond to 10.3 %/cm for L=7.0 cm and 13.6 %/cm for L=2.5 cm. An average value of the results for all μIMER lengths equaled 11.6 %H/cm with RSD of 10.3%. Taking into account the methodology of measurements (shortening of μIMER, collection of the eluates and finally their chromatographic analysis) and all factors which can cause an error (including the amount of μIMER preparation stages) it may be concluded that microreactor is homogenous over its length (L=8.5 cm). It should be added here that the μIMER was always connected to a

syringe at the same end. It may lead to a higher efficiency of each modification stage of the support surface at the inlet section of capillary. That is why, the greatest hydrolysis degree per μIMER length is observable (13.6 %/cm) for the length of 2.5 cm. 3.3 Protein digestion and MALDI-TOF identification The specificities of both microreactors (T1 and TS1) were evaluated using BSA as a model substrate. The eluates from T1 and TS1 as well as trypsin-digested bovine serum albumin MS standard were analyzed by MALDI-TOF MS. All of the spectra were recorded at the same spectrometric conditions and submitted to database search at identical set parameters. The average values of sequence coverage for BSA digested using T1 and TS1 were 35.7% (±2.8%) and 43.9% (±4.9%), respectively. By comparison, sequence coverage for commercially obtained tryptic digest of BSA standard was 65.6%. In the literature, a similar result for BSA digestion and MALDI-TOF MS identification was obtained using microreactor with trypsin immobilized on hydrophilized GMA-EDMA monolithic bed (sequence coverage of 44%) [14]. Application of monolithic microreactor prepared from AAm, MBA, and acryloylated trypsin for BSA digestion resulted in 29% sequence coverage [48]. In another study, laser radiation was employed for improvement of protein digestion which resulted in BSA sequence coverage increasing from 12% to 33% [49]. Significantly more peptide signals were recorded on the MS spectra of eluates compared to the results obtained for standard peptides mixture (Fig. 8) due to the high number of missed cleavage sites (see Table 2). Such differences between in-solution digestion of the proteins and using trypsin-immobilized IMER have already been described [50]. However, it is interesting that the disparities between the MS spectra of T1 and TS1 eluates were observed. Despite the lower TS1 activity toward the BAEE, the greater BSA sequence coverage was obtained using TS1 for digestion. This situation is probably connected with the enzyme immobilization strategy. In the case of T1, trypsin was directly attached to the HEMA hydroxyl groups. It resulted in a small distance between the support surface and enzyme molecules which probably led to the multipoint attachment. Consequently, the structure of trypsin active sites was probably disrupted and hence, its specificity was decreased after immobilization. It presumably caused the higher number of non-specific signals on MS spectrum as well as lower BSA sequence coverage compared to eluate from TS1. The high intensities of non-specific signals supressed the signals from trypsin-cleavage sites on the spectrum of eluate from T1 μIMER. From Table 2 a conclusion may be also drawn that in the case of BSA digestion using TS1 μIMER the values of intensity of signals from longer trypsin-cleaved peptides are greater than in the case of the eluate from T1 μIMER. Additionally, in eluate from TS1 μIMER greater amounts of larger peptides were detected. It suggests that trypsin immobilized via spacer arm has greater access for cleavage sites of longer polypeptides which are hydrolysed to fragments of high masses. In such cases the

signals from small peptides are supressed or they are not registered at all. It is important that the trypsin autolysis peaks were not recorded on the mass spectra of all the eluates. Fig. 8 Table 2 4. Conclusions The preparation procedure of N,N’-methylenebis(acrylamide)-co-2-hydroxyethyl methacrylate (HEMA-MBA) monolithic bed of high monomers content and good permeability was developed. The obtained results indicated that the most suitable proportion of the applied ternary porogen solvent is: 40% 1-decanol, 20% water and 20% 2-propanol. During the experiment, it was proved that BSA is less retained on HEMA-MBA monolithic bed compared to GMA-EDMA copolymer. It suggests that HEMA-MBA exhibits sufficiently hydrophilic character to avoid the hydrophobic protein retention in a capillary. Therefore, it seems to be a more preferable support for protease immobilization than the GMA-EDMA monolith, which is commonly used for enzymes attachment. Two different immobilization strategies were tested: direct immobilization on the hydroxyl groups contained in a support (T1) and trypsin attachment via spacer arm (TS1). In fact, the highly efficient and fast protein digestion may be achieved using T1 as well as TS1 microreactors. However, the microreactor T1 exhibited a higher activity with regard to low molecular weight substrate (BAEE) in comparison with microreactor TS1. What is the most interesting, the digestion of BSA using TS1 resulted in a higher sequence coverage than in the case of proteolysis in T1. All the above mentioned facts suggest that immobilization of trypsin via spacer arm resulted in higher enzyme specificity compared to the direct immobilization strategy. The preparation of monolithic microreactors is time-consuming, which is their major disadvantage. In the described method, polymerization of monolithic bed takes 24h while the stage of trypsin immobilization takes another 24h. However, efficient protein digestion may be achieved in 15 min using prepared μIMERs which may allow for performing at least 20 reactions in one working day. Taking into account that classical protein digestion in solution requires 16-24h, the application of microreactors seems to be more favorable. Acknowledgments This work was financially supported by the National Science Centre in the frame of the project Symfonia 1 No. 2013/08/W/NZ8/701 (2013-2016) and Maestro 6, No. 2014/14/A/ST4/00641 (2015-2017).

