Biofunctionalization and self-interaction chromatography in PDMS microchannels

Biochemical Engineering Journal 67 (2012) 111–119

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Biofunctionalization and self-interaction chromatography in PDMS microchannels Kedar S. Deshpande a,b , Shreyas Kuddannaya a , Judith Staginus c , Peter C. Thüne d , Louis C.P.M. de Smet c , Joop H. ter Horst b , Luuk A.M. van der Wielen a , Marcel Ottens a,∗ a

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628BC Delft, The Netherlands Intensified Reaction & Separation Systems, Delft University of Technology, Leeghwaterstraat 44, 2628CA Delft, The Netherlands c Department of Chemical Engineering, Delft University of Technology, Julianalaan 136, 2628BL Delft, The Netherlands d Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, 5600 MB Eindhoven, The Netherlands b

a r t i c l e

i n f o

Article history: Received 18 November 2011 Received in revised form 21 May 2012 Accepted 30 May 2012 Available online 9 June 2012 Keywords: Protein immobilization Self-interaction chromatography PDMS Osmotic second virial coefficient Microfluidic chip

a b s t r a c t In this paper we present an experimental protocol for protein immobilization on polydimethylsiloxane (PDMS) polymer surfaces and the subsequent application of a chromatographic PDMS microfluidic chip to measure protein–protein interactions. The PDMS surface modification steps are quantitatively and qualitatively experimentally analyzed using an array of techniques (water contact angle measurement, fluorescence spectroscopy and X-ray photoelectron spectroscopy). The protocol involves PDMS acidic surface activation using a potassium disulfite/potassium peroxidisulfate/acrylic acid mixture, followed by amination with 3-aminopropyl diethoxymethylsilane, followed by glutaraldehyde grafting and subsequent covalent protein binding. The applicability of such a miniaturized PDMS-based microfluidic system has been exemplified by measuring protein–protein interactions in a fast and accurate fashion for three model proteins, namely: hen egg white lysozyme, bovine ribonuclease-A and ␣-chymotrypsinogen. The protein interaction results align well with existing literature data using different materials and techniques. As the fabrication process for PDMS-based microstructures is relatively cheap, quick and requires limited lab expertise/access to specialized equipments, we consider that the implementation of such a flexible, easy to fabricate, PDMS-based microfluidic system for estimating protein interactions an important step toward quickly mapping protein phase behavior and measuring protein (self/cross) interactions in complex biological systems. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Proteins play a central role in cellular processes. For elucidating the biochemical role of a given protein, it is necessary to obtain its three-dimensional structure from X-ray diffraction, which in turn requires growing good quality crystals. More often than not, the actual bottleneck is determining solution conditions for protein crystal growth. There are no general guidelines for growing high-quality protein crystals and the steps followed are more often trail-and-error based. This has prompted interest in studying the behavior of protein solutions and to bring further rationale into this field. Amongst these efforts is the use of the osmotic second virial coefficient (B22 ) of a protein solution. This parameter B22 has been widely used to study weak protein–protein interactions in aqueous solutions. Many experimental studies have also demonstrated that the B22 parameter is closely related to protein solubility and protein

∗ Corresponding author. Fax: +31 15 278 2355. E-mail address: [email protected] (M. Ottens). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.05.012

crystallization behavior [1–4]. Successful protein crystallization has been reported for B22 values obtained in a slightly negative narrow range (−1 × 10−4 to −8 × 10−4 mol ml g−2 ), known as the “crystallization slot” [1–4]. For large positive B22 values, crystallization is unlikely due to high protein–protein repulsive forces, whereas for a large negative B22 , high protein–protein attractive forces result in the formation of amorphous precipitates. Thus the parameter B22 can be used as an initial screening tool towards finding solution conditions that promote protein–protein interactions for favorable crystallization behavior. Different techniques exist to measure B22 like static light scattering (SLS) [1], neutron scattering [5], small angle X-ray scattering (SAXS) [6] and membrane osmometry (OSM) [7], all with its specific characteristics and requirements. It has been demonstrated that self-interaction chromatography (SIC) is a relatively easy technique to screen the B22 [8]. Briefly, proteins immobilized on a stationary phase interact with the same protein molecules in the mobile phase and the retention time of the mobile protein pulse under isocratic elution conditions is measured. The retention behavior in such a SIC experiment reflects the average protein–protein interactions and is

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related to B22 [9]. Current applications, views on evaluating protein self-interactions through a B22 screening approach, the potential use of SIC in stable solution formulation and the crystallization of proteins have been well documented in a recent article [10]. The SIC technique has shown to be a reliable means to elucidate selfassociation of both native and denatured proteins in the presence of different cosolvents [11]. SIC has been mostly used on a ml scale, and recently some efforts have been devoted to miniaturize the approach [12,13]. Compared to the conventional ml-scale systems, microfluidic systems have several advantages including reduced sample consumption, short analysis time, faster mixing and the possibility for high degree of automation [14]. Due to their excellent optical properties and well-documented surface properties and chemistries, glass and silica have been predominantly used as the building material in the design and fabrication of microfluidic devices. Recently we presented a proof-of-concept of a miniaturized SIC technique where proteins were covalently linked to the microchip glass channel without the use of chromatographic resins [13]. However, the fabrication process of glass chips is time-consuming and requires access to specialized facilities. Also, certain stability issues persist while using silica as the base material for microchip fabrication limiting the applicability of the bio-functionalization protocols at specific conditions [15]. This has stimulated research on alternative materials, including cost effective, durable and easily processable polymeric materials as building blocks for microfluidic devices. Nowadays, polydimethylsiloxane (PDMS) is the most widely studied and used polymer for making chips [16,17]. PDMS has numerous advantages such as its feasibility for replica molding, controllability of surface chemistry, optical transparency and favorable elastomeric properties. The physical and chemical properties of PDMS have been widely exploited in the fabrication of microfluidic devices positioning PDMS as a practically feasible alternative to glass and silica [18,19]. Several strategies (covalent/non-covalent, site specific/non site specific) exist for immobilizing proteins onto the surface of PDMS substrate [20–22]. A microchip-based device for protein self-interaction chromatography using PDMS was reported [23], which comprised of normal preparative chromatography particles packed in a PDMS microchannel. In this work, we present a simple experimental approach for immobilizing proteins in PDMS microchannels, without the use of chromatographic particles. We have analyzed the protein immobilization process after every modification stage using multiple analytical tools to better understand and to quantitatively assess the effect of each surface modification step. We further demonstrate the use of the biofunctionalized PDMS microchip for SIC runs by measuring the B22 for several proteins (lysozyme, ribonuclease A and ␣-chymotrypsinogen). The overall biofunctionalization approach, as shown in this work, together with the possibility to measure protein–protein interactions, such as B22 as an example application, can be conveniently applied to other bioapplications and has potential to be scaled out in a high-throughput platform.

2. Materials and methods 2.1. Reagents and chemicals Potassium disulfite (K2 S2 O5 ) (P2522), potassium peroxidisulfate (K2 S2 O8 ) (P5592), anhydrous acrylic acid (147,230), triethoxysilane (APTES) (440,140), (3(3-aminopropyl) aminopropyl) diethoxymethylsilane (APDEMS) (371,890), glutaraldehyde (G5882) and ethanolamine (E0135) were purchased from Sigma–Aldrich, Germany. The salts, sodium citrate (0280), citric acid (2064) and sodium chloride (0278) were

purchased from JT Baker, Deventer, Netherlands, whereas ammonium sulfate (A801711727) and sodium phosphate (04629) were purchased from Merck Co., Germany and Sigma–Aldrich, Germany, respectively. Lysozyme from chicken egg white (L6876), ribonuclease-A (RNAse) from bovine serum (R5503), ␣-chymotrypsinogen type-II (C4879) were purchased from Sigma–Aldrich. Sylgard® 184 silicone elastomer kit was purchased from Dow Corning (USA).

2.2. Equipments The micro-SIC set-up consisted of a nano-pump (LC-20AD, Shimadzu), a splitter (MX7960- 000, Rheodyne), UV/vis spectrophotometer (GENios, Tecan) and the Shimadzu LC solutions (Version 1.22 sp1) software. Water contact angles were measured using a FM40 Easy Drop goniometer (Krüss GmbH, Germany) with droplet size controlled by a Gilmont syringe, fluorescence measurements were performed using a Leica DM5500B fluorescent microscope (Leica Microsystems Inc.) and image analysis software (Q-win Pro, 3.1.0, Leica). XPS measurements were performed on Kalpha (Thermo Fisher Scientific) equipped with a monochromatic AlK␣ X-ray source. Air plasma activation was performed with Harrick PDC-002 plasma cleaner.