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Figure captions Fig 1. Scheme of monolithic support preparation. Fig. 2 SEM images of monolithic beds synthesized using 27% monomers content (HEMA/MBA ratio of 3/2) and different percentage content of each co-solvent: A – %H2O =20, %D=60, %P= 20 (KF = 9.1·10-15 m2), B – %H2O =20, %D=40, %P= 40 (KF = 28·10-15 m2), C – %H2O =20, %D=20, %P= 60 (impermeable), D – %H2O =40, %D=20, %P= 40 (KF = 0.4·10-15 m2), E – %H2O =45, %D=20, %P= 35 (KF = 20·10-15 m2), F – %H2O =50, %D=20, %P= 30 (KF = 49·10-15 m2). Fig. 3 SEM images of monolithic beds synthesized using 35% monomers content (HEMA/MBA ratio of 3/2) and different percentage content of each co-solvent: A – %H2O =20, %D=32, %P= 48 (KF = 0.4·10-15 m2), B – %H2O =20, %D=40, %P= 40 (KF = 26·10-15 m2), C – %H2O =20, %D=55, %P= 25 (KF = 3.5·10-15 m2), D – %H2O =30, %D=20, %P= 50 (impermeable), E – %H2O =50, %D=20, %P= 30 (KF = 0.9·10-15 m2). Fig. 4 Comparison of FT-IR bands of BSA solution (red) and eluates from HEMA-MBA (blue) and GMA-EDMA (green) monolithic columns. Fig. 5 Scan of gel electropherogram obtained for BSA solution of concentration about 0.01 mg/ml (lane 1), eluate from HEMA-MBA monolithic column (lane 2), and eluate from GMAEDMA monolith (lane 3). Fig. 6 Reactions scheme of covalent trypsin immobilization on HEMA-MBA monolithic support using two strategies: A – direct enzyme attachment (T1 microreactor), B – enzyme attachment via spacer arm (TS1 microreactor). Fig. 7 Overlayed chromatograms of BAEE substrate solution (blue trace) and eluates after substrate digested in microreactors T1 (red trace) and TS1 (green trace) at room temperature and a flow rate of 1 μl/min. Fig. 8 MS spectra of: A – tryptic digest standard of BSA, B – eluate from T1 microreactor, C – eluate from TS1 microreactor.

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Fig.7

Fig.8

Table 1. The list of tested polymerization mixtures compositions and permeability values (KF) of obtained monolithic beds. %D – percentage content of 1-decanol in porogen solvent, %H2O – percentage content of water in porogen solvent, %P – percentage content of 2-propanol in porogen solvent, %M – percentage content of monomers in polymerization mixture. %M

27

35

%D 60 40 20 20 20 20 20 20 20 18 26 32 40 55

% H2O 20 20 20 40 45 50 30 40 50 20 20 20 20 20

%P 20 40 60 40 35 30 50 40 30 62 54 48 40 25

KF x 10-15 [m2] 9.1 28 0.4 20 49 0.9 0.4 26 3.5

RSD [%] 8.7 4.3 2.9 2.4 1.9 1.6 2.8 2.1 5.3

Table 2. The lists of signals recorded on the MS spectra of eluates (T1 and TS1). Signals of low (*), medium (**) and strong (***) intensity.

Sequence of identified polypeptide

MH+ calculated Missed cleavage sites

YLYEIAR ALKAWSVAR DTHKSEIAHR CASIQKFGER FKDLGEEHFK SLHTLFGDELCK RHPEYAVSVLLR FWGKYLYEIAR LRCASIQKFGER VTKCCTESLVNR DTHKSEIAHRFK LGEYGFQNALIVR FGERALKAWSVAR QTALVELLKHKPK DAFLGSFLYEYSR KFWGKYLYEIAR KQTALVELLKHKPK KVPQVSTPTLVEVSR MPCTEDYLSLILNR DDPHACYSTVFDKLK ALKAWSVARLSQKFPK RPCFSALTPDETYVPK LGEYGFQNALIVRYTR LFTFHADICTLPDTEK CCAADDKEACFAVEGPK QIKKQTALVELLKHKPK ADEKKFWGKYLYEIAR RHPYFYAPELLYYANK YTRKVPQVSTPTLVEVSR LKPDPNTLCDEFKADEKK HLVDEPQNLIKQNCDQFEK DTHKSEIAHRFKDLGEEHFK RPCFSALTPDETYVPKAFDEK QEPERNECFLSHKDDSPDLPK VHKECCHGDLLECADDRADLAK LKPDPNTLCDEFKADEKKFWGK

927.493 1001.589 1193.602 1195.589 1249.621 1419.694 1439.812 1445.758 1464.774 1466.709 1468.766 1479.795 1490.823 1504.921 1567.743 1573.853 1633.016 1639.938 1724.835 1795.832 1830.075 1880.921 1900.008 1907.921 1927.798 2002.253 2017.054 2045.028 2060.150 2148.064 2355.140 2424.205 2471.191 2541.167 2612.165 2666.328

0 1 1 1 1 0 1 1 2 1 2 0 2 1 0 2 2 1 0 1 3 0 1 0 1 3 3 1 2 2 1 3 1 2 2 3

m/z measured T1 927.489* 1001.585** 1193.599** 1195.591*** 1249.630* 1419.699* 1439.816* 1445.754* 1464.780* 1466.644* 1468.774*** 1479.800** 1490.750** 1504.923* 1567.755* 1573.857** 1633.026* 1639.942** 1830.078* 1927.797* 2002.259* 2017.043 2148.049* 2424.197* 2471.184* 2541.145* -

TS1 1001.621* 1195.624*** 1439.860* 1445.797* 1464.824*** 1468.804* 1479.849*** 1490.799** 1567.815** 1573.904** 1639.992* 1724.895* 1795.904* 1830.134** 1880.986*** 1900.071*** 1907.989* 2002.319* 2045.092* 2060.214* 2355.196* 2424.259* 2471.250*** 2541.223*** 2612.207* 2666.330*