2.3. Fabrication and modification of PDMS microchips Sylgard® 184 oligomer and curing agent were thoroughly mixed in the mass ratio of 10:1, degassed for 20 min and poured over the master (∼10 cm in diameter, 500 ␮m in thickness), then cured at 70 ◦ C for 150 min. The master was then kept overnight inside an oven maintained at 60 ◦ C. The cured PDMS layer was carefully pealed off the master and cut to the required dimensions with holes punched at the reagent inlet and exit. Similarly, a flat piece of PDMS without channel features was molded using a microscopic glass slide as master. This resulted in a serpentine design microchannel with 100 ␮m × 100 ␮m × 43.2 cm (diameter × width × length) dimensions. The PDMS slabs with microchannel features and flat PDMS onto the glass support were cleaned ultrasonically, subsequently with water, ethanol and water for 10 min, respectively, before drying them in a stream of nitrogen gas. Then both types of surfaces were treated with air plasma for 60 s at an RF coil power of 29.6 W and sealed together forming an irreversibly bonded PDMS microchip on a glass support.

2.4. Biofunctionalization of protein on PDMS microchannel The PDMS microchannel surface was further treated in a series of modification steps which involved surface activation, amination, cross-linking and finally protein attachment on the surface. The experiments were performed at room temperature unless stated otherwise.

2.4.1. Surface activation of microchannels Three different methods were analyzed to clean and activate the flat PDMS surfaces; air plasma, the use of piranha solution (1:3 vol.% mixture of H2 O2 and H2 SO4 for 10 min) and the use of an acrylic acid-containing salt mixture (here referred to as: acid mixture) (0.05 g of K2 S2 O5 , 0.1 g of K2 S2 O8 and 5 ml of acrylic acid dissolved in 50 ml water with a reaction time of 30 min). During all these processes, reactive silanol groups are generated on the PDMS surface [24], which facilitates subsequent surface modification. The resulting surface is also rendered hydrophilic and has therefore improved wettability.

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2.4.2. Amination reaction The activated microchannels were subsequently aminated using two aminating agents, APTES and APDEMS, with reaction times of 1 h, 24 h and 48 h at room temperature and at 2 vol.% concentration in water. After reaction, the surfaces were sonicated for 5 min in water to remove any physically adsorbed APTES or APDEMS molecules from the surface. 2.4.3. Aldehyde group grafting The aminated microchannel surfaces were then treated with 2.5 vol.% glutaraldehyde in water for 1 h at room temperature, followed by thorough washing with 5 mM sodium phosphate buffer at pH 7.0 [15,25,26]. After treatment the channels were sonicated for 5 min in water and dried with nitrogen gas. 2.4.4. Protein biofunctionalization A protein solution of 1 mg/ml in 5 mM sodium phosphate buffer (pH 7.0) was continuously passed through the aldehydefunctionalized microchannel surface overnight at +4 ◦ C [27]. Following this reaction, 0.2 M ethanolamine in water was passed through the microchannel to cap the unreacted glutaraldehyde groups [15]. Finally, the microchannel was thoroughly washed with 5 mM sodium phosphate buffer (pH 7.0) and stored at +4 ◦ C until further use. 2.5. Analytical techniques for surface characterization Different analytical techniques were deployed to characterize the protein immobilization process at every stage of the (bio)functionalization. 2.5.1. Water contact angle measurement Water contact angles were determined by measuring the angle formed between the solid and tangent to the drop surface. The typical droplet volume was in the order of 10 ␮l. Droplets of MilliQ water (resistivity >10 M) were contacted on several different spots on the surface, and the water contact angle was measured using the static sessile drop tangent method and analyzed with drop shape analysis software. At least three measurements were taken to obtain the average contact angle. 2.5.2. Fluorescence analysis 2.5.2.1. Silane labeling. Activated PDMS substrates were treated individually with two aminating agents, APTES and APDEMS, for 24 h at ambient temperature (Section 2.4.2). After amination, an activated amine-specific dye (5(6)-carboxyfluorescein, N-hydroxysuccinimide ester) (Pierce, USA), was chemically immobilized onto the surface. The conjugation protocol was adopted from the literature [28]. Following conjugation, the labeled substrates were sonicated for 10 min in water. Plain PDMS was also treated with the dye as the reference sample. Fluorescence intensities observed at 490 nm were compared with reference samples. 2.5.2.2. Protein labeling. Proteins immobilized in the microchannels were labeled to determine the success of immobilization as well as to monitor their stability at different pH conditions. A protein solution (1 mg/ml in phosphate buffer) was labeled with NHS Rhodamine (EZ-LabelTM Pierce NHS-Rhodamine protein labeling kit) maintaining a 1:2 mol ratio of protein: dye. This ratio was adequate with no free dye present as found after gradient elution using a D-SaltTM Dextran desalting column experiment. Fluorescence measurements were performed both on plain PDMS surfaces (0.5 cm × 0.5 cm) and also on protein-coated microchannels. Calibration of fluorescent intensities was performed by passing the protein labeled solutions in a concentration range of 0–200 ␮g/ml through the microchannel. Plain, unmodified PDMS also treated

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overnight with labeled protein solution following same protocol, washed several times with appropriate buffer, was used as the reference sample. 2.5.3. X-ray photoelectron spectroscopy (XPS) XPS spectra of samples (∼1 cm × ∼1 cm) were taken in normal emission at 10−9 mbar within 10 min. All C1s peaks corresponding to hydrocarbons were calibrated to a binding energy of 284.8 eV to correct for the energy shift caused by charging. Spectra were analyzed using the Casa-XPS. The surface elemental composition was derived from region scans taken at 50 eV pass energy using empirical Wagner sensitivity factors. 2.6. Determination of the immobilized protein concentration The amount of immobilized protein was determined using a micro-BCA (bicinchonic acid) assay (Pierce Inc.). For an efficient reaction of the surface-bound proteins with the BCA working reagent, the microchannel device was cut into shorter sections and incubated with the working reagent for 1 h at 60 ◦ C. After cooling of the reagent, the absorbance of the supernatant was measured at 562 nm. The amount of protein in the microchannel was calculated based on comparing the absorbance reading with the calibration curve obtained for the same protein. The calibration curve was generated using varying, but known, amounts of same protein solutions. 2.7. Self-interaction chromatography (SIC) and B22 calculation SIC experiments were performed for three proteins: lysozyme, ribonuclease A and ␣-chymotrypsinogen. For lysozyme, 5 mM sodium phosphate buffers with NaCl concentrations ranging from 0 to 0.8 M at pH 7.5 and 5 mM sodium acetate with NaCl concentrations ranging from 0 to 0.8 M at pH 4.5 were used. For RNAse, 5 mM sodium citrate buffers with NaCl concentrations ranging from 0 to 1.5 M at pH3.0 and for ␣-chymotrypsinogen, citric acid buffers with NaCl concentrations ranging from 0 to 0.4 M at pH 3.0 and pH 6.8 were used. Fig. 1 shows a schematic representation of the setup used in the micro-SIC runs for protein B22 estimation. Briefly, protein powder was dissolved in the appropriate buffers at final concentration of 1 mg/ml buffer. Prior to every SIC run, the microchip was equilibrated for 5–6 column volumes with the respective buffer. The buffer flow rate was maintained using the nano-pump at 10 ␮l/min. Precautions were taken that both the running buffer solution and the protein sample were filtered with 0.2 ␮m pore size filters before circulating in the system. It was also checked that there were no leaks, ensuring a constant flow rate and back pressure in the system. A protein pulse of 2 ␮l was injected by using a splitter arrangement. The protein pulse at the chip inlet and outlet was detected at 280 nm by means of (micro) UV detector. The retention times were computed from individual chromatograms using first moment analysis. The difference in retention time between protein pulse inlet and outlet was noted for individual background salt concentrations in the running buffer. B22 (mol ml g−2 ) was calculated from the chromatographic retention volume data obtained from the micro-SIC runs as [13] B22 =

Vo − Vr N × Mw

(1)

where Vo is the dead volume or retention volume in the proteinfree chip (ml); Vr is the retention volume in protein immobilized chip (ml); N is the amount of protein immobilized (g), Mw is the molecular weight of the protein (g mol−1 ). All the experiments were conducted at room temperature (22 ± 0.5 ◦ C).

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Fig. 1. PDMS microchip-based SIC experimental setup.

Table 1 Average water contact angles measured 5 min after using different activation techniques on plain PDMS surfaces. Substrate

Average water contact angle (degrees)

Plain PDMS Air plasma activated PDMS Acid mixture activated PDMS Piranha solution activated PDMS

108 18 88 80

± ± ± ±

1 1 1 1

3. Results and discussion During each surface modification step, we qualitatively and quantitatively characterized the modified surfaces. These results led to the optimal reaction conditions favorable for covalent protein attachment in the PDMS microchannels (Section 3.1). The microchannels were subsequently tested for self-interaction chromatography runs for B22 determination (Section 3.2). 3.1. Surface characterization PDMS substrates coated with APTES and APDEMS and modified by a glutaraldehyde treatment were characterized by several analytical techniques including water contact angle analysis, fluorescence spectroscopy and X-ray photoelectron spectroscopy measurements. Finally, the degree of protein immobilization was quantified by a micro-BCA analytical assay of proteins on the surface and compared with a theoretical estimation. 3.1.1. Water contact angle (WCA) measurements Effect of activation techniques. PDMS is inherently hydrophobic (WCA >100◦ ). Protein molecules directly immobilized on hydrophobic surfaces face risk of denaturation. PDMS surfaces can be temporarily rendered hydrophilic by exposing them to different environments, e.g. air plasma and aqueous acidic solutions, and protein molecules can then be immobilized with a lower risk of denaturation. Water contact angles were measured for PDMS surfaces that have been activated under different conditions (Table 1). At least three measurements were taken to obtain the average water contact angles. The changes in the water contact angles indicate chemical changes of the PDMS surfaces upon the different activation steps, i.e. more active groups will be present for lower WCAs. The data

presented in Table 1 shows that surfaces activated with air plasma have a lower contact angle (∼18◦ ) as compared to the surfaces activated with acid mixture activation (∼88◦ ) and piranha solution activation (∼80◦ ). Based on that, air plasma activation would be the method of choice, but this method is not easily compatible with substrates that have rather complex channel features. Since both wet-chemical activation methods are easy to be applied inside microchannels, we studied these routes for their activation effects on the PDMS microchannels. It is important to note that the contact angles of the activated PDMS surfaces were measured within 5 min after activation. For larger times, the contact angles were found to increase due to the hydrophobic recovery of PDMS, which is in agreement with literature [29,30]. Although the contact angles related to the wet-chemical methods are higher than those of plasma-activated samples, the reaction with silanes was possible (vide infra), indicating that active groups are present. 3.1.2. Fluorescence measurements 3.1.2.1. Silane reactivity measurements. Fluorescence measurements were performed to estimate the loading of two different silanes, APTES and APDEMS, on PDMS surfaces activated with acid mixture and piranha solution. After amination, the surfaces were fluorescently labeled with 5,6 carboxyfluorescein dye and the fluorescence intensity of the resulting substrates was measured. The mean gray values of intensities reported below are obtained as the average of three measurements. The obtained intensities provide important information on the relative reactivity of the surfacebound amino groups of the two silanes used. As shown in Table 2, irrespective of the activation technique used, higher intensities were observed in the samples modified with APDEMS (samples 2, 4) in comparison with APTES (samples 1, 3). This confirms the relatively higher reactivity of APDEMS molecule compared to APTES as also confirmed by XPS data Table 2 Mean gray intensities for 5,6 carboxyfluorescein dye measured on surfaces of PDMS samples. Samples

Description

Mean gray intensity

1 2 3 4 5

Acid mixture activated PDMS + APTES Acid mixture activated PDMS + APDEMS Piranha solution activated PDMS + APTES Piranha solution activated PDMS + APDEMS Plain PDMS + dye (reference sample)

23,976 26,993 22,664 27,147 150

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Fig. 2. pH stability test after protein immobilization; left: APTES route, right: APDEMS route.

(see Section 3.1.3). APTES has been widely used in the surface modification of glass substrates and silica surfaces and also the use of APDEMS has been reported [31]. It is however, difficult to directly compare the results on PDMS with the glass/silica-based modification, due to the different nature of the surfaces. The main difference is related to the possibility of the hydrophobic recovery of chemically modified PDMS surfaces. What can be said, however, is that APTES and APDEMS have a different number of reactive ethoxy groups; APTES has three ethoxy groups, whereas APDEMS has only two. Based on that, APTES is more prone to multilayer formation as compared to APDEMS due to a higher change of intermolecular cross-linking, which might cover some of the surface-bound amino groups. It must be noted that the structure and reactivity of the resulting multilayer is different from that of APTES monolayer [28]. Also it cannot be ruled out that APTES has different properties in terms of its ability to block the surface-bound amino groups as compared to APDEMS molecules, which might result in a reduced reactivity toward further functionalization in the case of APTES. In summary, the silanization reaction with APDEMS is easier to control due to the presence of double ethoxy moiety [32]. As a result, APDEMS molecules can firmly fix on the activated surface, forming a more uniform monolayer than any other silanes in aqueous solutions. Although both wet-based activation techniques, acid mixture and piranha solution, resulted in comparable results, we decided to discard piranha solution based activation route due to its potentially strong acidic and oxidizing properties.

3.1.2.2. pH stability analysis. Fluorescence measurements were performed on plain PDMS surfaces (0.5 cm × 0.5 cm) and also on microchannels onto which labeled protein molecules were immobilized (following the acid mixture-based, wet activation route). Plain PDMS treated overnight with individual labeled protein solution, sonicated and washed thoroughly was used as a reference sample. From Fig. 2, it can be seen that relatively higher intensities were observed for protein-immobilized substrates treated within the acidic pH range. The immobilization protocol where silanization was performed using APDEMS (Fig. 2, right) showed relatively higher fluorescence intensities together with marginal drop in intensities at alkaline pH range in comparison to its APTES counterpart (Fig. 2, left). The decrease in the fluorescent intensity at alkaline pH (>8.5) is the indicator of silane layer dissolution and thereby protein detachment from the surface [15], while the limited stability of imines at acidic pH could explain the reduced fluorescent intensities at the lower pH values (which is found to be most apparent for lysozyme).

3.1.3. X-ray photoelectron spectroscopy (XPS) In order to study the changes in elemental composition on the acid mixture activated PDMS surface during the protein immobilization steps, we performed XPS analysis. From Table 3, it can be seen that plain PDMS shows an atomic ratio of C/O/Si close to 2/1/1, which is typical for a clean surface. For both the silane agents used, the % loading of nitrogen and N/C ratio significantly increases upon addition of lysozyme, which is in line with the presence of amino groups on the surface. The N/C ratio for the top protein layer (excluding the carbon content of the siloxane layer) was estimated to be 0.21 post APTES modification and 0.24 post APDEMS modification. The resultant N/C ratios are close to theoretical N/C ratio of 0.29, calculated using amino-acid sequence for native lysozyme. The XPS data shows that anchoring of the protein was successful for both the routes. 3.1.4. Experimental protocol for protein immobilization The investigated reaction conditions for derivatization of the amine layer, a homo-bi-functional glutaraldehyde layer and immobilized protein were selected based on all analysis results. The final protocol was selected to be activation using an acid mixture followed by amination by 2 vol.% APDEMS solution (24 h), followed by 2.5 vol.% glutaraldehyde (1 h) grafting and finally covalently linking the protein molecule onto the surface (overnight at +4 ◦ C). All the PDMS microchannels were treated using this experimental protocol. 3.1.5. Determination of the amount of immobilized protein The amount of each protein for a monolayer coverage (␮g/cm2 ) was calculated theoretically by estimating the number of immobilized molecules which could occupy the total inner surface area of the microchannel (1.105 cm2 , channel diameter 100 ␮m) compared to the area occupied by a single protein molecule.

Fig. 3. Orientation possibilities of immobilized protein molecules inside a microchannel surface.

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Table 3 XPS elemental analysis of PDMS substrates after acid mixture activation and different subsequent surface functionalization steps. Sample

%Carbon (C)

%Nitrogen (N)

%Oxygen (O)

%Silicon (Si)

%Total

Ratio (N/C)

Plain PDMS PDMS + APTES PDMS + APTES + glutaraldehyde PDMS + APTES + glutaraldehyde + lysozyme PDMS + APDEMS PDMS + APDEMS + glutaraldehyde PDMS + APDEMS + glutaraldehyde + lysozyme

53.5 51.9 58.9 56.7 79.3 64.7 57.8

0.5 1.5 2.2 6.7 2.5 3.8 8.8

23.1 24.2 22.5 24.1 13.6 22.1 23.1

22.9 22.4 16.4 12.6 4.7 9.5 10.4

100.0 100.0 100.0 100.1 100.1 100.1 100.1

0.01 0.03 0.04 0.12 0.03 0.06 0.15

Table 4 Example of a theoretical calculation of a monolayer coverage (␮g/cm2 ) for lysozyme. Description

Units

Values

a. Molecular weight b. Hydrodynamic diameter [33] c. Avogadro number d. Surface area of single protein molecule e. Diameter of the microchannel f. Length of the microchannel g. Area of the microchannel (area of hemi circular cylinder + area of flat surface) h. Theoretical number of immobilized proteins (g/d) i. Theoretical amount of immobilized proteins (h × a/c × g)

␮g mol−1 cm mol−1 cm2 cm cm cm2 – ␮g/cm2

14,400 × 106 3.11 × 10−6 6.023 × 1023 7.59 × 10−14 0.01 43.2 1.10 1.45 × 1013 0.314

Fig. 3 outlines a schematic representation of three possible orientation schemes post protein immobilization. Based on the ellipsoid geometry consideration, it can be noted that scheme A (site specific attachment) would give highest amount of surface coverage and scheme B (surface amino based attachment) would give the lowest amounts immobilized. Although, with a proper selection of pH conditions during the immobilization process, a particular scheme (A or B) can be realized, practically it is difficult to control and achieve an exclusive scheme (A or B). Since the second virial coefficient is a measure of protein interactions averaged over all possible orientations, we rely on the fact that immobilization occurs at multiple sites on the protein surface, leading toward multiple immobilized orientations. Hence, for calculation purposes, we have considered scheme C with an average hydrodynamic configuration of the protein molecule. This, in principle, gives the amount of surface coverage values in between scheme A and scheme B. Similar calculations (as in Table 4) were performed for ribonuclease A

Table 5 Comparison of theoretical and experimental (micro-BCA assay) amounts of immobilized protein in a PDMS microchannel. Protein

Theoretical amount for monolayer coverage (␮g/cm2 )

Experimental amount estimated by a micro-BCA assay (␮g/cm2 )

Lysozyme Ribonuclease A ␣-Chymotrypsinogen

0.314 0.201 0.367

0.252 ± 0.0095 0.145 ± 0.021 1.431 ± 0.016

and ␣-chymotrypsinogen for the theoretical amount immobilized for a monolayer coverage. Table 5 shows the amount of protein immobilized in the microchannel estimated experimentally by a micro-BCA technique in comparison to the theoretically calculated (monolayer) values. The micro-BCA approach of estimating the amount of

Fig. 4. Left: Chromatograms for SIC measurements on lysozyme at pH 7.5 at different NaCl concentrations; right: B22 trend of lysozyme at pH range 7.0–7.6. Red triangle with red solid line, pH 7.5, micro-SIC (this work), black circle with long dash line, pH 7.5, micro-SIC [13], black square with dash-dot-dot line, pH 7.6, SIC, [34], open circle with dash-dot line, pH 7.0, SIC [33], open triangle with dotted line, pH 7.6, SIC [35]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 5. Left: Chromatograms for SIC measurements on lysozyme at pH 4.5 at different NaCl concentrations; right: B22 trend of lysozyme at pH 4.5. Red triangle with red solid line, pH 4.5, micro-SIC (this work), black circle with long dash line, pH 4.5, micro-SIC [13], open circle with dash-dot line, pH 4.5, SIC [33], black square with dash-dot-dot line, pH 4.5, SIC [34]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

protein immobilized on surfaces gives more insight into a submono/mono/multilayer immobilization tendency of a particular protein using a specified immobilization chemistry approach. The knowledge of surface coverage is important as the use of Eq. (1) in estimating the B22 is valid for sub-mono/mono layer coverage conditions. The experimental surface coverage values by micro-BCA assay in Table 5 for lysozyme and ribonuclease A indicates sub-monolayer coverage of proteins on the surface, whereas a possible multilayer coverage could be seen for the ␣chymotrypsinogen case. The surface coverage, and thereby a mono layer coverage possibility, could be controlled and achieved by using appropriate immobilization parameters. Multiple monolayer (multilayer) immobilizations might lead to higher protein consumption, potential protein denaturation/aggregation and thereby measuring multiple protein interactions.

of NaCl, a typical salting-out behavior. The correlation of micro-SIC data (red solid line) with literature data indicates reproducibility and validity of the micro-SIC approach with respect to larger scale B22 measurement techniques. Fig. 6 shows the mapping of ribonuclease A B22 profile from the microchip data (red solid line) compared to literature data obtained under the same or similar conditions [33]. At pH 3, the virial coefficients indicate repulsive interactions at low ionic strength and attractive interactions at high ionic strength. Hence there is an increase in the retention volume with increasing salt concentration and thus a seemingly decreasing trend for B22 values. Nevertheless, the principle message here is that the micro-SIC technique employed can be used for accurate and fast estimation of crystallization condition via protein–protein interaction parameters, like the B22 , with minimal consumption of tested proteins.

3.2. Self-interaction chromatography and B22 measurements The proteins were immobilized in the PDMS microchannels using the optimized protocol (Section 3.1.4). Before sample injection, the microchannel was equilibrated to 5–6 column volumes for each running buffer condition. The SIC experiments were carried out to monitor protein retention times as a function of background salt concentration in the mobile phase under different pH conditions. The retention volume of the protein free column Vo was calculated at the condition in which no protein–protein interaction is seen. This value was calculated by measuring the retention volume (Vr = Vo ) at a condition (pH, salt concentration) mentioned in literature [1,8,23] for which B22 was calculated to be 0. B22 was determined from the obtained values of retention volume (Vr ), corrected for extra column effects, by using the Eq. (1). A typical time frame, excluding equilibration, for a single B22 run is in the range of 3–5 min. For each condition, at least five B22 measurements were taken and average values are reported. Figs. 4 and 5 show the mapping of lysozyme’s B22 profile from the microchip data, at different pH values, compared to literature data obtained under the same or similar conditions. The values, overall trend and magnitude of error were found to be in good agreement with the reported B22 values from open literature at similar conditions. The B22 measured for lysozyme at pH 7.5 and pH 4.5, both show a decreasing trend with increasing ionic strength

Fig. 6. B22 trend of ribonuclease-A at pH 3.0 for different NaCl concentrations. Red triangle with red solid line, pH 3.0, micro-SIC (this work), open circle with dashdot line, pH 3.0, SIC [33]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 7. Left: B22 trend of ␣-chymotrypsinogen at pH 6.8 for different (NH4 )2 SO4 concentrations; red triangle with red solid line, pH 6.8, micro-SIC (this work, N estimated from micro-BCA assay), open square with long dash line, pH 6.8, micro-SIC (this work, N calculated theoretically), open circle with dash-dot line, pH 6.8, SIC [33], black circle with dotted line, pH 6.5, OSM [36]. Right: B22 trend of ␣-chymotrypsinogen at pH 3.0 for different (NH4 )2 SO4 concentrations; red triangle with red solid line, pH 3.0, micro-SIC (this work, N estimated from micro-BCA assay), open square with long dash line, pH 3.0, micro-SIC (this work, N calculated theoretically), open circle with dash-dot line, pH 3.0, SIC [33], open triangle with dash-dot-dot line, pH 3.0, SEC [37]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 7 shows the mapping of ␣-chymotrypsinogen’s B22 profile from the microchip data (red solid line), at different pH values, compared to literature data obtained under the same or similar conditions. Chymotrypsinogen is known to be susceptible to cleavage due to autocatalytic activation of chymotrypsinogen to chymotrypsin above pH 5.0. Nevertheless, the enzymatic activity of chymotrypsin solutions monitored for 24 h was not found to vary above 5% in a recent study [33]. In view of this observation, to restrict the autocatalytic cleavage process, all the B22 measurements were performed within a time frame of 12 h. In the Fig. 7, the B22 measured for ␣-chymotrypsinogen show rather flat profiles for pH 6.8 and for pH 3.0. The flat B22 profile, or no change in interaction between protein molecules upon changing the salt concentration, could be possibly due to multiple layers of immobilized ␣-chymotrypsinogen which would lead to a too high N value in Eq. (1). It can be observed (Table 5) that the amount of ␣chymotrypsinogen is about 3.8 times higher than that calculated for a monolayer coverage. The higher amounts of ␣-chymotrypsinogen measured could be due to the fact that micro-BCA analytical assay was performed with lysozyme as the standard and hence may have lead to discrepancies in interpreted values of absorbance figures in case of ␣-chymotrypsinogen. The B22 measurements are best reliable in conditions where the immobilized protein amounts are ideally lower or close to monolayer coverage. For comparison purposes, we estimated the ␣-chymotrypsinogen B22 trends using the theoretical amount for monolayer coverage (Table 5). The trends obtained (Fig. 7, open square with long dash lines) suggest that controlling the amount of immobilized protein to a sub/monolayer coverage value does have a significant impact on the overall B22 trends.

4. Conclusions We demonstrate a miniaturized flexible version of selfinteraction chromatography by immobilizing proteins on PDMS microchannel. The fabrication of PDMS microchannels is straightforward and immobilization chemistry for functionalizing biomolecules, such as proteins, has been shown to be

successful at micron-dimensions. We consider that such a flexible, easy to fabricate, miniaturized microchip to probe protein–protein interactions in solutions by the SIC approach is an important step toward quickly mapping protein phase behavior of more complex systems. The application of such microscale fabricated devices can be applied in a range of applications, such as a pre-screening tool in drug delivery, estimating cross-interactions in solutions and studying the behavior of protein-drug molecules for bio-pharma applications.

Acknowledgement This research was financially supported by the Delft Research Centers, SIP (Sustainable Industrial Processes) and LST (Life Science and Technology). Dr. Michel Rosso (TU Delft) is kindly acknowledged for his help with the initial surface modifications and stimulating discussions. Judith Staginus and Louis C.P.M. de Smet would like to thank Wetsus, Centre of Excellence for Sustainable Water Technology, for financial support.

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